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Factors Influencing Adenovirus-Mediated Airway Transduction in Fetal Mice
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doi:10.1016/j.ymthe.2005.02.020
Factors Influencing Adenovirus-Mediated Airway Transduction in Fetal Mice S. M. K. Buckley,1,* S. N. Waddington,1 S. Jezzard,1 L. Lawrence,2 H. Schneider,3 M. V. Holder,1 M. Themis,1 and C. Coutelle1 1
Gene Therapy Research Group, Department of Cell and Molecular Biology, SAF Building, Imperial College, South Kensington, London SW7 2AZ, United Kingdom 2 Leukocyte Biology, Department of Cell and Molecular Biology, SAF Building, Imperial College, South Kensington, London SW7 2AZ, United Kingdom 3 Childrens Hospital, University of Erlangen, Loschgetr. 15, D-91054 Erlangen, Germany *To whom correspondence and reprint requests should be addressed.
Available online 12 April 2005
Intra-amniotic injection of adenovirus allows transduction of the fetal airways following natural fetal breathing movements. This administration method is promising for use in gene therapy for cystic fibrosis and other diseases for which the main target for exogenous gene expression is the lung. Here we have investigated factors that may affect the efficacy of gene transfer to the murine fetal lung. We examined marker compound distribution and transgene expression (from a firstgeneration adenoviral vector) at different stages of development. This demonstrated that fetal breathing movements at 15–16 days of gestation are of sufficient intensity to carry marker/vector into the fetal lungs. These movements can be significantly stimulated by the combination of intraamniotic theophylline administration and postoperative exposure of the dam to elevated CO2 levels. However, the most important factor for efficient and consistent pulmonary transgene delivery is the dose of adenoviral vector used, as both the degree of transduction and the percentage of lungs transduced increases with escalating viral dose.
INTRODUCTION Gene transfer to the developing fetal airways in utero may provide an approach to overcoming the barriers to efficient gene transfer encountered, including excessively sticky mucus and ongoing inflammatory reactions, that have been problematic in all clinical trials conducted so far for cystic fibrosis in adult patients [1]. Application to the fetal airways may also benefit from the presence and accessibility of still-expanding epithelial progenitor or stem cells and the potential to induce tolerance against the transgenic protein [2]. In connection with early prenatal diagnosis, this approach may thus allow the prevention of early-onset disease manifestation and provide a life-long cure. Successful delivery of a marker gene to the fetal airways, by minimally invasive ultrasound-guided transcutaneous vector application directly to the fetal trachea, has recently been demonstrated by our team in sheep [3]. In the mouse, the one species for which animal models of cystic fibrosis are currently available, the only feasible route of application is the amniotic cavity. Intra-amniotic injection has previously been used to deliver adenovirus to murine fetuses [4–7], but marker gene expression in
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the airway epithelia was limited and, in our experience, very variable even in different fetuses from the same litter. We also have observed previously that gene transfer to the airways coincides with the onset of occasional fetal breathing movements in the later stages of pregnancy [8]. In order to achieve optimal transduction of the fetal airway epithelia, with a view to future application of a cystic fibrosis transmembrane conductance regulator (CFTR)-expressing construct in CFTR-knockout mice, we have investigated several parameters that we expect will contribute to the efficiency of in utero airway gene transfer after intra-amniotic injection. We have chosen a first-generation adenoviral vector (AdRSVhgal) for these investigations, as we know that this vector works efficiently in this system. This will not be the ultimate vector of choice for therapeutic approaches, but it serves to dissect the role of non-virus-related parameters and to define the conditions for future use of more appropriate vector systems, such as helper-dependent adenovirus and chimeric adeno/retrovirus vectors. The investigations presented here confirm that fetal breathing movements are essential for airway transduction and also demonstrate that stimulation of these
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movements significantly increases vector intake and pulmonary transgene expression. However, the most important factor for consistent gene transfer and expression is the application of high-titer vector preparations, as up to 97% of fetal lungs were highly transduced after intra-amniotic injection of 2 1011 particles of adenovirus without the stimulation of breathing movements.
RESULTS
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DISCUSSION
Fetal lung transduction in the mouse after intra-amniotic injection of a first-generation adenovirus expressing hgalactosidase has previously been found to be highly variable among litters, fetuses, and even lobes from the same fetal lung (data not shown). For an effective therapy, any method of gene therapy application must be consistent in the amount and location of transgene delivery achieved. Ideally, when investigating possible therapies for lung diseases such as cystic fibrosis, widespread pulmonary transduction should be achieved in 100% of treated subjects. With this aim in mind, the various factors suspected of producing this wide variation in murine fetal lung transduction were examined in order to establish a reliable transduction procedure for future therapeutic experiments in CFTR-knockout mice. After Intra-amniotic Injection at 13 and 16 Days of Gestation, Transgene Expression is Predominantly Seen in the Fetal Skin and Lung, Respectively To investigate the potential vector distribution after intra-amniotic injection, an inert marker compound, colloidal carbon, was used to ascertain which fetal organs were accessible via this route of administration. Injections were performed using 50 Al of colloidal carbon at 13 and 16 days of gestation (Figs. 1A and 1B, respectively). Colloidal carbon is extremely adhesive, thus all areas of the fetus that come into contact with the amniotic fluid can be determined. Fetuses were harvested after 1 h and cleared (see Materials and Methods) so that we could visualize distribution of the carbon. At 13 days of gestation, the carbon covered the skin but also penetrated the buccal and nasal cavities. However, no carbon could be detected in any of the internal organs. In contrast, at 16 days of gestation, as well as staining the buccal and nasal cavities, carbon reached the trachea, lung, stomach, and gut. It is clearly visible in the airway space but does not reach the lung parenchyma to a significant extent (see also Fig. 4A). We then performed intra-amniotic injections of 5 1010 particles of first-generation adenovirus, AdRSVhgal, at various stages of fetal development (from 12 to 17 days of gestation) to determine the distribution of adenovirusmediated transgene expression. Fetuses were harvested after 48 h and stained for h-galactosidase expression (Figs. 1C–1F). No h-galactosidase staining was observed in
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untreated littermates (data not shown). At 17 days of gestation, all dams aborted within 24 h of the operation, hence no data could be obtained. At the other gestational ages investigated there were no differences in the survival rates of the injected and noninjected fetuses (~95%). There was, however, significant variation in the localization and extent of h-galactosidase expression between individual fetuses, particularly with respect to airway transduction, which, overall, mirrored the distribution pattern seen with colloidal carbon. In general, h-galactosidase expression in the skin was strong and widespread in the earlier gestational ages (Fig. 1C), while staining of the internal organs such as lung and gut was negligible (Fig. 1D). Minimal expression was detected in the nasal and buccal cavities, but this did not extend any further. In contrast, fetuses injected at the later stages presented almost the opposite pattern of expression. No skin staining was seen (Fig. 1E), probably due to keratinization of the fetal skin at this developmental stage [9]. However, widespread and intense staining was present in the epithelia of both the nasal and the buccal cavities, extending down the trachea and esophagus to the airways and gut, respectively (Fig. 1F). More detailed analysis of the lungs (of a highly transduced fetus) injected at 16 days of gestation revealed transgene expression throughout the bronchioles of all pulmonary lobes (Figs. 1G–1I), as well as extensive expression in the trachea (Fig. 1J), stomach (Fig. 1K), and gut (Fig. 1L). It is important to note that transgene expression did not extend into the alveolar bud spaces of the undeveloped fetal lung. This is consistent with the pattern of colloidal carbon deposition and suggests that the fetal breathing movements are not of sufficient intensity to move amniotic fluid to the extremities of the lungs, as they would also have to force out the endogenous lung liquid present in these spaces. However, we cannot exclude the possibility that lack of transduction may be caused by an inability of this adenovirus to transduce these cells. To quantitate the degree of transgene expression obtained in the fetal skin and lung at 13 and 16 days of gestation, intra-amniotic injections were performed as above and both fetal lungs and samples of fetal skin were taken at 48 h and analyzed for the presence of hgalactosidase protein by ELISA (Fig. 2). The distribution of h-galactosidase was again similar to that seen by histological staining. At 13 days of gestation, skin transduction was significantly higher than that seen at 16 days of gestation ( p b 0.0001), which may be important when considering in utero gene therapy strategies for skin disorders, such as epidermolysis bullosa. The opposite pattern was seen with respect to the lung ( p b 0.0001). We have previously observed that spontaneous fetal breathing movements occur in mice at 15–16 days of gestation [8]. This is consistent with earlier observations in rats that show these movements to begin at the same point in development, at 16 days of gestation [10]. These
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FIG. 1. Qualitative comparison of colloidal carbon distribution and transgene expression after intra-amniotic injection at 13 and 16 days of gestation in the fetal mouse. Intra-amniotic injections were performed with 50 Al of colloidal carbon at 13 (A) and 16 (B) days of gestation; fetuses were harvested after 1 h and cleared to allow visualization of carbon penetration. At 13 days of gestation, carbon is internalized only into the buccal and nasal passages. At 16 days of gestation, carbon can also be found in the trachea and lungs, demonstrating the onset of fetal breathing movements by this stage of development. Injections were repeated with 5 1010 particles of AdRSVhgal and fetuses stained for the presence of h-galactosidase protein. At 13 days of gestation, the skin is highly transduced (C), whereas the internal organs are not infected (D). At 16 days of gestation the opposite pattern occurs, as the skin is not transduced (E), while both the lung and gut show transgene expression (F) (L, lung; G, gut). Further analysis of the fetal lungs at 16 days of gestation reveals transgene expression throughout the bronchioles of all pulmonary lobes (G–I), as well as extensive expression in the trachea (J), gut (K), and stomach (L).
