Original Article

Gene Therapy (2012) 19, 936–946; doi:10.1038/gt.2011.157; published online 10 November 2011

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression

A A Rahim1,6, A M Wong2,6, S Ahmadi2, K Hoefer2, S M K Buckley3, D A Hughes3, A N Nathwani3, A H Baker4, J H McVey5, J D Cooper2 and S N Waddington1

  1. 1Gene Transfer Technology Group, Institute for Women's Health, University College London, London, UK
  2. 2Paediatric Storage Disorder Laboratory, MRC Centre for Neurodegeneration, Institute of Psychiatry, Kings College London, London, UK
  3. 3Department of Haematology, University College London, London, UK
  4. 4BHF Glasgow Cardiovascular Centre, Univeristy of Glasgow, Glasgow, UK
  5. 5Molecular Medicine, Thrombosis Research Institute, London, UK

Correspondence: Dr SN Waddington, Institute for Women's Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, UK. E-mail: s.waddington@ucl.ac.uk

6These authors contributed equally to this work.

Received 18 July 2010; Revised 15 July 2011; Accepted 17 August 2011
Advance online publication 10 November 2011



The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development.


adenovirus; adeno-associated virus; fetal brain; pseudotypes; gene delivery



The efficient delivery of genetic material to cells of the central nervous system (CNS) during fetal development has useful applications and represents a powerful research tool. Transduction of discrete areas of the brain and subsequent expression of cytotoxic molecules, short hairpin RNA or dominant-negative forms of proteins could mimic neurodegenerative human disease in animals without the need to generate transgenic or knockout models. Furthermore, transduction of specific cell types during gestation may allow for informative studies of fetal brain development.

A number of CNS diseases present progressive pathology during gestation in patients and animal models.1, 2 Delivery of therapeutic genes to the fetal brain of such models would potentially answer a number of fundamental questions that need addressing for potential therapies to be devised. For example, does expression of therapeutic protein during gestation increase lifespan and is neonatal intervention too late? In the case of neurodegenerative disease, this is of particular importance because damaged neurons of the CNS have a very limited capacity to regenerate. The vast majority of viral vector-mediated gene-delivery studies to the CNS have been in adult3, 4, 5 and, to a lesser extent, neonatal6, 7, 8, 9 animal models. This has been done using a broad range of viral vectors with differential serotypes and pseudotypes resulting in variable tropisms and transduction efficiencies. These studies have highlighted the need for specific cellular receptors to be available on cell membranes for transduction by a particular viral vector to occur. However, the availability or levels of expression of such receptors has been shown to vary significantly during different stages of development.10 Therefore, studies of transduction/tropisms in adult or neonates may have no relevance in the fetal brain.

The concept of viral-mediated gene delivery to the fetal brain is not a new one and dates back to the 1980s.11 While providing interesting data, these investigations invariably included only one viral vector type. This makes independent comparisons between vector tropisms and gene-delivery efficiencies in the fetal brain difficult. We have shown previously that an integration-deficient lentivirus can mediate efficient long-term gene delivery to the fetal brain with no significant immune response and enhanced viral spread compared with the adult brain.12 These findings support the use of non-integrating vectors for CNS gene delivery to neurons given their postmitotic status and the reduced risk of insertional mutagenesis. However, for the application of gene delivery to the fetal brain to be fully realised, more studies are required that address the above issues and build upon the advantages that this technique offers. In this study, we present data that expand upon our previous work to include other commonly used non-integrating viral vectors and their various pseudotypes. This provides the opportunity for direct comparisons and selection of appropriate vectors for a variety of downstream applications.



Widespread and differential patterns of gene delivery to the fetal brain using non-integrating viral vectors

To assess the ability of a range of non-integrating viral vectors to transduce cells of the fetal murine brain, vector preparations of adenovirus serotype 5 (Ad5), self-complementary adeno-associated virus pseudotyped with capsids from AAV5, AAV8 and AAV9 were produced, concentrated and titered. All vectors express enhanced green fluorescent protein (EGFP) driven by the cytomegalovirus promoter (Figure 1). Although titres between Ad5 and AAV vectors were not standardised, the different serotypes of AAV were normalised to allow for direct comparisons between the different pseudotypes. The titre of the Ad5 virus stock used was 2 × 1011 viral genomes per ml and AAV2/5, AAV2/8 and AAV2/9 was 7 × 1011 viral genomes per ml.

