Targeted Gene Transfer to Fetal Rat Lung Interstitium by Ultrasound-guided Intrapulmonary Injection
Authors: Tiago Henriques-Coelho, Sílvia Gonzaga, Masayuki Endo, Philip W Zoltick, Marcus Davey, Adelino F Leite-Moreira, Jorge Correia-Pinto, Alan W Flake
Journal: Molecular Therapy (2007)

In utero gene transfer to the developing lung may have clinical or research applications. In this study, we developed a new method for specifically targeting the fetal rat lung with adeno and lentiviral vectors encoding the enhanced green fluorescence protein (EGFP) marker gene at E15.5 using ultrasound biomicroscopy (UBM). Survival rate, morphometric parameters, viral biodistribution, and lung transduction efficiency were analyzed and compared to the intra-amniotic route of administration. Expression of EGFP started as early as 24 and 72 h after the injection of adenoviral and lentiviral vectors, respectively. Both vectors transduced lung parenchyma with gene expression limited to interstitial cells of the injected region, in contrast to intra-amniotic injection, which targeted the pulmonary epithelium. Expression of EGFP was most intense at E18.5 and E21.5 for adenoviral and lentiviral vectors, respectively. In contrast to lentivirus, adenoviral expression significantly declined until final analysis at 1 week of age. This study demonstrates the feasibility of targeting the fetal rat lung interstitium with viral vectors under UBM guidance during the pseudoglandular stage. This model system may facilitate in vivo studies of dynamic lung morphogenesis and could provide insight into the efficacy of prenatal gene transfer strategies for treatment of specific lung disorders. In utero gene transfer to the developing lung may have clinical or research applications. In this study, we developed a new method for specifically targeting the fetal rat lung with adeno and lentiviral vectors encoding the enhanced green fluorescence protein (EGFP) marker gene at E15.5 using ultrasound biomicroscopy (UBM). Survival rate, morphometric parameters, viral biodistribution, and lung transduction efficiency were analyzed and compared to the intra-amniotic route of administration. Expression of EGFP started as early as 24 and 72 h after the injection of adenoviral and lentiviral vectors, respectively. Both vectors transduced lung parenchyma with gene expression limited to interstitial cells of the injected region, in contrast to intra-amniotic injection, which targeted the pulmonary epithelium. Expression of EGFP was most intense at E18.5 and E21.5 for adenoviral and lentiviral vectors, respectively. In contrast to lentivirus, adenoviral expression significantly declined until final analysis at 1 week of age. This study demonstrates the feasibility of targeting the fetal rat lung interstitium with viral vectors under UBM guidance during the pseudoglandular stage. This model system may facilitate in vivo studies of dynamic lung morphogenesis and could provide insight into the efficacy of prenatal gene transfer strategies for treatment of specific lung disorders. IntroductionPrenatal gene transfer may offer a number of unique biological advantages relative to postnatal gene transfer.1Porada CD et al.Gestational age of recipient determines pattern and level of transgene expression following in utero retroviral gene transfer.Mol Ther. 2005; 11: 284-293Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 2Zanjani ED Anderson WF Prospects for in utero human gene therapy.Science. 1999; 285: 2084-2088Crossref PubMed Scopus (126) Google Scholar, 3Waddington SN Kennea NL Buckley SM Gregory LG Themis M Coutelle C Fetal and neonatal gene therapy: benefits and pitfalls.Gene Ther. 2004; 11: S92-S97Crossref PubMed Scopus (38) Google Scholar, 4Flake AW Stem cell and genetic therapies for the fetus.Semin Pediatr Surg. 2003; 12: 202-208Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar The relatively high frequency and accessibility of stem cells and progenitors and their rapid proliferation in the fetus may offer efficiency advantages for stem-cell-targeted gene transfer. The low total cell number in the fetus allows relatively high vector to target cell ratios, allowing the use of small amounts of vector. The immunologic immaturity of the early gestational fetus may induce tolerance to vector-associated or transgene-encoded proteins. Finally, in utero gene transfer has the potential to treat a disease before its clinical manifestations. Advances in prenatal diagnosis of genetic and congenital disorders with progressively more sensitive techniques may increase opportunities for consideration of prenatal gene therapy.5Sylvester KG Yang EY Cass DL Crombleholme TM Adzick NS Fetoscopic gene therapy for congenital lung disease.J Pediatr Surg. 1997; 32: 964-969Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 6Surbek DV Tercanli S Holzgreve W Transabdominal first trimester embryofetoscopy as a potential approach to early in utero stem cell transplantation and gene therapy.Ultrasound Obstet Gynecol. 