breathing movements are probably even more pronounced in fetuses at 17–19 days of gestation, although this has not been investigated here due to increased abortion rates at these later stages of pregnancy. It is therefore evident that the optimal time for a fetal gene therapy strategy in the mouse, one that aims to deliver a therapeutic gene to the lung by intra-amniotic injection, is at 16 days of gestation.
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Adenoviral Dose and Fetal Lung Transduction Show a Significant Positive Correlation In in vitro experiments to investigate the effect of amniotic fluid on adenovirus stability, we incubated adenovirus in amniotic fluid at 378C and removed aliquots after different time points for the transduction of HT 1080 cells in culture (these cells were used because they are highly permissive for adenovirus infection). We
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and Fig. 4), these experiments demonstrate that the high variability observed in fetal lung transduction, even at the same gestational age, is not due to a loss/depletion of virus or to a rapid reduction in its infectious ability but instead must be caused by other factors. To further investigate these factors, the effect of the dose of AdRSVhgal on fetal pulmonary transduction was examined. A range of viral doses, from 6.4 107 to 2 1011 particles/fetus, was administered via intra-amniotic injection, and the h-galactosidase content of fetal lungs was determined by ELISA after 48 h (Fig. 3). Viral dose could not be further increased due the limitations of viral production. There is clearly a significant positive correlation between the dose of AdRSVhgal and the degree of pulmonary transgene expression ( p b 0.001). Interestingly, the relative incidence of nontransduced airways decreased with higher viral doses.
FIG. 2. Quantitative comparison of transgene expression in the lung and skin after intra-amniotic injection at 13 and 16 days of gestation in the fetal mouse. Intra-amniotic injections were performed with 5 1010 particles AdRSVhgal/ fetus at 13 and 16 days of gestation; fetuses were harvested after 48 h and lungs and skin analyzed for h-galactosidase content by ELISA (n = 12 for each data set). At 13 days of gestation, skin transduction is significantly higher than that seen at 16 days of gestation (***p b 0.0001). In addition, lung transduction is significantly increased at 16 days of gestation compared with the earlier stage of development due to the initiation of fetal breathing movements (***p b 0.0001).
found that an increase in the extent of adenovirus transduction occurred briefly during the first few minutes of incubation (up to 140% of transduction observed compared to preincubation in PBS). However, after 30 min incubation, transduction fell to 60% at 1 h and reached a plateau at 5% at 2 h (data not shown). In a separate experiment, fetuses were injected with 5 1010 particles AdRSVhgal, and small aliquots of amniotic fluid were immediately removed; samples were also taken from the same fetuses after 1 h and placed on cells in vitro to assess the amount of infectious virus present. In this experiment, a reduction in the number of blue cells transduced after infection was observed with samples taken immediately after injection (values are means F SD, 161 F 48) compared to those taken after 1 h (140 F 10). However, this difference was not statistically significant ( p = 0.097), probably due to the high degree of variation in the extent and frequency of breathing movements between fetuses in vivo. Nevertheless, in combination with our demonstration of fetal breathing within, at the longest, 1 h after injection (see text below
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Fetal Breathing Can Be Stimulated by Intra-amniotic Injection of Theophylline and Administration of Postoperative CO2 As uptake of virus into the fetal airways is one of the most important factors for airway transduction, we hypothesized that one reason for the observed variability could be variation in the frequency and/or intensity of fetal breathing movements. In addition, one of the major disadvantages of administering gene therapy vectors via intra-amniotic injection is the large dilution of vector in amniotic fluid. This makes it necessary to use higher titers of virus than would be required by direct intrapulmonary application. While these large doses are feasible with adenovirus due to the production of high-titer preparations, they present a significant hurdle for future in utero gene therapy strategies with alternative vectors that aim to target airway epithelia. Stimulation of fetal breathing movements at the time of injection could perhaps increase virus uptake and transduction, making it possible to reduce the amount of virus needed to achieve significant pulmonary transduction. In general, the net flow of fluid within the respiratory system of the fetus is from the lungs, since airway epithelial cells secrete liquid at a steady rate [11]. Various exogenous factors have previously been shown to inhibit (adenosine [12,13], smoking [14], alcohol [15], depressant drugs [16,17], hypoxia [18]) or stimulate (glucose [19,20], theophylline [21–24], hypercapnia [18]) fetal breathing movements in both animal models and humans. More importantly, with respect to this study, disturbance of the fetus by vigorous palpation or amniocentesis has also been shown to have an inhibitory effect on fetal breathing movements [25]. Intra-amniotic injection is a technique very similar to amniocentesis, thus it is likely that fetal breathing movements will be decreased after vector application, the precise time that they are desired for uptake of vectors into the airways.
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FIG. 3. Dose of AdRSVhgal in intra-amniotic injections and lung transgene expression show a positive correlation. A range of doses of AdRSVhgal, from 6.4 107 to 2 1011 particles/fetus, was applied by intra-amniotic injections; fetuses were harvested after 48 h and lungs analyzed for h-galactosidase content by ELISA. There is a significant positive correlation between the two factors ( p b 0.0001); no transgene expression is detected until the viral dose reaches 1.6 109 particles/fetus, after which point the h-galactosidase content increases with increasing viral dose. (Note: a larger number of fetuses treated with 2 1011 particles/fetus are also analyzed in Fig. 5; under this scenario one fetal lung does contain no detectable h-galactosidase).