Figure 1.
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Schematic representation of viral vector constructs. Gene-delivery and -expression studies in the fetal brain were conducted using AAV pseudotypes 2/5, 2/8 and 2/9 and Ad5. All AAV vectors were in the self-complementary (sc) format and encoded GFP driven by the cytomegalovirus (CMV) promoter (a). Two Ad5 viruses were used; one expressing GFP driven by the CMV promoter and the other expressing luciferase (Luc), also driven by the CMV promoter (b and c, respectively). ITR, inverted terminal repeat; Poly A, polyadenylation sequence; TRS, terminal repeat.

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In all, 5μl of each virus (Ad5=1 × 109 viral genomes and AAV2/5, AAV2/8 and AAV2/9=3.5 × 109 viral genomes) was injected, in utero, into the brains of fetal MF1 mice at embryonic (E) day 15 (n=3 per dam). The injected mice were labelled, subcutaneously, with colloidal carbon to differentiate them from their uninjected littermates. Once born, these mice were reared until post-gestation (P) day 25 after which they were culled and the brains were removed, fixed and sectioned. All mice that received injections of virus were accounted for and easily identifiable by the colloidal carbon marking. No procedure-related mortality was observed at any point. EGFP expression was detected using anti-EGFP antibodies and 3,3′-diaminobenzidine (DAB) staining. Examination of the brain sections by light microscopy showed varying levels of cell transduction present in both the cerebral hemispheres (Figure 2). One of the brains injected with AAV2/9 showed no GFP immunoreactivity and so was classified as a failed injection. Representative images were taken from the level of the pre-frontal cortex, striatum, caudal hippocampus and cerebellum of uninjected control brain (Figures 2a–d) and brains injected with Ad5 (Figures 2e–h), AAV2/5 (Figures 2i–l), AAV2/8 (Figures 2m–p) and AAV2/9 (Figures 2q–t). Efficient and widespread transduction was seen in the brains transduced with AAV2/8 and AAV2/9. Gene expression was apparent in the brains injected with Ad5 but was visibly less widespread. In comparison, AAV2/5-transduced cells were sparsely distributed and this vector was clearly less efficient at transducing neural cells.

Figure 2.
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Immunohistochemical detection of GFP expression 1 month post in utero and neonatal intracranial injection of viral vectors. Fetal mice at embryonic day 15 (E15) were administered with Ad5 (1 × 109 vector genomes) and AAV pseudotypes (3.5 × 109 vector genomes) expressing GFP via in utero intracranial injection (n=3 per dam). The mice were born and reared until post-gestation day 25 (P25). The brains were harvested, sectioned and immunohistochemically analysed for detection of GFP. Representative images were taken from the pre-frontal cortex, striatum, hippocampus and the cerebellum. All brains administered with virus demonstrated varying levels of GFP immunoreactivity except for one of the AAV2/9 brains that was designated as a failed injection. Images are taken from the brain sections of uninjected mice as negative controls (n=3) (ad), mice injected with Ad5 (n=3) (eh), AAV2/5 (n=3) (il), AAV2/8 (n=3) (mp) and AAV2/9 (n=2) (qt). P1 neonatal mice were also intracranially injected with the same volume and number of viral particles of Ad5 (n=3). Age-matched uninjected mice were used as a negative control. The mice were culled 1 month post injection and immunohistochemically stained, as above. Representative images were taken from the brain sections of uninjected mice (ux) and Ad5-injected mice (yB).