2000; 15: 302-307Crossref PubMed Scopus (25) Google ScholarThe fetal lung is an attractive target organ for fetal gene transfer. There are a number of genetic and acquired disorders with peri- or postnatal pulmonary manifestations. These include monogenetic diseases like cystic fibrosis7Larson JE et al.Gene transfer into the fetal primate: evidence for the secretion of transgene product.Mol Ther. 2000; 2: 631-639Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar or surfactant protein B (SP-B) deficiency that would presumably require long-term expression of the deficient or defective gene. However, there are also abnormalities of lung growth, such as congenital diaphragmatic hernia, or lung maturation, such as respiratory distress syndrome of prematurity, that could potentially benefit from strategies that achieve transient gene expression in specific pulmonary distributions. Additionally, an important advantage of the use of non-integrating viral vectors with transient gene expression is the avoidance of many of the current concerns regarding integrating vectors in the fetus, i.e., insertional mutagenesis, developmental abnormalities, and germline alteration. We therefore anticipate the future need for a variety of lung-targeting strategies using different gene transfer technologies to achieve optimal therapeutic results.In this study, we describe a new method for the direct injection of viral vectors into the rat fetal lung as early as the pseudoglandular phase of lung development. Using this technique, we assessed the efficiency and distribution of transduction in the lung, the biodistribution of transduction, and the toxicity of equine infectious anemia virus (EIAV) and adenovirus vectors expressing the enhanced green fluorescence protein (EGFP) marker gene after in utero injection. In addition, we compare the direct injection technique to intra-amniotic injection with respect to the efficiency of lung transduction, and organ, and cellular distribution.ResultsThe technique of ultrasound-guided injectionsUltrasound biomicroscopy (UBM) was used to scan the fetus to identify the lung Figure 1). E15.5 was the earliest stage that we could perform in utero injections with a high survival rate and with accurate targeting of the developing lung. At E13.5 and E14.5, the lung bud was very difficult to visualize and mortality was unacceptably high. Important technical components of this procedure were the performance of a maternal laparotomy to expose the gravid uterus, and the application of pre-warmed sterile ultrasound gel over the uterus. This allowed manipulation of the fetal position into the axial orientation, which we found was the optimal plane for injection. The lung was best visualized between the level of the cardiac apex and upper part of the liver. Applying this imaging technique, we could inject small volumes (25–350 nL) of a viral vector suspension into the fetal lung parenchyma without any visually apparent injury to the lung or fetus. Injection of larger volumes was associated with the disruption of fetal lung architecture. The fetus and micropipette were manipulated so that the tip of the micropipette traversed only the uterine wall, amniotic membrane, amniotic cavity, fetal skin, and thoracic cavity (Figure 1 and Supplementary Video S1), avoiding the placenta, limbs, heart, or liver.Safety of in utero gene transferViral vectors used in this study were first-generation adenoviral vector and second-generation EIAV. To determine whether there was inherent toxicity from the viral preparation or from the early expression of the transgene, we compared survival after the intrapulmonary injection of viral vectors with injection of an equivalent volume of phosphate-buffered saline (PBS). The overall survival rate was 81±6, 82±5, and 87±4% for the PBS, EIAV, and adeno groups, respectively. Survival rates were calculated per dam by the formula: (number of injected fetuses alive/total number of injected fetuses per dam) × 100. There was no statistical difference among the groups at the studied time points (Table 1).Table 1Survival and somatic growthPBSEIAVAdenoE16.5 Survival (%)85±982±778±12 Body weight (g)0.49±0.020.53±0.010.51±0.02 Lung weight (mg)1.82±0.081.98±0.041.78±0.14 Heart weight (mg)0.94±0.070.86±0.050.98±0.8E18.5 Survival (%)94±587±689±1 Body weight (g)1.60±0.041.74±0.101.52±0.09 Lung weight (mg)3.19±0.103.27±0.123.23±0.12 Heart weight (mg)0.75±0.040.62±0.020.72±0.05E21.5 Survival (%)72±1685±1096±4 Body weight (g)6.10±0.245.85±0.135.35±0.10 Lung weight (mg)2.50±0.102.56±0.102.66±0.09 Heart weight (mg)0.76±0.060.69±0.060.77±0.02P7 Survival (%)72±1182±1282±7 Body weight (g)15.19±0.1314.00±0.6816.43±0.64 Lung weight (mg)2.26±0.052.15±0.082.29±0.06 Heart weight (mg)0.67±0.010.76±0.020.70±0.02EIAV, equine infectious anemia virus; PBS, phosphate-buffered saline. Survival is presented as mean±SE of survival rate per dam calculated by the formula: number of injected fetuses alive/total number of injected fetuses per dam × 100. Open table in a new tab In total, 53 dams were used and 527 fetuses were injected. There were no maternal deaths. There was no evidence of transplacental transfer of vector or transfected cells when maternal blood was analyzed for EGFP by flow cytometry (data not shown), although maternal tissues were not analyzed in detail. Fetuses delivered by cesarean section or at term were viable and without gross anomalies. Morphometric parameters, namely body weight, lung-to-body and heart-to-body weight ratios were similar and there were no statistical differences among the groups (Table 1). Macroscopic structure of the lung lobes, diaphragm, heart, and thoracic cavity was normal. Pups that survived until the seventh day after birth displayed overtly normal development and activity levels.Efficiency of gene transfer to the lung and kinetics of gene expressionTo determine the overall pattern of gene expression, we used stereoscopic fluorescence microscopy. After intrapulmonary injections, we observed that expression of EGFP, with the volumes of injectate utilized, was almost entirely limited to the injected lung and the ipsilateral pleural space after intrapulmonary injection (Table 2 and Figure 2). The time course of EGFP expression differed for the two vectors. EGFP expression was detected as early as 24 h after injection (E16.5) for adenoviral vector whereas the first expression did not appear until 72 h after injection for the EIAV-derived lentiviral vector (Figure 2). Maximum expression was observed at E16.5–E18.5 for adenovirus and at E21.5–P7 for lentivirus. For adenovirus, the intensity of lung EGFP fluorescence gradually decreased until P7 when it was barely discernable. For EIAV at P7, the intensity of the fluorescence was maintained, but the area of fluorescence was smaller presumably owing to the growth of the non-transduced lung and the relatively small area of initial transduction. In the adenoviral group, expression of EGFP was also seen in some fetuses in the skin and eyes, whereas in both groups the contralateral non-injected lung was positive in a few fetuses (Table 2).Table 2Viral vector biodistribution by fluorescence stereomicroscopyE16.5E18.5E21.5P7EIAVAdenoEIAVAdenoEIAVAdenoEIAVAdenon injected1420312422261923Lung injectedaNumbers represent the number of injected lungs that were negative or positive for EGFP fluorescence at the time points stated. Positive animals were subjectively ranked on a scale of + to +++ for intensity of fluorescence. −71211010 +511831111512 ++188108131010 +++1103112231Lung non-injected02144410Brain00000000Diaphragm00000000Eye06050902Gonads00000000Heart00010000Intestine00000000Kidney00000000Liver00000000Muscle00000000Skin02030410Spleen00000000Stomach00000000Trachea00000000EGFP, enhanced green fluorescence protein; EIAV, equine infectious anemia virus.a Numbers represent the number of injected lungs that were negative or positive for EGFP fluorescence at the time points stated. Positive animals were subjectively ranked on a scale of + to +++ for intensity of fluorescence. Open table in a new tab Figure 2Fluorescence stereomicroscopy and lung immunostaining for EGFP. Injected fetuses at E15.5 were analyzed at E16.5, E18.5, E21.5, and P7. Representative examples of fluorescence stereomicroscopy of the lungs at each analysis time after injection with (a–d) lentiviral vector and (e–h) adenoviral vector are shown. Viral vector transduction can be detected by green fluorescence in an area around the location of the injection. Representative images of immunostaining for EGFP from the lungs injected with (i–l) lentiviral vector and (m–p) adenoviral vector at the same analysis times are presented. (i–l) Lentiviral vector was only detected at (j) E18.5, with more EGFP expression being detected at (k) E21.5 and maintained until (l) P7. (m–p) Adenoviral vector induced an earlier expression of EGFP with maximal expression at (m, n) E16.5–E18.5. EGFP expression was analyzed by immunoperoxidase (brown staining) and both vectors were expressed in interstitial cells. No epithelial expression was detected. Bar=50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm the fluorescence observations and to provide a