In order to stimulate fetal breathing, we chose to combine two effectors that have been shown to act synergistically. Moss and Scarpelli demonstrated that theophylline increases the sensitivity of the respiratory center to CO2 in fetal sheep [24]. Intravascular administration of adenosine inhibits fetal breathing, and this effect is disrupted by theophylline [12,13]. It is thought that theophylline produces its effect through antagonism to endogenous adenosine, as it is structurally similar and thus may act as a competitive inhibitor. Additional putative mechanisms, including the alteration of cAMP levels via the inhibition of phosphodiesterase [26] and calcium release [27], have also been proposed to explain the stimulatory effect of theophylline on fetal breathing. The effect of CO2 is probably mediated by the effect of acid ions on the fetal brain stem. Acidification of the blood, or cerebral spinal fluid, increases fetal breathing movements, while the addition of bicarbonate to increase the pH has the opposite effect [28,29]. In initial semiquantitative experiments using coinjection of theophylline and AdRSVhgal into the amniotic fluid, along with postoperative CO2, fetal lungs were stained and graded blind on a scale of 0–5 depending on the intensity of h-galactosidase expression observed. These data gave promising results, with the best transduction seen at a dose of theophylline of 1.6 mg/ml, but,
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due to the semiquantitative nature of this method of analysis, in which small differences in h-galactosidase expression could not be observed, enhancement of transduction with stimulation was not statistically significant. In all further studies, bstimulationQ refers to the optimal conditions found: intra-amniotic injection of 1.6 mg/ml theophylline and postoperative administration of 10% CO2 in air for 1 h. To study the mechanical effects of fetal breathing on the intake of amniotic fluid, without the interference of other variables involved in the infectability of the airway epithelia in individual fetuses, we injected colloidal carbon into the amniotic cavity instead of adenovirus, either with or without theophylline (1.6 mg/ml). Fetuses were harvested after 1 h, and carbon could already be seen at varying degrees in virtually all fetal lungs, demonstrating that fetal breathing movements occur at least within this time period. Fig. 4A shows the histology from an intensely stained lung after intra-amniotic injection with colloidal carbon. The carbon is clearly present in all bronchi and bronchioles but does not penetrate the vasculature of the fetus. Lungs were then graded blind on a scale of 0–5 (Figs. 4B–4G), depending on the degree of carbon present. This is an arbitrary scale defined in our laboratory. Fig. 4H shows that, overall, theophylline and CO2 stimulation increases the degree of
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FIG. 4. Intra-amniotic injection of theophylline combined with postoperative CO2 administration increases fetal breathing and the intake of colloidal carbon into fetal lungs. Colloidal carbon was administered by intra-amniotic injection with (stimulation, n = 44) or without (control, n = 34) co-injection of 1.6 mg/ml theophylline and administration of postoperative 10% CO2 in air for 1 h. Fetuses were harvested after 1 h. Histological examination of an intensely stained lung shows that carbon is present in all bronchi and bronchioles but does not penetrate the vasculature of the fetus (A). Lungs were graded blind on a scale of 0–5 (B–G), depending on the degree of carbon present. Theophylline and CO2 stimulation increases the degree of pulmonary staining with colloidal carbon ( p b 0.001) (H).
pulmonary staining with colloidal carbon ( p b 0.001). This is most dramatically seen by the twofold increase in the percentage of fetuses with grade-5-stained lungs. Under stimulation, 93% of fetal lungs exhibited strong colloidal carbon staining (grades 3–5), whereas the injection of carbon without stimulation resulted in only 59% staining at these grades. We then investigated whether this stimulation of fetal breathing would increase pulmonary transgene expression. Intra-amniotic injections were first performed using the maximal dose of 2 1011 particles AdRSVhgal/fetus. Fetuses were harvested after 48 h, and the presence of h-galactosidase protein in the lungs was determined by ELISA. Fig. 5 demonstrates that, despite the wide range of transgene expression levels seen, stimulation of fetal breathing movements significantly increased the h-galactosidase expression of the fetal lungs ( p b 0.05). When these experiments were
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repeated with a lower dose of virus (5 10 10 particles/fetus), a greater degree of enhancement was seen ( p b 0.01). This disparity is most likely due to the saturation of airway epithelial cells by transgene expression at the higher adenoviral dose, leaving less scope for an increase after stimulation. As a further test of the hypothesis that theophylline increases particle uptake into the lung by acting on the fetal breathing centers, we coapplied colloidal carbon and adenosine, a known inhibitor of these fetal breathing movements. Varying concentrations of adenosine (based on a previous study in fetal sheep [13]) were injected into the murine peritoneal cavity of fetuses at 16 days of gestation, followed by intra-amniotic injections of colloidal carbon. Control fetuses were injected with PBS before marker dye administration. Fetuses were culled after 1 h, and the degree of colloidal carbon staining in the fetal lung was graded after clearing (Fig. 6). Adenosine admin-
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FIG. 5. Intra-amniotic injection of theophylline combined with postoperative CO2 administration increases fetal breathing and adenoviral-mediated transgene expression. Intra-amniotic injections were performed with 5 1010 or 2 1011 particles AdRSVhgal/fetus with (stimulation) or without (control) coinjection of 1.6 mg/ml theophylline and administration of postoperative 10% CO2 in air for 1 h (n = 24 and 30 for each condition at 5 1010 and 2 1011 particles/fetus, respectively). Fetuses were harvested after 48 h and pulmonary h-galactosidase content was analyzed by ELISA. At both viral doses, stimulation significantly increased fetal breathing and thus the amount of h-galactosidase present in fetal lungs, but the enhancement was more pronounced at the lower dose (**p b 0.01) than at the higher dose (*p b 0.05).
istration significantly reduced the amount of colloidal carbon present in the fetal lungs ( p b 0.01). These experiments were repeated using AdRSVhgal, but although an inhibitory trend was observed after adenosine administration, the data were not statistically significant. This was probably due to the high degree of variability between fetuses, and power calculations showed that an unreasonable number of animals would be needed to obtain a statistically significant result. Although the stimulation of breathing movements clearly had a positive effect on transduction efficiency, it did not decrease the degree of variation and did not abolish the occasional transduction failure. These events could still have been due to lack of breathing in these fetuses or to other still unknown factors. One such factor, which we have not investigated here, is the
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expression of suitable receptors on the airway epithelia during fetal development. The main receptor for adenovirus, the coxsackie adenovirus receptor (CAR), is expressed on the basolateral surface of airway epithelial cells. CAR mRNA has been shown to be expressed in developing mouse airways at 12 days of gestation [30]. Various studies in primary human airway epithelial cells [31], rabbit or human epithelia [32], and rodent trachea and lung [33,34] have employed chemical agents, such as sodium caprate or EGTA, to transiently disrupt tight junctions between the epithelial cells, allowing adenovirus access to receptors. Data presented here clearly show that adenovirus is capable of infecting all areas of the fetal airway without these treatments, although, at lower viral doses, the opening of tight junctions may enhance transgene expression. However, it is not clear whether there may be a variation in expression at this time of fetal life, whether the murine fetal airways contain tight junctions at this stage of development, or, even if they do, to what extent agents such as sodium caprate would be able to exert their effect after intraamniotic injection. In summary, we have investigated three major factors that influence the degree of pulmonary transgene expression achieved after intra-amniotic injection in utero: gestational age, adenoviral dose, and the extent of fetal breathing movements. In the mouse, fetal breathing movements begin at day 15–16 of gestation, so access to the fetal lung via the amniotic fluid is only possible from this stage of development onward. The combination of intra-amniotic application of 1.6 mg/ml theophylline and postoperative CO2 stimulates fetal breathing and thus can be used to significantly increase the degree of pulmonary transgene expression achieved. However, it appears that the dose of AdRSVhgal used is the most critical factor, as the percentage of the lungs transduced (to any degree) increases with the dose of virus used. Nevertheless, in cases in which lower viral titers must be used, stimulation of fetal breathing movements by theophylline and CO2 may be helpful.