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To gauge whether fetal administration can enhance dissemination of the vector through the brain compared with neonatal administration, we administered three P1 neonates with 5μl of Ad5-GFP via intracranial injection. One month post injection, the brains were harvested and analysed by immunohistochemistry for GFP expression, as performed on the fetal-administered brains. A direct comparison between fetal (Figures 2e–h) and neonatal Ad5-GFP-administered brains (Figures 2y–B) revealed less GFP staining in the latter group within the contralateral hemisphere when compared with the fetal-injected brains. Furthermore, there was also less spread towards rostral and caudal areas of the brain in these neonatally injected mice. This was best exemplified in the cerebellum where in utero injection resulted in gene expression around the fourth ventricle but also extensive staining in the cerebellar lobes (Figure 2h). However, neonatal administration resulted only in staining restricted to around the fourth ventricle (Figure 4b). The limited spread of virus in neonatal-administered brains compared with fetal-administered brains was consistent in all the brains examined (Supplementary Figure S1).

Further inspection of brain sections at higher magnification revealed both stark and subtle differences in the cellular tropisms of the different vectors. Figure 3 shows transduction patterns in the hippocampus, somatosensory barrel field, piriform cortex and the cerebellar lobes. The cornu ammonis (CA1–3) subfields of the hippocampus were transduced to variable levels of efficiency, dependent upon the vector used, by Ad5 and AAV pseudotypes 2/5, 2/8 and 2/9 (Figures 3a–d). However, while the AAV vectors preferentially transduced neurons, Ad5 also transduced cells of glial morphology (indicated by white arrows). Transduction patterns in somatosensory barrel field showed subtle differences in transduction of cortical layers. Ad5 transduction was restricted to cortical layer II neurons and scattered glial cells (Figure 3e), whereas AAV pseudotypes 2/5, 2/8 and 2/9 transduced neurons throughout cortical layers II–VI (Figures 3f–h). These cellular tropisms were also observed in the motor and cingulate cortex (data not shown). Examination of the piriform cortex revealed no transduction by Ad5 (Figure 3i). However, all three AAV serotypes mediated gene delivery to cells in this area (Figures 3j–l). Differential transduction patterns were also seen in the cerebellum (Figures 3m–p). Ad5 transduced cells in lobes 1–5 but not the simple lobule (sim) whereas AAV2/5 transduction was restricted to lobes 3–5, but also sim. Both AAV pseudotypes 2/8 and 2/9 transduced lobes 1–5 and sim.

Figure 3.
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GFP expression in discrete areas of the brain. Immunohistochemically stained brains for the detection of GFP expression were examined by light microscopy. Representative images were taken from the following discrete regions: hippocampus, somatosensory barrel field (S1BF), piriform cortex and cerebellar lobes. Images show the regions of the brains that were administered with Ad5 (ae), AAV2/5 (fj), AAV2/8 (ko) and AAV2/9 (pt). All vectors transduced cells of neuronal morphology although Ad5 also transduced larger glial cells, as indicated by white arrows. A quantitative comparison of GFP expression by threshold analysis of brain sections was conducted in the following discrete regions: CA1 region of the hippocampus (CA1), striatum (CPu), primary motor cortex (M1), medial septal nucleus (MS), piriform cortex (Pir), VPL/VPM and the cerebellar nodule (10Cb) (u). The data are plotted as the mean±s.e.m., n=3 for Ad5-, AAV2/5- and AAV2/8-injected brains, and n=2 for AAV2/9.

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By selecting discrete areas of these sections and measuring GFP immunostaining intensity by threshold analysis, a semiquantitative comparison of viral vector-mediated gene expression was conducted. GFP staining was measured in the CA1 region of the hippocampus (CA1), striatum (CPu), primary motor cortex (M1), medial septal nucleus (MS), piriform cortex (Pir), ventral posteromedial and ventral posterolateral thalamic nucleus (VPM/VPL) and the cerebellar nodule (10Cb). Similar to what we had qualitatively seen, the levels and patterns of gene expression varied depending upon the vector used (Figure 3u). All vectors mediated higher levels of gene expression in the CA1 region of the hippocampus and the MS, but comparatively lower levels in the CPu and M1. All vectors produced high levels of expression in the Pir except for Ad5, which produced no detectable GFP expression. Lower amounts of GFP were detected in the VPL/VPM, except for in AAV2/5-administered brains where expression was higher. Conversely, AAV2/5 produced the lowest levels of GFP measured in the 10Cb when compared with the other vectors. Interestingly, the amounts of GFP expression produced by AAV2/8 and 2/9 were comparable in all brain regions.

