1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven
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Carlon, M. S., Toelen, J., da Cunha, M. M., Vidović, D., Van der Perren, A., Mayer, S., et al. A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model. J. Vis. Exp. (68), e4219, doi:10.3791/4219 (2012).
Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and α1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.
1. Mating of Mice to Obtain Desired Pregnancy Stage
Time mate pregnant NMRI mice so that they are 18 days (E18) pregnant (total gestation E19.5) at the time of surgery. Prior to and after surgery they are housed in filter top cages at normal room temperature and normal daylight with free access to water and chow.
2. Fetal Intratracheal (I.T.) Injection (Figure 1)
3. Fetal Intra-amniotic (I.A.) Injection
4. Assessment of Injected Fetuses and Cross-fostering
5. Representative Results
The overall scheme of the experiment is depicted in Figure 2.
Determination of the optimal volume for intratracheal injection
To determine the optimal volume for I.T. injection, we empirically chose different volumes ranging from 10, 20 to 30 μl (n=3/volume). For easy detection, we chose to inject red fluorescent molecules (fluospheres, Molecular Probes, Leiden, The Netherlands) sized 100 nm. After I.T. injection in E18 old fetuses, the lungs were harvested 24 hr later, fixed in 4% paraformaldehyde overnight at 4 °C and 6 μm frozen sections were made. Nuclei and actin filaments were stained with Hoechst 33258 (Sigma-Aldrich, Bornem, Belgium) and Alexa Fluor 488 phalloidin (Invitrogen, Merelbeke, Belgium) respectively for 20 min at room temperature. Confocal images were made using a Biorad Radiance 2100 Confocal microscope with LaserSharp2000.6 software by Carl Zeiss. The relative fluorescence (ratio of red to blue fluorescence representing fluospheres and nuclear staining, respectively) was quantified using ImageJ online software (Figure 3). Although at the time of fetal surgery, a backflow was detected only after injection of 30 μl, indicating an excess of fluids injected, 30 μl gave the highest amount of fluorescent signal in the lung parenchyma as quantified by measuring the relative fluorescence (analysis of variance, comparisons for each pair using Student's t-test, *p < 0.05, ***p < 0.001).
Quantitative assessment of fluospheres in pulmonary tissue and biodistribution to the gastro-intestinal tract
Next, we wanted to compare the efficiency of targeting the fetal mouse lung after I.T. versus I.A. injection. To do so, 30 μl of fluospheres were delivered to the fetal mouse lung after either I.T. or I.A. injection in E18 pregnant NMRI mice (n=5 per group). I.T. injection resulted in a significantly higher delivery of fluospheres to the fetal lung compared to the I.A. route (1.43 ±0.56 and 0.05 ± 0.02 relative fluorescence (ratio of fluospheres to Hoechst respectively, analysis of variance, Student's t-test, *** p<0.001) (Figures 4 a-c). Untreated control fetuses were used for normalization of fluorescent background signal. The gastro-intestinal tract was positive for both the I.T. and the I.A. injected animals (Figure 4 d). No red fluorescence was observed in other tissues from treated fetuses or in the negative control animals (data not shown).
Comparison of intratracheal and intra-amniotic injection following rAAV2/6.2 mediated gene delivery in the fetal lung
After comparing both delivery methods by injecting fluorescent molecules, we wanted to evaluate the efficiency of viral transduction and subsequent gene expression after I.T. and I.A. injection using rAAV vectors. rAAV2/6.2 encoding firefly luciferase (fLuc) (3x1010 GC/fetus) under the control of the chicken-β-actin (CBA) promoter was injected I.T. (n=8) or I.A. (n=6) in fetal NMRI mice at E18. After caesarean section and fostering, surviving pups were followed up by non-invasive bioluminescence imaging (BLI) and monitored for fLuc activity (photons/second, p/s) at 1 week of age (Figure 5). The total photon flux for the I.T. group was significantly higher than that in the I.A. group and the negative control (analysis of variance, comparisons for each pair using Student's t-test, *p<0.05). The average BLI signal in the I.A. group was not significantly higher than that in the negative control group.