MATERIALS
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METHODS
In vivo procedures. Pregnant MF1 female mice were anesthetized by the inhalation of isofluorane (Abbot Laboratories, Queensborough, UK). A midline laparotomy was performed using sterile techniques, and both horns of the gravid uterus were exposed. Approximately six fetuses were injected per litter. Each amniotic cavity was injected (50-Al vol) by penetration of the uterus wall, the yolk sac, and amniotic membranes with a 33-gauge Hamilton Microliter Syringe (EssLab, Hadleigh, UK). Amniotic sacs were palpated after injection to ensure thorough mixing of the injectate. The amniotic fluid volume increases with fetal age; in the mouse it is approximately 300 Al at 16 days of gestation, which would dilute the injected virus amount about sixfold. Care was taken to ensure that injections were performed into the amniotic cavity and not into the extraembryonic coelom [35]. 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
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FIG. 6. Intraperitoneal injection of adenosine inhibits fetal breathing and intake of colloidal carbon into fetal lungs. Colloidal carbon was administered by intra-amniotic injection with (inhibition, n = 14) or without (control, n = 14) preceding intraperitoneal injection of 18 Ag adenosine/fetus. Fetuses were harvested after 1 h and lungs were graded blind on a scale of 0–5 (Figs. 4B–4G), depending on the degree of carbon present. Adenosine administration inhibits fetal breathing and hence decreases the degree of colloidal carbon observed in fetal lungs ( p b 0.01).
warmed cage until awake and active and housed in an undisturbed environment until the harvesting of fetuses. Mice treated with theophylline (Sigma-Aldrich, St. Louis, MO, USA; 1.6 mg/ml) were placed in a chamber containing 10% CO2 in air for approximately 1 h after surgery. Fetal and maternal survival was greater than 95%. Adenovirus vector. First-generation (E1 and E3 regions deleted) serotype 5 adenoviral vector (AdRSVhgal) containing the h-galactosidase reporter gene (driven by an RSV promoter) was prepared as previously described [36]. Virus was freshly prepared in PBS prior to injection. Virus titer was determined as particle number/ml by spectrophotometry using a coefficient of extinction of 1.1 1012 virus/OD260 unit [37]. Primers that amplify from within the E1A region of adenovirus were used to test viral batches for the presence of replication-competent virus. The vectors used were E1A deleted so that positive E1A PCR products would only arise from replication-competent virus. No E1A PCR product was obtained from any of the applied virus preparations. Colloidal carbon marker. Colloidal carbon suspension was prepared by first mixing 5 g of gum arabic (Sigma) thoroughly with 30 ml of dH20. The mixture was ground against a glass plate until a smooth paste was formed. Two grams of Printex P beads, furnace black (Degussa-Huls, Frankfurt, Germany), were added and ground until thoroughly mixed. The solution was sonicated (Branson Sonifier 450) and then autoclaved to ensure sterility. Visualization of colloidal carbon and reporter gene expression. Fetuses were harvested 48 h after in utero injection, and those to be analyzed for reporter gene expression were placed in 100% ethanol for 1 h. Approximately 200 Al of 100% ethanol was injected into the peritoneal cavity to ensure complete internal fixation before the fetuses were sliced sagitally. Samples were thoroughly washed in PBS and placed in X-gal solution (0.4 mg/ml 5-bromo-4-chloro-3-indolyl-h-d-galactosidase in DMSO, 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, 0.1 mM MgCl2). To better observe staining in the lungs, samples were cleared by dehydration in 100% methanol and transferred to a benzyl benzoate and benzyl alcohol mixture (2:1 v/v) [38]. The specimens were photographed under a dissection microscope (S7-CTV, Olympus, Japan) using a digital camera (DP10, Olympus). Brightness and contrast were optimized using Adobe Photoshop CS. Reporter gene quantification. h-Galactosidase levels were determined by ELISA using a commercially available kit (Boehringer Mannheim, Mannheim, Germany). Fetal tissues were dissected 48 h after in utero injection and stored at 208C in 200 Al of kit lysis buffer before analysis. The protein content of samples was analyzed using the bicinchoninic acid protein assay system (Pierce, Rockford, IL, USA). Levels of h-galactosidase
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activity were standardized on the basis of protein content, and values are presented as picogram of h-galactosidase per milligram of protein. The limit of h-galactosidase detection with this assay is z30 pg/ml (according to the manufacturer’s instructions). Statistical analysis. Data were analyzed by Mann-Whitney nonparametric tests, except for the analysis of transgene expression versus viral dose (Fig. 3), which was analyzed by parametric regression analysis after logarithmic transformation using MINITAB (Minitab Inc., State College, PA, USA). Where data are displayed as box plots, the top of the box is the third quartile (Q3, 75% of the data values are less than or equal to this value), the bottom of the box is the first quartile (Q1, 25% of the data values are less than or equal to this value), and the line bisecting the box is the median. In addition, the upper whisker extends to the highest data value within the upper limit (upper limit, Q3 + 1.5(Q3–Q1)) and the lower whisker extends to the lowest value within the lower limit (lower limit = Q1–1.5(Q3–Q1)). Values beyond the whiskers are outliers (denoted by black circles).
ACKNOWLEDGMENTS We thank Andrea Pavirani (Transgene, Strasbourg, France) for the AdRSVhgal construct and S. R. Underwood (Imperial College, London, UK) for the kind gift of adenosine. Sport Aiding Medical Research for Kids (SPARKs), the Katharine Dormandy Trust for Haemophilia and Allied Disorders, and the Medical Research Council (MRC) contributed to funding. HS was funded by German Research Foundation (SCHN 569/3-1). RECEIVED FOR PUBLICATION DECEMBER 9, 2004; ACCEPTED FEBRUARY 22, 2005.
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7. Senoo, M., et al. (2000). Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqManpolymerase chain reaction assay [in-process citation]. Mol. Genet. Metab. 69: 269 – 276. 8. Douar, A. M., et al. (1997). Foetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus. Gene Ther. 4: 883 – 890. 9. Kashiwagi, M., Kuroki, T., and Huh, N. (1997). Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin. Dev. Biol. 189: 22 – 32. 10. Kobayashi, K., Lemke, R. P., and Greer, J. J. (2001). Ultrasound measurements of fetal breathing movements in the rat. J. Appl. Physiol. 91: 316 – 320. 11. Olver, R. E., and Strang, L. B. (1974). Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J. Physiol. 241: 327 – 357. 12. Bissonnette, J. M., Hohimer, A. R., and Knopp, S. J. (1991). The effect of centrally administered adenosine on fetal breathing movements. Respir. Physiol. 84: 273 – 285. 13. Koos, B., and Matsuda, K. (1990). Fetal breathing, sleep state, and cardiovascular responses to adenosine in sheep. J. Appl. Physiol. 68: 489 – 495. 14. Manning, F. A., and Feyerabend, C. (1976). Cigarette smoking and fetal breathing movements. Br. J. Obstet. Gynaecol. 83: 262 – 270. 15. Fox, H. E., et al. (1978). Maternal ethanol ingestion and the occurrence of human fetal breathing movements. Am. J. Obstet. Gynecol. 132: 354 – 358. 16. Boddy, K., et al. (1976). The effects of pentobarbitone and pethidine on foetal breathing movements in sheep. Br. J. Pharmacol. 57: 311 – 317. 17. Worthington, D., Piercy, W. N., and Smith, B. T. (1978). Modification of ovine fetal respiratory-like activity by chronic diazepam administration. Am. J. Obstet. Gynecol. 131: 749 – 754. 18. Boddy, K., et al. (1974). Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxaemia and hypercapnia in sheep. J. Physiol. 243: 599 – 618. 19. Natale, R., Patrick, J., and Richardson, B. (1978). Effects of human maternal venous plasma glucose concentrations on fetal breathing movements. Am. J. Obstet. Gynecol. 132: 36 – 41. 20. Schreyer, P., et al. (1991). Ovine fetal breathing and swallowing activity in response to plasma glucose changes. Gynecol. Obstet. Invest. 32: 15 – 19. 21. Bissonnette, J. M., et al. (1990). Theophylline stimulates fetal breathing movements during hypoxia. Pediatr. Res. 28: 83 – 86. 22. Carnielli, V. P., et al. (2000). Metabolic and respiratory effects of theophylline in the preterm infant. Arch. Dis. Child Fetal Neonatal Ed. 83: F39 – F43.