A broader qualitative analysis of transduction patterns in discrete areas of the brain was conducted and scored with (−) indicating no transduction, (+) a few transduced cells, (++) a high number of transduced cells and (+++) a region that was saturated with transduced cells (Table 1).

Confirmation of viral vector neural cell-type tropism by immunofluorescence and scanning confocal microscopy

Based on the morphology of GFP immunoreactive cells, all AAV vectors preferentially transduced neurons, but Ad5 transduced cells of both neuronal and glial morphology. To confirm these cell tropisms, brain sections were immunofluorescently stained for both GFP- and cell-specific phenotypic markers, and examined by multi-channel scanning confocal microscopy. Ad5-transduced brain sections were stained with TO-PRO-3 to label cell nuclei (Figure 4e), NeuN to label neurons (Figure 4f) and anti-GFP antibodies to identify transduced cells (Figure 4g), revealing the expression of GFP in a subset of NeuN-positive neurons (Figure 4h). The larger GFP-positive cells with glial morphology were negative for NeuN staining (Figure 4f). These GFP-positive cells were confirmed as astrocytes by colocalisation with for the astrocyte-specific marker S100β (Figure 4l). To confirm the predominantly neuronal tropisms of the AAV pseudotypes, brain sections from injected mice were also immunofluorescently stained with TO-PRO-3, NeuN and anti-GFP antibodies. Very few GFP-positive cells were detected in sections from AAV2/5-injected brains (Figure 4o), which reflected the low levels of GFP expression observed with DAB staining in somatosensory barrel field (Figure 3c) and measured in other areas of the cortex such as M1 (Figure 3u). However, those GFP-positive cells that were detected were also NeuN stained (Figure 4p). A similar colocalisation of GFP and NeuN staining was also evident in brains injected with AAV2/8 and 2/9 (Figures 4p and t). In contrast, uninjected brains showed no detectable GFP staining (Figure 4c).

Figure 4.
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Immunofluorescent and scanning confocal microscopy studies of viral vector neural cell tropisms. Brain sections from injected mice were immunofluorescently stained using cell-specific markers and anti-GFP antibodies. Sections from uninjected brains were used as a negative control and stained with TO-PRO-3 to label cell nuclei (blue), NeuN to label neurons (red), anti-GFP antibodies (green) and a merged image (ad, respectively). No non-specific anti-GFP staining could be detected (c). Ad5-administered brain sections were stained with TO-PRO-3 (e) and NeuN (f). Anti-GFP staining revealed expression in two morphologically distinct cell types; the vast majority in smaller cells with neuronal morphology and fewer larger cells with glial morphology (g). The merging of images revealed colocalisation of GFP and NeuN resulting in yellow signal and confirming them as neurons (h). Examples of colocalisation within cells are highlighted with white arrows. The larger glial cells were negative for NeuN staining. To identify these cells, sections were stained with TO-PRO-3 (i) and S100β to label astrocytes (red). When images of S100β labelled cells (j) were merged with images of GFP expressing cells (k), colocalisation of signals confirmined these cells to be astrocytes (l). AAV2/5-injected brain sections were also stained with TO-PRO-3 (m) and revealed low levels of GFP staining and occasional bright cells (o) that colocalised with NeuN-stained cells (n) and confirmed neuronal tropism (p). AAV2/8 and AAV2/9-administered brain sections were stained with TO-PRO-3 (q and u, respectively) and revealed strong staining with anti-GFP antibodies (s and w, respectively) that colocalised with NeuN-stained cells (r and v, respectively) producing yellow signal (t and x, respectively) and confirming the neuronal tropism of these vectors.