better quantitative assessment of changes in gene expression over time in the injected lungs, we performed real-time polymerase chain reaction (PCR) (Figure 3a). Evaluation of the number of vector copies per lung revealed the presence of the vector at E16.5 in both EIAV and adenoviral groups. However, the number of vector copies was statistically higher for adenoviral vector at E16.5 and E18.5. Whereas adenoviral-associated copy number progressively decreased after E16.5, copy number from EIAV gene transfer increased after E18.5 and was statistically greater than adenovirus at P7. It is important to note that this is not a direct comparison of transduction efficiency between the two viral vectors owing to the differing volumes and titers injected. Approximately 25 nL of adenoviral vector at a titer of 2–8 × 1011 i.p./mL was injected versus 350 nL of EIAV vector at a titer of 108–109 i.p./mL. Thus, even at the extremes of volume allowed by the model, there was at least a 100-fold greater dose of adenoviral vector injected. However, this quantitative data confirm the fluorescence observation of the rapid diminution in EGFP expression for adenoviral-transduced lungs over the interval between E16.5 and P7. In contrast, copy numbers, although relatively low, were maintained in the EIAV group throughout the duration of the study.Figure 3Quantitative PCR for EGFP. (a) Number of vector copies detected by quantitative PCR in lung samples after PBS, adenoviral, and lentiviral injections, at different time points of gestation. (b) Number of vector copies detected by quantitative PCR in non-pulmonary tissues and non-injected lung samples after PBS, adenoviral, and lentiviral injections at E21.5. Lv, liver; B, brain; St, stomach; Sp, spleen; I, intestine; M, muscle; K, kidney; H, heart; Tx, thorax; Tr, trachea; D, diaphragm; E, eye; G, gonads; Sk, skin; niL, non-injected lung of injected fetuses. *Significant differences=P⩽0.05 compared to PBS control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biodistribution of viral vectorsTo confirm our impression that EGFP gene expression was relatively limited to the lung after intraparenchymal injection and to more globally assessed gene expression in the injected fetuses, we performed quantitative PCR on several tissues at E21.5 (Figure 3b). A statistically higher number of vector copies relative to PBS controls were observed in the heart and in the tract of the micropipette through the thoracic wall in both the EIAV-EGFP and Adeno-EGFP groups. For all other tissues analyzed, there were no statistical differences in EGFP expression above the PBS-injected controls.Cellular distribution of gene transduction within the lungEGFP-positive cells were predominantly found in the pulmonary interstitium for both the EIAV and adenoviral groups, after intrapulmonary injections (Figure 2i–p). There was no expression of reporter gene in the surface epithelium of airways or in the vascular endothelium. EGFP-positive cells neither stained for vimentin, a mesenchymal-derived cell marker (Figure 4a and b), nor for SP-B, a surfactant protein expressed in Type II pneumocytes (Figure 4c and d and Supplementary Figure S2). Therefore, we demonstrated that transduced cells were located within the interstitial compartment of the lung and that they were not epithelial. This is in distinct contrast to the epithelial-restricted expression seen with intra-amniotic vector injections (Figure 4e and f).Figure 4Interstitial localization of tranduced cells after intraparenchymal injection. (a, b) Lung double immunofluorescence for EGFP (green) and vimentin (red) in fetuses injected with (a) lentiviral and (b) adenoviral vectors. Lungs were harvested at P7. (c, d) Lung immunoperoxidase staining for EGFP (brown) and immunofluorescence for SP-B (green). Images represent an overlay of bright light and fluorescent images at original magnification × 60 and × 40 for c and d, respectively. Merged images were created with Adobe Photoshop software and adjusted for brightness, contrast, and opacification to allow optimal visualization of both markers (original images before manipulation can be seen in Supplementary Figure S2). EGFP-positive cells did not colocalize with vimentin, or SP-B, and were localized within the interstitial region of the parenchyma. (e, f) Epithelial localization of transduced cells after intra-amniotic injection. Intra-amniotic injections were performed at E16.5 and the fetuses were harvested at E21.5. (e) Fluorescence stereomicroscopy and (f) lung immunostaining for EGFP are presented demonstrating the transduction of only epithelial cells. Bar=50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionWe report for the first time, the feasibility of using