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23. Ishikawa, M., et al. (1996). Maternal theophylline administration and breathing movements in late-gestation human fetuses. Obstet. Gynecol. 88: 973 – 978. 24. Moss, I. R., and Scarpelli, E. M. (1981). Stimulatory effect of theophylline on regulation of fetal breathing movements. Pediatr. Res. 15: 870 – 873. 25. Manning, F. A. (1997). Fetal breathing movements as a reflection of fetal status. Postgrad. Med. 61: 116 – 122. 26. Butcher, R. W., and Sutherland, E. W. (1962). Adenosine 3V, 5V-phosphate in biological materials. J. Biol. Chem. 237: 1244 – 1250. 27. Blinks, J. R., et al. (1972). Influence of caffeine and other methylxanthines on mechanical properties of isolated mammalian heart muscle: evidence for a dual mechanism of action. Circ. Res. 30: 367 – 392. 28. Hohimer, A. R., et al. (1983). Central chemical regulation of breathing movements in fetal lambs. Respir. Physiol. 52: 99 – 111. 29. Moore, P. J., and Hanson, M. A. (1994). Control of breathing: central influences. In Fetus and Neonate: Physiology and Clinical Applications (M. A. Hanson, J. A. D. Spencer, C. H. Rodeck, D. Walters, Eds.), pp. 109 – 136, Vol. 2. Cambridge Univ. Press, Cambridge. 30. Schachtner, S., et al. (1999). Temporally regulated expression patterns following in utero adenovirus- mediated gene transfer. Gene Ther. 6: 1249 – 1257. 31. Coyne, C. B., et al. (2000). Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am. J. Respir. Cell Mol. Biol. 23: 602 – 609. 32. Wang, G., et al. (2000). Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo. Am. J. Respir. Cell Mol. Biol. 22: 129 – 138. 33. Chu, Q., et al. (2001). EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo. Hum. Gene Ther. 12: 455 – 467. 34. Gregory, L. G., et al. (2003). Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo. Mol. Ther. 7: 19 – 26. 35. Jollie, W. P. (1990). Development, morphology, and function of the yolk-sac placenta of laboratory rodents. Teratology 41: 361 – 381. 36. Stratford-Perricaudet, L., et al. (1992). Widespread long-term gene transfer to mouse skeletal muscles and heart. J. Clin. Invest. 90: 626 – 630. 37. Maizel, J. V., Jr., White, D. O., and Scharff, M. D., (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36: 115 – 125. 38. Turkay, A., Saunders, T., and Kurachi, K. (1999). Intrauterine gene transfer: gestational stage-specific gene delivery in mice. Gene Ther. 6: 1685 – 1694.
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Factors Influencing Adenovirus-Mediated Airway Transduction in Fetal Mice S. M. K. Buckley,1,* S. N. Waddington,1 S. Jezzard,1 L. Lawrence,2 H. Schneider,3 M. V. Holder,1 M. Themis,1 and C. Coutelle1 1
Gene Therapy Research Group, Department of Cell and Molecular Biology, SAF Building, Imperial College, South Kensington, London SW7 2AZ, United Kingdom 2 Leukocyte Biology, Department of Cell and Molecular Biology, SAF Building, Imperial College, South Kensington, London SW7 2AZ, United Kingdom 3 Childrens Hospital, University of Erlangen, Loschgetr. 15, D-91054 Erlangen, Germany *To whom correspondence and reprint requests should be addressed.
Available online 12 April 2005
Intra-amniotic injection of adenovirus allows transduction of the fetal airways following natural fetal breathing movements. This administration method is promising for use in gene therapy for cystic fibrosis and other diseases for which the main target for exogenous gene expression is the lung. Here we have investigated factors that may affect the efficacy of gene transfer to the murine fetal lung. We examined marker compound distribution and transgene expression (from a firstgeneration adenoviral vector) at different stages of development. This demonstrated that fetal breathing movements at 15–16 days of gestation are of sufficient intensity to carry marker/vector into the fetal lungs. These movements can be significantly stimulated by the combination of intraamniotic theophylline administration and postoperative exposure of the dam to elevated CO2 levels. However, the most important factor for efficient and consistent pulmonary transgene delivery is the dose of adenoviral vector used, as both the degree of transduction and the percentage of lungs transduced increases with escalating viral dose.
INTRODUCTION Gene transfer to the developing fetal airways in utero may provide an approach to overcoming the barriers to efficient gene transfer encountered, including excessively sticky mucus and ongoing inflammatory reactions, that have been problematic in all clinical trials conducted so far for cystic fibrosis in adult patients [1]. Application to the fetal airways may also benefit from the presence and accessibility of still-expanding epithelial progenitor or stem cells and the potential to induce tolerance against the transgenic protein [2]. In connection with early prenatal diagnosis, this approach may thus allow the prevention of early-onset disease manifestation and provide a life-long cure. Successful delivery of a marker gene to the fetal airways, by minimally invasive ultrasound-guided transcutaneous vector application directly to the fetal trachea, has recently been demonstrated by our team in sheep [3]. In the mouse, the one species for which animal models of cystic fibrosis are currently available, the only feasible route of application is the amniotic cavity. Intra-amniotic injection has previously been used to deliver adenovirus to murine fetuses [4–7], but marker gene expression in
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the airway epithelia was limited and, in our experience, very variable even in different fetuses from the same litter. We also have observed previously that gene transfer to the airways coincides with the onset of occasional fetal breathing movements in the later stages of pregnancy [8]. In order to achieve optimal transduction of the fetal airway epithelia, with a view to future application of a cystic fibrosis transmembrane conductance regulator (CFTR)-expressing construct in CFTR-knockout mice, we have investigated several parameters that we expect will contribute to the efficiency of in utero airway gene transfer after intra-amniotic injection. We have chosen a first-generation adenoviral vector (AdRSVhgal) for these investigations, as we know that this vector works efficiently in this system. This will not be the ultimate vector of choice for therapeutic approaches, but it serves to dissect the role of non-virus-related parameters and to define the conditions for future use of more appropriate vector systems, such as helper-dependent adenovirus and chimeric adeno/retrovirus vectors. The investigations presented here confirm that fetal breathing movements are essential for airway transduction and also demonstrate that stimulation of these
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movements significantly increases vector intake and pulmonary transgene expression. However, the most important factor for consistent gene transfer and expression is the application of high-titer vector preparations, as up to 97% of fetal lungs were highly transduced after intra-amniotic injection of 2 1011 particles of adenovirus without the stimulation of breathing movements.