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Analysis of immune system activation

As immune responses to vectors and to the high levels of GFP expression may occur, we looked for any evidence of activated microglia in all brains using antibodies against CD68. As a positive control, brains were taken from Ppt1/ mice that display intense microglial activation as part of their pathological neuronal ceroid lipofuscinosis phenotype.13 As previously reported,13 the motor cortex of Ppt1/ mice revealed intense CD68 immunoreactivity (Figure 5a), but there was no obvious increase in staining for this marker of microglial activation in the brains injected with any of the viruses (Figures 5c–f), compared with uninjected brains (Figure 5b). Higher-power examination of the M1 motor cortex revealed microglia in a quiescent state in uninjected and vector-administered brains compared with activated and swollen microglia in the Ppt1/ brains (inset boxes). To quantify the levels of staining, threshold analysis was performed on these CD68-stained sections. Analysis of variance followed by Bonferoni correction revealed that microglial activation was significantly higher in Ppt1/ mice than in uninjected or virus-injected brains. No significant difference in the levels of microglial activation was seen between the brains that received viruses and uninjected control brains (Figure 5g).

Figure 5.
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Analysis of microglia activation by CD68 immunohistochemistry. Brain sections from injected and control uninjected mice were probed with antibodies against the activated microglia marker CD68 and detected using DAB staining. As a positive control, sections of brains from Ppt1/ mice that are known to have an activated microglia response were also included. Representative images were taken of the M1 motor cortex from the positive control Ppt1/ mouse (a), an uninjected negative control mouse (b) and mice injected with Ad5 (c), AAV2/5 (d), AAV2/8 (e) and AAV2/9 (f). High magnification representative images of microglia are shown in the inset boxes. Quantitative measurement of staining intensity of the sections was conducted by threshold image analysis (g). Data are plotted as the mean±s.d., n=3 for Ad5-, AAV2/5- and AAV2/8-injected brains, n=2 for AAV2/9. The Ppt1/ mice exhibited significantly higher microgliosis compared with all injected mice, which were indistinguishable from the uninjected control (*P<0.05, one-way analysis of variance with Bonferroni post hoc correction).

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To further evaluate whether perinatal administration induced immune tolerance, fetal and neonatal mice were intracranially injected with Ad5-GFP and uninjected mice served as naive controls. Three months later all mice were challenged by intraperitoneal injection of Ad5-GFP. Mice that received neither intracranial nor intraperitoneal Ad5-GFP served as negative controls. One month later mice were culled and blood plasma was collected and analysed for antibodies against GFP using SPR-biosensor immunoassay shown in Supplementary Figure S2. There was no evidence of anti-GFP antibodies in serum of mice challenged at 3 months following fetal or neonatal intracranial administrations of Ad5-GFP compared with serum from uninjected unchallenged controls. However, even naive mice challenged by intraperitoneal injection of Ad5-GFP developed no detectable antibody response. As a positive control, commercial anti-GFP antibodies produced a large shift in relative binding and enhancement in the SPR immunoassay.

Sustained long-term expression using non-integrating viral vectors

To assess long-term vector-driven gene expression in the brain, fetal mice were intracranially injected with AAV2/9 and Ad5 vectors expressing GFP and firefly luciferase, respectively (Figures 1a and c). The mice injected with Ad5 were imaged at P21, 2.5 months and 12 months post injection by whole body bioluminescence imaging (in vivo imaging system (IVIS); Caliper Life Sciences, Hopkinton, MA, USA). Although there was a marked drop in the intensity of luciferase expression between P21 and 2.5 months, the expression was sustained and localised within the brain (Figure 6a). The mice administered with AAV2/9 were culled 9 months post injection. The brains were harvested, fixed and sectioned for GFP immunohistology, as described earlier. GFP-stained cells were still present throughout the brain (Figures 6b–f), although a qualitative microscopic survey revealed a marked decrease in expression when compared with that seen after 1 month using this vector (Figures 3p–t). Although this general decrease in expression was present throughout the brain, it was region specific with the piriform cortex retaining relatively high levels of expression compared with the somatosensory barrel field.

Figure 6.
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Long-term gene expression in brains administered with Ad5 and AAV2/9. Fetal mice administered with Ad5 expressing luciferase and AAV2/9 expressing GFP (n=3) via in utero intracranial injection were reared after birth for periods of 1 year and 9 months, respectively. The mice administered with Ad5 were examined using the IVIS to detect luciferase expression at P21, 2.5 months and 12 months post injection and representative images are shown (a). Mice that received AAV2/9 were culled and the brains were harvested and sectioned for detection of GFP expression by immunohistochemistry. Representative images are shown of lower magnification of sections showing the cerebral cortex, hippocampus and the thalamus (b) and higher magnification images of the hippocampus (c), somatosensory barrel field (S1BF) (d), pirifom cortex (e) and cerebellar lobes (f).