Distinction between a correct and an incorrect fetal intratracheal injection
Distinction between a correct and an incorrectI.T. injection can be assessed at several levels. At the time point of surgery, during injection into the fetal trachea, no resistance will be noticed when the needle is positioned in the trachea. However, a higher resistance will be noticed when injecting in the paratracheal space. Second, at caesarean section as the fetus is semi-transparent, it is possible to see the lungs and subsequently the presence of a visible dye (e.g. Chinese ink, fluorescent molecules). A last option to assess a correct injection is by optical imaging and more specifically bioluminescence imaging. BLI is an elegant system to non-invasively follow-up gene expression of the reporter gene firefly luciferase, but the spatial resolution and anatomic information are limited. Magnetic resonance imaging (MRI) provides high resolution, tomographic images containing detailed anatomical information. Therefore we investigated the combination of BLI with MRI to obtain an overlay image, which combines the surface BLI signal with a visualization of the deeper anatomical structures (internal organs). Our aim was to obtain more detailed in vivo information of the localization of gene expression to be able to distinguish a correct from a wrong I.T. injection.
Combined BL-MR images were acquired on several animals injected I.T. with rAAV2/6.2 CBA-fLuc and CBA-LacZnls (3x1010 GC/fetus for each vector, n=10) at one week of age (Figure 6). BL imaging revealed a signal emanating from the neck and the thoracic region. Co-registration of MRI with BLI located luciferase gene expression in the pulmonary region following a correct injection (Figure 6 a), but in the neck and abdominal area after an incorrect injection (Figure 6 b). Histological analysis by X-gal staining confirmed the in vivo co-registration.
Survival after intratracheal and intra-amniotic injection
Figure 1. Intratracheal injection in E18 fetal mice. In this figure the main steps of the surgical procedure for fetal I.T. injection are depicted. In a first step one uterine horn is exteriorized. In a next step, a purse string suture is passed through the uterine wall and the fetal membranes (amniotic membrane and parietal yolk sac) over the area where later on the fetal head will be exposed through. Next, the head and the neck of the fetus are exteriorized through the hysterotomy, following which the fetal head is kept in hyperextension by a 5-0 polyglactin 910 suture on two forceps between the jaws. Under stereoscopic zoom microscopy (x10 magnification) the fetal trachea is visualized by making a vertical neck incision using sharp and blunt dissection. In a last step, a total volume of 30 μl of substance is injected into the trachea under direct vision through the stereoscopic zoom microscope.
Figure 2. General overview of the experiment.
Figure 3. Determination of the optimal volume for intratracheal injection. To determine the optimal volume for I.T. injection, 10, 20 or 30 μl (n=3/volume) of red fluospheres sized 100 nm were administered in E18 old fetuses and the lungs were harvested 24 hr later. Nuclei and actin filaments were stained with Hoechst 33258 and Alexa Fluor 488 phalloidin respectively. The relative fluorescence (ratio of red to blue fluorescence representing fluospheres and nuclear staining respectively) was quantified using ImageJ online software. Mean ± SD, analysis of variance, comparisons for each pair using Student's t-test, *p<0.05, ***p<0.001.
Figure 4. Quantitative assessment of fluospheres in pulmonary tissue and biodistribution to the gastro-intestinal tract. 30 μl of red fluospheres were delivered to the fetal mouse lung after (a) I.T. or (b) I.A. injection in E18 pregnant NMRI mice to compare the efficiency of targeting the fetal mouse lung. Untreated control fetuses were used for normalization of fluorescent background signal. Nuclei and actin filaments were stained with Hoechst 33258 and Alexa Fluor 488 phalloidin, respectively. (c) The relative fluorescence (ratio of red to blue fluorescence representing fluospheres and nuclear staining respectively) was quantified using ImageJ online software. (d) The gastro-intestinal tract was positive for both the I.T. and the I.A. injected animals. Mean ± SD, analysis of variance, Student's t-test, ***p<0.001. Click here to view larger figure.