RESULTS
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DISCUSSION
Fetal lung transduction in the mouse after intra-amniotic injection of a first-generation adenovirus expressing hgalactosidase has previously been found to be highly variable among litters, fetuses, and even lobes from the same fetal lung (data not shown). For an effective therapy, any method of gene therapy application must be consistent in the amount and location of transgene delivery achieved. Ideally, when investigating possible therapies for lung diseases such as cystic fibrosis, widespread pulmonary transduction should be achieved in 100% of treated subjects. With this aim in mind, the various factors suspected of producing this wide variation in murine fetal lung transduction were examined in order to establish a reliable transduction procedure for future therapeutic experiments in CFTR-knockout mice. After Intra-amniotic Injection at 13 and 16 Days of Gestation, Transgene Expression is Predominantly Seen in the Fetal Skin and Lung, Respectively To investigate the potential vector distribution after intra-amniotic injection, an inert marker compound, colloidal carbon, was used to ascertain which fetal organs were accessible via this route of administration. Injections were performed using 50 Al of colloidal carbon at 13 and 16 days of gestation (Figs. 1A and 1B, respectively). Colloidal carbon is extremely adhesive, thus all areas of the fetus that come into contact with the amniotic fluid can be determined. Fetuses were harvested after 1 h and cleared (see Materials and Methods) so that we could visualize distribution of the carbon. At 13 days of gestation, the carbon covered the skin but also penetrated the buccal and nasal cavities. However, no carbon could be detected in any of the internal organs. In contrast, at 16 days of gestation, as well as staining the buccal and nasal cavities, carbon reached the trachea, lung, stomach, and gut. It is clearly visible in the airway space but does not reach the lung parenchyma to a significant extent (see also Fig. 4A). We then performed intra-amniotic injections of 5 1010 particles of first-generation adenovirus, AdRSVhgal, at various stages of fetal development (from 12 to 17 days of gestation) to determine the distribution of adenovirusmediated transgene expression. Fetuses were harvested after 48 h and stained for h-galactosidase expression (Figs. 1C–1F). No h-galactosidase staining was observed in
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untreated littermates (data not shown). At 17 days of gestation, all dams aborted within 24 h of the operation, hence no data could be obtained. At the other gestational ages investigated there were no differences in the survival rates of the injected and noninjected fetuses (~95%). There was, however, significant variation in the localization and extent of h-galactosidase expression between individual fetuses, particularly with respect to airway transduction, which, overall, mirrored the distribution pattern seen with colloidal carbon. In general, h-galactosidase expression in the skin was strong and widespread in the earlier gestational ages (Fig. 1C), while staining of the internal organs such as lung and gut was negligible (Fig. 1D). Minimal expression was detected in the nasal and buccal cavities, but this did not extend any further. In contrast, fetuses injected at the later stages presented almost the opposite pattern of expression. No skin staining was seen (Fig. 1E), probably due to keratinization of the fetal skin at this developmental stage [9]. However, widespread and intense staining was present in the epithelia of both the nasal and the buccal cavities, extending down the trachea and esophagus to the airways and gut, respectively (Fig. 1F). More detailed analysis of the lungs (of a highly transduced fetus) injected at 16 days of gestation revealed transgene expression throughout the bronchioles of all pulmonary lobes (Figs. 1G–1I), as well as extensive expression in the trachea (Fig. 1J), stomach (Fig. 1K), and gut (Fig. 1L). It is important to note that transgene expression did not extend into the alveolar bud spaces of the undeveloped fetal lung. This is consistent with the pattern of colloidal carbon deposition and suggests that the fetal breathing movements are not of sufficient intensity to move amniotic fluid to the extremities of the lungs, as they would also have to force out the endogenous lung liquid present in these spaces. However, we cannot exclude the possibility that lack of transduction may be caused by an inability of this adenovirus to transduce these cells. To quantitate the degree of transgene expression obtained in the fetal skin and lung at 13 and 16 days of gestation, intra-amniotic injections were performed as above and both fetal lungs and samples of fetal skin were taken at 48 h and analyzed for the presence of hgalactosidase protein by ELISA (Fig. 2). The distribution of h-galactosidase was again similar to that seen by histological staining. At 13 days of gestation, skin transduction was significantly higher than that seen at 16 days of gestation ( p b 0.0001), which may be important when considering in utero gene therapy strategies for skin disorders, such as epidermolysis bullosa. The opposite pattern was seen with respect to the lung ( p b 0.0001). We have previously observed that spontaneous fetal breathing movements occur in mice at 15–16 days of gestation [8]. This is consistent with earlier observations in rats that show these movements to begin at the same point in development, at 16 days of gestation [10]. These
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FIG. 1. Qualitative comparison of colloidal carbon distribution and transgene expression after intra-amniotic injection at 13 and 16 days of gestation in the fetal mouse. Intra-amniotic injections were performed with 50 Al of colloidal carbon at 13 (A) and 16 (B) days of gestation; fetuses were harvested after 1 h and cleared to allow visualization of carbon penetration. At 13 days of gestation, carbon is internalized only into the buccal and nasal passages. At 16 days of gestation, carbon can also be found in the trachea and lungs, demonstrating the onset of fetal breathing movements by this stage of development. Injections were repeated with 5 1010 particles of AdRSVhgal and fetuses stained for the presence of h-galactosidase protein. At 13 days of gestation, the skin is highly transduced (C), whereas the internal organs are not infected (D). At 16 days of gestation the opposite pattern occurs, as the skin is not transduced (E), while both the lung and gut show transgene expression (F) (L, lung; G, gut). Further analysis of the fetal lungs at 16 days of gestation reveals transgene expression throughout the bronchioles of all pulmonary lobes (G–I), as well as extensive expression in the trachea (J), gut (K), and stomach (L).
breathing movements are probably even more pronounced in fetuses at 17–19 days of gestation, although this has not been investigated here due to increased abortion rates at these later stages of pregnancy. It is therefore evident that the optimal time for a fetal gene therapy strategy in the mouse, one that aims to deliver a therapeutic gene to the lung by intra-amniotic injection, is at 16 days of gestation.
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Adenoviral Dose and Fetal Lung Transduction Show a Significant Positive Correlation In in vitro experiments to investigate the effect of amniotic fluid on adenovirus stability, we incubated adenovirus in amniotic fluid at 378C and removed aliquots after different time points for the transduction of HT 1080 cells in culture (these cells were used because they are highly permissive for adenovirus infection). We
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and Fig. 4), these experiments demonstrate that the high variability observed in fetal lung transduction, even at the same gestational age, is not due to a loss/depletion of virus or to a rapid reduction in its infectious ability but instead must be caused by other factors. To further investigate these factors, the effect of the dose of AdRSVhgal on fetal pulmonary transduction was examined. A range of viral doses, from 6.4 107 to 2 1011 particles/fetus, was administered via intra-amniotic injection, and the h-galactosidase content of fetal lungs was determined by ELISA after 48 h (Fig. 3). Viral dose could not be further increased due the limitations of viral production. There is clearly a significant positive correlation between the dose of AdRSVhgal and the degree of pulmonary transgene expression ( p b 0.001). Interestingly, the relative incidence of nontransduced airways decreased with higher viral doses.
FIG. 2. Quantitative comparison of transgene expression in the lung and skin after intra-amniotic injection at 13 and 16 days of gestation in the fetal mouse. Intra-amniotic injections were performed with 5 1010 particles AdRSVhgal/ fetus at 13 and 16 days of gestation; fetuses were harvested after 48 h and lungs and skin analyzed for h-galactosidase content by ELISA (n = 12 for each data set). At 13 days of gestation, skin transduction is significantly higher than that seen at 16 days of gestation (***p b 0.0001). In addition, lung transduction is significantly increased at 16 days of gestation compared with the earlier stage of development due to the initiation of fetal breathing movements (***p b 0.0001).
found that an increase in the extent of adenovirus transduction occurred briefly during the first few minutes of incubation (up to 140% of transduction observed compared to preincubation in PBS). However, after 30 min incubation, transduction fell to 60% at 1 h and reached a plateau at 5% at 2 h (data not shown). In a separate experiment, fetuses were injected with 5 1010 particles AdRSVhgal, and small aliquots of amniotic fluid were immediately removed; samples were also taken from the same fetuses after 1 h and placed on cells in vitro to assess the amount of infectious virus present. In this experiment, a reduction in the number of blue cells transduced after infection was observed with samples taken immediately after injection (values are means F SD, 161 F 48) compared to those taken after 1 h (140 F 10). However, this difference was not statistically significant ( p = 0.097), probably due to the high degree of variation in the extent and frequency of breathing movements between fetuses in vivo. Nevertheless, in combination with our demonstration of fetal breathing within, at the longest, 1 h after injection (see text below
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Fetal Breathing Can Be Stimulated by Intra-amniotic Injection of Theophylline and Administration of Postoperative CO2 As uptake of virus into the fetal airways is one of the most important factors for airway transduction, we hypothesized that one reason for the observed variability could be variation in the frequency and/or intensity of fetal breathing movements. In addition, one of the major disadvantages of administering gene therapy vectors via intra-amniotic injection is the large dilution of vector in amniotic fluid. This makes it necessary to use higher titers of virus than would be required by direct intrapulmonary application. While these large doses are feasible with adenovirus due to the production of high-titer preparations, they present a significant hurdle for future in utero gene therapy strategies with alternative vectors that aim to target airway epithelia. Stimulation of fetal breathing movements at the time of injection could perhaps increase virus uptake and transduction, making it possible to reduce the amount of virus needed to achieve significant pulmonary transduction. In general, the net flow of fluid within the respiratory system of the fetus is from the lungs, since airway epithelial cells secrete liquid at a steady rate [11]. Various exogenous factors have previously been shown to inhibit (adenosine [12,13], smoking [14], alcohol [15], depressant drugs [16,17], hypoxia [18]) or stimulate (glucose [19,20], theophylline [21–24], hypercapnia [18]) fetal breathing movements in both animal models and humans. More importantly, with respect to this study, disturbance of the fetus by vigorous palpation or amniocentesis has also been shown to have an inhibitory effect on fetal breathing movements [25]. Intra-amniotic injection is a technique very similar to amniocentesis, thus it is likely that fetal breathing movements will be decreased after vector application, the precise time that they are desired for uptake of vectors into the airways.