UBM to perform in utero intrapulmonary injections in rats at the pseudoglandular stage of lung development. Both adenoviral and lentiviral vectors efficiently transduced lung parenchyma, although different expression patterns were observed following injection. As expected, adenoviral vector expression appeared quickly and was transient, whereas lentiviral vector expression was relatively delayed and persisted through the time period of this study. Unexpectedly, in contrast to intra-amniotic injections, both vectors selectively transduced interstitial cells and not alveolar or airway epithelial cells, or vascular endothelial cells.Several routes of vector administration have been utilized to achieve prenatal gene transfer to the lung including intra-amniotic,8Buckley SM et al.Factors influencing adenovirus-mediated airway transduction in fetal mice.Mol Ther. 2005; 12: 484-492Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 9Holzinger A Trapnell B Weaver TE Whitsett JA Iwamoto HS Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues.Pediatr Res. 1995; 38: 844-850Crossref PubMed Scopus (79) Google Scholar, 10Boyle MP Enke RA Adams RJ Guggino WB Zeitlin PL In utero AAV-mediated gene transfer to rabbit pulmonary epithelium.Mol Ther. 2001; 4: 115-121Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 11Garrett DJ Larson JE Duna D Marrero L Cohen JC In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.BMC Biotechnol. 2003; 30: 3-16Google Scholar systemic,12Schachtner S Buck C Bergelson J Baldwin H Temporally regulated expression patterns following in utero adenovirus-mediated gene transfer.Gene Ther. 1999; 6: 1249-1257Crossref PubMed Scopus (52) Google Scholar, 13Bouchard S et al.Long-term transgene expression in cardiac and skeletal muscle following fetal administration of adenoviral or adeno-associated viral vectors in mice.J Gene Med. 2003; 5: 941-950Crossref PubMed Scopus (34) Google Scholar, 14Waddington SN et al.Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.Gene Ther. 2003; 10: 1234-1240Crossref PubMed Scopus (61) Google Scholar intratracheal,15Vincent MC Trapnell BC Baughman RP Wert SE Whitsett JA Iwamoto HS Adenovirus-mediated gene transfer to the respiratory tract of fetal sheep in utero.Hum Gene Ther. 1995; 6: 1019-1028Crossref PubMed Scopus (69) Google Scholar, 16Peebles D et al.Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero.Gene Ther. 2004; 11: 70-78Crossref PubMed Scopus (52) Google Scholar, 17Skarsgard ED Huang L Reebye SC Yeung AY Jia WW Lentiviral vector-mediated, in vivo gene transfer to the tracheobronchial tree in fetal rabbits.J Pediatr Surg. 2005; 40: 1817-1821Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar and intrapulmonary.18Tarantal AF et al.Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): lung-targeting approaches.Mol Ther. 2001; 4: 614-621Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 19Tarantal AF et al.Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery.Mol Ther. 2005; 12: 87-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Intra-amniotic and systemic approaches share the same limitations of being nonspecific for fetal lung. Intratracheal and intrapulmonary delivery, although more specific to the lung, have previously only been performed in large animal models. Tarantal et al.18Tarantal AF et al.Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): lung-targeting approaches.Mol Ther. 2001; 4: 614-621Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar demonstrated, in a non-human primate model, that intrapulmonary injections during the pseudoglandular stage allowed specific targeting of the lung relative to injections performed during the embryonic stage. In this study, we used UBM to perform in utero intrapulmonary injections. The use of this relatively new imaging technology made injection of the fetal rat lung at E15.5. UBM utilizes high-frequency (20–100 MHz), pulse-echo ultrasound for imaging live tissues and organs, allowing near microscopic resolution and has been previously utilized for fetal brain injections.20Turnbull DH Bloomfield TS Baldwin HS Foster FS Joyner AL Ultrasound backscatter microscope analysis of early mouse embryonic brain development.Proc Natl Acad Sci USA. 1995; 92: 2239-2243Crossref PubMed Scopus (123) Google Scholar, 21Foster FS Pavlin CJ Harasiewicz KA Christopher DA Turnbull DH Advances in ultrasound biomicroscopy.Ultrasound Med Biol. 2000; 26: 1-27Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar Using UBM, we could successfully target the rat lung during the mid-point of the pseudoglandular stage of lung development with a high fetal survival rate.First-generation adenovirus and EIAV lentivirus-based vectors were used in this study. We chose to study first-generation adenoviral vectors rather than adeno-associated viral vectors because we wished to see rapid, high-level expression of the marker gene. We used EIAV owing to the fact that human immunodeficiency virus-1-based lentiviral vectors have not shown high efficiency transduction22Hofmann W et al.Species-specific, postentry barriers to primate immunodeficiency virus infection.J Virol. 