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The efficient delivery of genetic material to the fetal brain has many valuable downstream applications. MicroRNAs have a crucial role in CNS development and neurodegenerative diseases.14, 15, 16 Expression of candidate miRNAs, through viral vector delivery to the brain, is a method of elucidating their roles in these events. For example, AAV-mediated overexpression of miR-134 in postnatal brains confirmed its role in dendritic arborisation of pyramidal neurons in cortical layer V.17 Such an approach could be used to elucidate the role of different miRNA sequences in the developing fetal brain. Conversely, vectors expressing ‘microRNA sponges’ could be used to inhibit miRNA sequences.18 In a similar manner, vectors expressing short hairpin RNA could be administered to the fetal brain to knockdown a gene's expression and evaluate its function in development. This would have particular relevance if the subsequent phenotype provided a useful model for neurodegenerative disease, circumventing the need for producing complex transgenic models. A similar strategy could be applied by delivering a dominant-negative form of a gene.

Gene delivery to the fetal brain is also a powerful tool for investigating the feasibility of therapeutic approaches in early neonatal lethal neurodegenerative diseases. Acute neuronopathic type II Gaucher disease is such an example; irreversible disease pathology is present in the patient's brain during gestation2, 19, 20 and death usually occurs before 2 years of age.21 The delivery of therapeutic genes to the brains of mouse models of such diseases during gestation could provide vital information; is in utero intervention required to prevent irreversible brain damage and whether lifespan can be enhanced? The unique advantages that in utero gene delivery offers for the study of neonatal lethal diseases have been discussed in the following reviews.22, 23

For all the above applications to be realised, the correct gene-delivery vector needs to be utilised. This requires a thorough understanding of viral transduction efficiency, spread, tropism (tissue type, regional and cellular) and immune response. On the basis of our previous work using non-integrating lentiviral vectors, the post mitotic status of neurons and reduced risk of insertional mutagenesis, we decided to expand the study to other non-integrating viral vectors, adenovirus and AAV.

Viral transduction is dependent upon the expression of suitable receptors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is the primary site of cell interaction for Ad5. Studies have shown that the levels of CAR expression in the brain decline significantly from the embryonic stage to adulthood in mice.10, 24 Coxsackievirus B5, which also uses CAR as its primary receptor, demonstrated higher levels of transduction in embryonic immature neurons than mature neurons and correlated with the downregulation of CAR with age.25 Examination of CAR expression in human adult brains revealed low levels to no expression with only the choroid plexus and pituitary gland showing higher concentrations.26 On the basis of this evidence, Ad5 could be viewed as a poor choice for gene delivery to the adult brain, but well suited to the fetal brain. We observed efficient gene delivery with long-term expression detectable a year after administration that is consistent with similar duration of expression seen in adult mouse brains.27 However, expression was less widespread in the brain compared with the AAV pseudotypes and more confined to specific areas. Interestingly, gene delivery to the cerebellum and CA1 hippocampus using Ad5 was comparable to that observed with the most efficient AAV2/8 and AAV2/9 vectors. No significant Ad5 microglia-mediated immune response was detected in comparison with the AAV pseudotypes and uninjected control mice. Although transduction was predominantly neuronal, sparse astrocyte transduction could also be seen with this vector.

The receptors for AAV serotypes are comparatively less studied, primarily due to their more recent introduction as gene delivery vectors. All AAV pseudotypes used in this study resulted in widespread gene delivery with AAV2/8 and 2/9 being the most efficient. Gene expression was detected from rostral to caudal regions of the brain and in both the hemispheres. Furthermore, both pseudotypes had similar transduction profiles. This may be attributed to the common putative laminin receptor that they share.28 More recently, N-terminal linked galactose has been characterised as a major receptor for AAV9.29 AAV serotypes 8 and 9 have been shown to efficiently transduce cells in the neonatal mouse7 and adult non-human primate brains.30 These data, taken together with our findings, suggest that the receptor required for AAV2/8 and 2/9 transduction is present from gestation through to adulthood in mice.