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FIG. 3. Dose of AdRSVhgal in intra-amniotic injections and lung transgene expression show a positive correlation. A range of doses of AdRSVhgal, from 6.4 107 to 2 1011 particles/fetus, was applied by intra-amniotic injections; fetuses were harvested after 48 h and lungs analyzed for h-galactosidase content by ELISA. There is a significant positive correlation between the two factors ( p b 0.0001); no transgene expression is detected until the viral dose reaches 1.6 109 particles/fetus, after which point the h-galactosidase content increases with increasing viral dose. (Note: a larger number of fetuses treated with 2 1011 particles/fetus are also analyzed in Fig. 5; under this scenario one fetal lung does contain no detectable h-galactosidase).
In order to stimulate fetal breathing, we chose to combine two effectors that have been shown to act synergistically. Moss and Scarpelli demonstrated that theophylline increases the sensitivity of the respiratory center to CO2 in fetal sheep [24]. Intravascular administration of adenosine inhibits fetal breathing, and this effect is disrupted by theophylline [12,13]. It is thought that theophylline produces its effect through antagonism to endogenous adenosine, as it is structurally similar and thus may act as a competitive inhibitor. Additional putative mechanisms, including the alteration of cAMP levels via the inhibition of phosphodiesterase [26] and calcium release [27], have also been proposed to explain the stimulatory effect of theophylline on fetal breathing. The effect of CO2 is probably mediated by the effect of acid ions on the fetal brain stem. Acidification of the blood, or cerebral spinal fluid, increases fetal breathing movements, while the addition of bicarbonate to increase the pH has the opposite effect [28,29]. In initial semiquantitative experiments using coinjection of theophylline and AdRSVhgal into the amniotic fluid, along with postoperative CO2, fetal lungs were stained and graded blind on a scale of 0–5 depending on the intensity of h-galactosidase expression observed. These data gave promising results, with the best transduction seen at a dose of theophylline of 1.6 mg/ml, but,
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due to the semiquantitative nature of this method of analysis, in which small differences in h-galactosidase expression could not be observed, enhancement of transduction with stimulation was not statistically significant. In all further studies, bstimulationQ refers to the optimal conditions found: intra-amniotic injection of 1.6 mg/ml theophylline and postoperative administration of 10% CO2 in air for 1 h. To study the mechanical effects of fetal breathing on the intake of amniotic fluid, without the interference of other variables involved in the infectability of the airway epithelia in individual fetuses, we injected colloidal carbon into the amniotic cavity instead of adenovirus, either with or without theophylline (1.6 mg/ml). Fetuses were harvested after 1 h, and carbon could already be seen at varying degrees in virtually all fetal lungs, demonstrating that fetal breathing movements occur at least within this time period. Fig. 4A shows the histology from an intensely stained lung after intra-amniotic injection with colloidal carbon. The carbon is clearly present in all bronchi and bronchioles but does not penetrate the vasculature of the fetus. Lungs were then graded blind on a scale of 0–5 (Figs. 4B–4G), depending on the degree of carbon present. This is an arbitrary scale defined in our laboratory. Fig. 4H shows that, overall, theophylline and CO2 stimulation increases the degree of
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FIG. 4. Intra-amniotic injection of theophylline combined with postoperative CO2 administration increases fetal breathing and the intake of colloidal carbon into fetal lungs. Colloidal carbon was administered by intra-amniotic injection with (stimulation, n = 44) or without (control, n = 34) co-injection of 1.6 mg/ml theophylline and administration of postoperative 10% CO2 in air for 1 h. Fetuses were harvested after 1 h. Histological examination of an intensely stained lung shows that carbon is present in all bronchi and bronchioles but does not penetrate the vasculature of the fetus (A). Lungs were graded blind on a scale of 0–5 (B–G), depending on the degree of carbon present. Theophylline and CO2 stimulation increases the degree of pulmonary staining with colloidal carbon ( p b 0.001) (H).
pulmonary staining with colloidal carbon ( p b 0.001). This is most dramatically seen by the twofold increase in the percentage of fetuses with grade-5-stained lungs. Under stimulation, 93% of fetal lungs exhibited strong colloidal carbon staining (grades 3–5), whereas the injection of carbon without stimulation resulted in only 59% staining at these grades. We then investigated whether this stimulation of fetal breathing would increase pulmonary transgene expression. Intra-amniotic injections were first performed using the maximal dose of 2 1011 particles AdRSVhgal/fetus. Fetuses were harvested after 48 h, and the presence of h-galactosidase protein in the lungs was determined by ELISA. Fig. 5 demonstrates that, despite the wide range of transgene expression levels seen, stimulation of fetal breathing movements significantly increased the h-galactosidase expression of the fetal lungs ( p b 0.05). When these experiments were
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repeated with a lower dose of virus (5 10 10 particles/fetus), a greater degree of enhancement was seen ( p b 0.01). This disparity is most likely due to the saturation of airway epithelial cells by transgene expression at the higher adenoviral dose, leaving less scope for an increase after stimulation. As a further test of the hypothesis that theophylline increases particle uptake into the lung by acting on the fetal breathing centers, we coapplied colloidal carbon and adenosine, a known inhibitor of these fetal breathing movements. Varying concentrations of adenosine (based on a previous study in fetal sheep [13]) were injected into the murine peritoneal cavity of fetuses at 16 days of gestation, followed by intra-amniotic injections of colloidal carbon. Control fetuses were injected with PBS before marker dye administration. Fetuses were culled after 1 h, and the degree of colloidal carbon staining in the fetal lung was graded after clearing (Fig. 6). Adenosine admin-
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FIG. 5. Intra-amniotic injection of theophylline combined with postoperative CO2 administration increases fetal breathing and adenoviral-mediated transgene expression. Intra-amniotic injections were performed with 5 1010 or 2 1011 particles AdRSVhgal/fetus with (stimulation) or without (control) coinjection of 1.6 mg/ml theophylline and administration of postoperative 10% CO2 in air for 1 h (n = 24 and 30 for each condition at 5 1010 and 2 1011 particles/fetus, respectively). Fetuses were harvested after 48 h and pulmonary h-galactosidase content was analyzed by ELISA. At both viral doses, stimulation significantly increased fetal breathing and thus the amount of h-galactosidase present in fetal lungs, but the enhancement was more pronounced at the lower dose (**p b 0.01) than at the higher dose (*p b 0.05).