1999; 73: 10020-10028PubMed Google Scholar, 23Romano G Michell P Pacilio C Giordano A Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications.Stem Cells. 2000; 18: 19-39Crossref PubMed Scopus (139) Google Scholar in many tissues and owing to the successful application of EAIV vectors in fetal rodent models by Waddington et al.14Waddington SN et al.Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.Gene Ther. 2003; 10: 1234-1240Crossref PubMed Scopus (61) Google Scholar Adenovirus has been one of the most extensively studied recombinant viral systems because of its high transduction efficiency, rapid expression, accommodation of large transgene inserts, and high titers.23Romano G Michell P Pacilio C Giordano A Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications.Stem Cells. 2000; 18: 19-39Crossref PubMed Scopus (139) Google Scholar, 24Senoo M et al.Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay.Mol Genet Metab. 2000; 69: 269-276Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 25Breyer B et al.Adenoviral vector-mediated gene transfer for human gene therapy.Curr Gene Ther. 2001; 1: 149-162Crossref PubMed Scopus (132) Google Scholar, 26Driskell RA Engelhardt JF Current status of gene therapy for inherited lung diseases.Annu Rev Physiol. 2003; 65: 585-612Crossref PubMed Scopus (64) Google Scholar The primary problem with adenovirus is its high immunogenicity and propensity to invoke strong immune responses. We previously documented that this is a problem with intratracheal administration in late gestational fetal lambs; however, in the same model, no significant inflammation is observed with adenoviral administration during the preimmune phase of lamb immunologic development.27Yang EY Cass DL Sylvester KG Wilson JM Adzick NS BAPS Prize—1997. Fetal gene therapy: efficacy, toxicity, and immunologic effects of early gestation recombinant adenovirus. British Association of Paediatric Surgeons.J Pediatr Surg. 1999; 34: 235-241Abstract Full Text PDF PubMed Scopus (38) Google Scholar Similarly, as we would predict from the stage of rat immune development at E15.5, we saw no overt inflammatory response in the lung by histology in these experiments. However, as the purpose of this study was not induction of immune tolerance, we did not perform a detailed analysis of immune response to adenoviral products or transgene. In contrast to adenovirus, lentiviral vectors are relatively non-toxic and minimally immunogenic and can stably integrate transgene into dividing and non-dividing cells with subsequent long-term gene expression.28Naldini L et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (3979) Google Scholar, 29Zufferey R Nagy D Mandel RJ Naldini L Trono D Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat Biotech. 1997; 15: 871-875Crossref PubMed Scopus (1550) Google Scholar, 30Sena-Esteves M Tebbets JC Steffens S Crombleholme T Flake AW Optimized large-scale production of high titer lentivirus vector pseudotypes.J Virol Methods. 2004; 122: 131-139Crossref PubMed Scopus (228) Google Scholar The main disadvantages are the low titers that are usually obtained in vitro.23Romano G Michell P Pacilio C Giordano A Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications.Stem Cells. 2000; 18: 19-39Crossref PubMed Scopus (139) Google Scholar This proved somewhat limiting in the current study. With EIAV vector titers of only 107–108 infectious particles per mL, we needed to inject the maximal tolerated volume of 350 nL of EIAV to achieve significant transduction.The rapid loss of EGFP expression in the adenoviral-transduced lungs is in keeping with the known episomal location of adenoviral gene expression, the small volume of vector administered (25 nL), and the rapid proliferation of fetal lung tissue. Our data are also consistent with the known capacity of EIAV to stably integrate transgene into the host genome. In the EIAV-transduced lungs, there was, if anything, a slight increase in copy number at P7. We would conclude from this analysis that adenovirus would be the most appropriate vector to use when the goal is to induce rapid and transient overexpression of a gene in this model, whereas, lentivirus would be a more suitable vector to induce sustained and long-term gene expression.Gene transfer was localized to the injected lung with the only exceptions b