Gene delivery was comparatively inefficient using pseudotype AAV2/5. This is consistent with the findings of Lubansu et al. where AAV serotype 5 was shown to be ineffective in transducing rat embryonic (E15) ganglionic eminence cultures. These data are in contrast to studies showing that AAV serotype 5 was efficient at transducing cells in adult mouse31, 32 and non-human primate brain.33, 34 This discrepancy suggests that the availability of AAV5-binding sites, such as 2,3-linked sialic acid,35 and potential receptors may vary from gestation through to adulthood.

The mechanism by which AAV2/8 and AAV2/9 are able to efficiently transduce widespread areas of the brains, when compared with Ad5 or AAV2/5, remain unclear. Two plausible explanations for these observations can be proposed. The first is that the receptors required for transduction by either AAV2/8 or AAV2/9 are more prevalent and expressed at higher concentrations on the neuronal cell membranes, although this needs to be confirmed experimentally in the fetal brain. The second possibility is that these viruses have the ability to transport their genomes along axonal projections to areas distal to the site of injection. Cearley et al.36 showed that stereotaxic injection of AAV9 into the hippocampus of adult mice brains in one hemisphere resulted in detection of marker gene RNA and protein in the contralateral hippocampus. Furthermore, this was also detected in other distal areas of the brain that were connected to the ipsilateral injected hippocampus via axonal projections such as the septal nuclei and entorhinal cortex.

The potential immune response to any viral vector administered to the fetal brain is an important consideration. Whether the vector is being utilised to investigate the brain development or a disease model, microglial activation is not desirable as this could interfere with the readout and limit efficacy. Importantly, we did not see any significant microglia activation in the brains injected with viruses when compared with uninjected control brains.

Our SPR immunoanalysis of GFP antibody production, following fetal and neonatal inoculation by intracranial injection of Ad5-GFP followed by challenging via intraperitoneal injection of the vector 3 months later, was inconclusive. No anti-GFP antibodies were detected in those mice receiving intracranial Ad5-GFP in the fetal or neonatal period and challenged at 3 months. However, even naive mice challenged at 3 months showed no antibody response either. Therefore, this suggests that to interrogate the immune status of mice injected intracranially in the fetal or neonatal period, it would be important to choose a more immunogenic transgene from which an immune response in challenged naive mice is guaranteed, as we have shown for human factor IX.37

IVIS whole body imaging at P21 and 2.5 months, and 12 months post injection revealed sustained Ad5-mediated luciferase gene expression. Interestingly, there was a marked drop in luciferase expression between the P21 and 2.5 months time point. Although the reason for this is unknown apart from the possibility that this represents real loss of expression, progressive thickening of the cranium may be one explanation. An alternative explanation is that even if gene expression was maintained at constant levels, the increasing volume of the brain from 21 days to 12 months would cause an increase in photon absorption, and thus a dampening of signal. Examination of AAV2/9-mediated GFP expression 9 months post injection also revealed GFP-positive cells present in all areas examined at 1 month post injection. However, there was a visible decrease in staining intensity. Given the preferential transduction of neurons by all viruses and the postmitotic status of these cells, we propose that methylation of the cytomegalovirus promoter may be responsible for this reduced GFP staining. Although the cytomegalovirus promoter is known to induce high levels of gene expression, methylation of CpG motifs leading to gene silencing and loss of expression over time is well documented.38, 39 Therefore, if longer periods of sustained expression are required, then alternative, recently described promoters such as the mammalian β-glucuronidase minimal promoter40 or the ubiquitous chromatin-opening element39 may be more suitable. Furthermore, although we have looked at the levels of expression mediated by each virus in discrete areas of the brain, the actual number of viral particles in each of these areas is unknown. This information would be useful in gauging any differential promoter activity in terms of both levels and duration of gene expression.