istration significantly reduced the amount of colloidal carbon present in the fetal lungs ( p b 0.01). These experiments were repeated using AdRSVhgal, but although an inhibitory trend was observed after adenosine administration, the data were not statistically significant. This was probably due to the high degree of variability between fetuses, and power calculations showed that an unreasonable number of animals would be needed to obtain a statistically significant result. Although the stimulation of breathing movements clearly had a positive effect on transduction efficiency, it did not decrease the degree of variation and did not abolish the occasional transduction failure. These events could still have been due to lack of breathing in these fetuses or to other still unknown factors. One such factor, which we have not investigated here, is the
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expression of suitable receptors on the airway epithelia during fetal development. The main receptor for adenovirus, the coxsackie adenovirus receptor (CAR), is expressed on the basolateral surface of airway epithelial cells. CAR mRNA has been shown to be expressed in developing mouse airways at 12 days of gestation [30]. Various studies in primary human airway epithelial cells [31], rabbit or human epithelia [32], and rodent trachea and lung [33,34] have employed chemical agents, such as sodium caprate or EGTA, to transiently disrupt tight junctions between the epithelial cells, allowing adenovirus access to receptors. Data presented here clearly show that adenovirus is capable of infecting all areas of the fetal airway without these treatments, although, at lower viral doses, the opening of tight junctions may enhance transgene expression. However, it is not clear whether there may be a variation in expression at this time of fetal life, whether the murine fetal airways contain tight junctions at this stage of development, or, even if they do, to what extent agents such as sodium caprate would be able to exert their effect after intraamniotic injection. In summary, we have investigated three major factors that influence the degree of pulmonary transgene expression achieved after intra-amniotic injection in utero: gestational age, adenoviral dose, and the extent of fetal breathing movements. In the mouse, fetal breathing movements begin at day 15–16 of gestation, so access to the fetal lung via the amniotic fluid is only possible from this stage of development onward. The combination of intra-amniotic application of 1.6 mg/ml theophylline and postoperative CO2 stimulates fetal breathing and thus can be used to significantly increase the degree of pulmonary transgene expression achieved. However, it appears that the dose of AdRSVhgal used is the most critical factor, as the percentage of the lungs transduced (to any degree) increases with the dose of virus used. Nevertheless, in cases in which lower viral titers must be used, stimulation of fetal breathing movements by theophylline and CO2 may be helpful.
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In vivo procedures. Pregnant MF1 female mice were anesthetized by the inhalation of isofluorane (Abbot Laboratories, Queensborough, UK). A midline laparotomy was performed using sterile techniques, and both horns of the gravid uterus were exposed. Approximately six fetuses were injected per litter. Each amniotic cavity was injected (50-Al vol) by penetration of the uterus wall, the yolk sac, and amniotic membranes with a 33-gauge Hamilton Microliter Syringe (EssLab, Hadleigh, UK). Amniotic sacs were palpated after injection to ensure thorough mixing of the injectate. The amniotic fluid volume increases with fetal age; in the mouse it is approximately 300 Al at 16 days of gestation, which would dilute the injected virus amount about sixfold. Care was taken to ensure that injections were performed into the amniotic cavity and not into the extraembryonic coelom [35]. 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
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FIG. 6. Intraperitoneal injection of adenosine inhibits fetal breathing and intake of colloidal carbon into fetal lungs. Colloidal carbon was administered by intra-amniotic injection with (inhibition, n = 14) or without (control, n = 14) preceding intraperitoneal injection of 18 Ag adenosine/fetus. Fetuses were harvested after 1 h and lungs were graded blind on a scale of 0–5 (Figs. 4B–4G), depending on the degree of carbon present. Adenosine administration inhibits fetal breathing and hence decreases the degree of colloidal carbon observed in fetal lungs ( p b 0.01).
warmed cage until awake and active and housed in an undisturbed environment until the harvesting of fetuses. Mice treated with theophylline (Sigma-Aldrich, St. Louis, MO, USA; 1.6 mg/ml) were placed in a chamber containing 10% CO2 in air for approximately 1 h after surgery. Fetal and maternal survival was greater than 95%. Adenovirus vector. First-generation (E1 and E3 regions deleted) serotype 5 adenoviral vector (AdRSVhgal) containing the h-galactosidase reporter gene (driven by an RSV promoter) was prepared as previously described [36]. Virus was freshly prepared in PBS prior to injection. Virus titer was determined as particle number/ml by spectrophotometry using a coefficient of extinction of 1.1 1012 virus/OD260 unit [37]. Primers that amplify from within the E1A region of adenovirus were used to test viral batches for the presence of replication-competent virus. The vectors used were E1A deleted so that positive E1A PCR products would only arise from replication-competent virus. No E1A PCR product was obtained from any of the applied virus preparations. Colloidal carbon marker. Colloidal carbon suspension was prepared by first mixing 5 g of gum arabic (Sigma) thoroughly with 30 ml of dH20. The mixture was ground against a glass plate until a smooth paste was formed. Two grams of Printex P beads, furnace black (Degussa-Huls, Frankfurt, Germany), were added and ground until thoroughly mixed. The solution was sonicated (Branson Sonifier 450) and then autoclaved to ensure sterility. Visualization of colloidal carbon and reporter gene expression. Fetuses were harvested 48 h after in utero injection, and those to be analyzed for reporter gene expression were placed in 100% ethanol for 1 h. Approximately 200 Al of 100% ethanol was injected into the peritoneal cavity to ensure complete internal fixation before the fetuses were sliced sagitally. Samples were thoroughly washed in PBS and placed in X-gal solution (0.4 mg/ml 5-bromo-4-chloro-3-indolyl-h-d-galactosidase in DMSO, 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, 0.1 mM MgCl2). To better observe staining in the lungs, samples were cleared by dehydration in 100% methanol and transferred to a benzyl benzoate and benzyl alcohol mixture (2:1 v/v) [38]. The specimens were photographed under a dissection microscope (S7-CTV, Olympus, Japan) using a digital camera (DP10, Olympus). Brightness and contrast were optimized using Adobe Photoshop CS. Reporter gene quantification. h-Galactosidase levels were determined by ELISA using a commercially available kit (Boehringer Mannheim, Mannheim, Germany). Fetal tissues were dissected 48 h after in utero injection and stored at 208C in 200 Al of kit lysis buffer before analysis. The protein content of samples was analyzed using the bicinchoninic acid protein assay system (Pierce, Rockford, IL, USA). Levels of h-galactosidase
MOLECULAR THERAPY Vol. 12, No. 3, September 2005 Copyright C The American Society of Gene Therapy
activity were standardized on the basis of protein content, and values are presented as picogram of h-galactosidase per milligram of protein. The limit of h-galactosidase detection with this assay is z30 pg/ml (according to the manufacturer’s instructions). Statistical analysis. Data were analyzed by Mann-Whitney nonparametric tests, except for the analysis of transgene expression versus viral dose (Fig. 3), which was analyzed by parametric regression analysis after logarithmic transformation using MINITAB (Minitab Inc., State College, PA, USA). Where data are displayed as box plots, the top of the box is the third quartile (Q3, 75% of the data values are less than or equal to this value), the bottom of the box is the first quartile (Q1, 25% of the data values are less than or equal to this value), and the line bisecting the box is the median. In addition, the upper whisker extends to the highest data value within the upper limit (upper limit, Q3 + 1.5(Q3–Q1)) and the lower whisker extends to the lowest value within the lower limit (lower limit = Q1–1.5(Q3–Q1)). Values beyond the whiskers are outliers (denoted by black circles).
ACKNOWLEDGMENTS We thank Andrea Pavirani (Transgene, Strasbourg, France) for the AdRSVhgal construct and S. R. Underwood (Imperial College, London, UK) for the kind gift of adenosine. Sport Aiding Medical Research for Kids (SPARKs), the Katharine Dormandy Trust for Haemophilia and Allied Disorders, and the Medical Research Council (MRC) contributed to funding. HS was funded by German Research Foundation (SCHN 569/3-1). RECEIVED FOR PUBLICATION DECEMBER 9, 2004; ACCEPTED FEBRUARY 22, 2005.
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