Figure 5. Comparison of intratracheal and intra-amniotic injection following rAAV2/6.2 mediated gene delivery in the fetal lung. BLI signal at 1 week after injection of rAAV2/6.2 (3 × 1010 GC/fetus CBA-fLuc) with corresponding quantification of total photon flux. All animals were scanned, separated by black partitions, to avoid scattering of photons to neighboring animals. The pseudocolor scale depicts the photon flux per second, per square centimeter per steradian (p/s/cm2/sr). Measurements were obtained in a 4.3 cm2 rectangular region of interest. Mean ± SD, analysis of variance, comparisons for each pair using Student's t-test, *p<0.05. Figure adapted from Carlon et al., 2010. Reprinted by permission from Macmillan Publishers Ltd: [Molecular Therapy] (doi:10.1038/mt.2010.153), copyright (2010). Click here to view larger figure.
Figure 6. Distinction between a correct and an incorrect fetal intratracheal injection following rAAV2/6.2 mediated gene delivery in the fetal lung. Combined BL-MR images were acquired on several animals injected I.T. with rAAV2/6.2 CBA-fLuc and CBA-LacZnls (3x1010 GC/fetus for each vector) at one week of age. BL imaging revealed a signal emanating from the neck and the thoracic region. Co-registration of MRI with BLI located luciferase gene expression in the pulmonary region following a correct injection (a), but in the neck and abdominal area after an incorrect injection (b). Histological analysis confirmed the in vivo co-registration. Scale bar = 100 μm. Figure adapted from Carlon et al., 2010. Click here to view larger figure.
|Injection substance||Injection method||Survival to delivery a||Survival rate of fostering b||Early neonatal survival rate c|
|rAAV2/6.2||I.T.||85,3 (64/75)||62,5||53,3 (40/75)|
|I.A.||86,3 (44/51)||86,4||74,5 (38/51)|
Table 1. Survival after FETAL intratracheal AND intra-amniotic injection. a Survival to delivery, i.e. after fetal surgery and at caesarean section, before fostering. b Pups were only fostered if they were pink, moving and breathing normally. c The early neonatal survival rate is expressed as a function of the initial number of pups injected. Abbreviations: I.T. Intratracheal injection; I.A. Intra-amniotic injection; n.a. not applicable.
Possible modifications and trouble-shooting
Significance of the technique with respect to existing methods
No conflicts of interest declared.
M.C. and A.V.d.P. are doctoral fellows supported by grants from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). J.T. holds a part-time Clinical Research Fellowship (KOOR) from UZ Leuven. D.V. is a doctoral fellow supported by a grant from KU Leuven, DBOF/10/062. M.M.d.C is a doctoral fellow supported by a grant from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and Erasmus Mundus. Research was funded by IWT-Vlaanderen, by the EC grant DIMI (LSHB-CT-2005-512146) and by the In vivo Molecular Imaging Research group (IMIR) from the KU Leuven. We would like to acknowledge the UPenn Vector Core founded by James M. Wilson for their kind gift of the AAV6.2 packaging plasmid for rAAV vector production.
|NMRI mice||Janvier, Le Genest St Isle, France|
|Isoflurane||Isoba, Intervet / Schering-Plough Animal Health, Milton Keynes, UK|
|Prolene 6-0||Ethicon, Groot Bijgaarden, Belgium|
|Vicryl 5-0||Ethicon, Groot Bijgaarden, Belgium|
|50 μl Hamilton Glass Syringe, Model 1710.5 TLLX SYR||Hamilton, Reno, NV, USA||5495-20|
|30G sharp needle||Hamilton, Reno, NV, USA||7762-03|
|2% xylocaine||AstraZeneca, Zoetermeer, The Netherlands|