The issue of what the corresponding developmental stage in humans is in relation to an E15 mouse is a difficult question to answer. There is little doubt that human and mouse CNS development are vastly different and this is further complicated by the choice of marker used as a comparison, for example, anatomical, immunological or physiological. If we were to use an anatomical marker as an example, then diffusion sensor magnetic resonance imaging has shown that the cortical and subplate in humans thickens in the second trimester of human fetal development.41 However, this occurs later at E14-18 in the fetal mouse gestational period.42 Again, this raises the importance of larger animal studies, in particular non-human primates, to provide a more representative model. However, this does not reduce the importance of studies in mice given the wide range of CNS disease models available.

Our data suggest greater viral dissemination through the fetal brain than the neonatal brain. These data show that greater vector distribution and expression can be achieved with the same amount of vector by administration in the fetal versus neonatal period. Although this study suggests that AAV2/8 and 2/9 are the most suitable vectors for widespread delivery of genetic material to mouse fetal brain, they may not be suitable for all applications. The limited packaging capacity of self-complementary adeno-associated viruses means that insertion of large genes over ~1.4kb would not be feasible. In such a case, adenoviral vectors would need to be considered, given their much larger packaging capacity. Therefore, selection of vector will ultimately depend upon the desired application. Over the last few years, AAV9 has received much attention, primarily due to its ability to cross the blood–brain barrier following intravenous administration and mediate efficient gene delivery to the brain.43 Although this is important, intravenous administration also results in gene delivery to the visceral organs and is not suitable for those studies where gene expression is required to be restricted to the brain. Future development of the AAV9 vector may yield a version where intravenous administration produces gene expression only within the CNS. However, until such developments have been achieved, intracranial administration is the most suitable approach for gene expression restricted to the brain. We believe that this study provides essential information on viral vector spread, tropisms, immune tolerance and longevity of gene expression in the fetal brain. This is vital for novel approaches in studying brain development and neonatal lethal neurodegenerative diseases.


Materials and methods

Viral vector production

Ad5-GFP and Ad5-Luc were produced as previously described by Nicklin et al.44 The titre was calculated using a micro-bicinchoninic acid assay and following the established formula; 1μg of protein=4 × 109 viral particles.45 The self-complimentary AAV2/5-GFP and AAV2/8-GFP were produced using the methods previously described by Nathwani et al.46 Vector genomes were calculated using the previously described slot-blot analysis with supercoiled plasmid DNA as standards.47 The University of Pennsylvania Vector Core Facility supplied the self-complimentary AAV2/9-GFP virus and the details of vector production, titration, and quality control can be found on their website (http://www.med.upenn.edu/gtp/vectorcore/). The titres of the viral preparations used in the study were: Ad5=2 × 1011 viral genomes per ml, and AAV2/5, AAV2/8 and AAV2/9=7 × 1011 viral genomes per ml.

Fetal and neonatal intracranial injection of viruses into mice and staining of sections

We have previously described the in utero intracranial injection technique that was used in this study.12 Briefly, pregnant MF1 mice at 15 days gestation were placed under isofluorane anaesthesia, and a midline laparotomy was conducted to expose the uterus. In all, 5μl of vector was administered to three fetuses per dam via transuterine injection directed towards the anterior horn of the lateral ventricle on the left side of the brain using a 33-gauge needle (Hamilton, Reno, NV, USA). Fetuses administered with virus were labelled by subcutaneous injection of 3μl of colloidal carbon ink solution in the flank to identify them from un-administered fetuses. The laparotomy was closed using 6–0 silk sutures and the mouse was permitted to recover in a warm cage. One day post gestation (P1), neonatal mice were also administered with 5μl of the Ad5-GFP vector. The mice were anaesthetised on ice for 3min before receiving the injection and were also directed towards the anterior horn of the lateral ventricle on the left side of the brain using a 33-gauge needle. The injected mice were labelled in the paw pad using colloidal carbon ink. The injected neonates were then returned to their littermates. End-point analysis was conducted 1 month post injection by anaesthetising fetal- and neonatal-injected mice with isoflurane and performing transcardial perfusion fixation with heparinised PBS followed by 4% paraformaldehyde. The brains were removed and cryoprotected in 30% sucrose in 50mM Tris-buffered saline (TBS) before serial cryosectioning at 40μm-thick sections using a Microm HM 430 Freezing Microtome (Thermo Fisher Scientific, Loughborough, UK).

Immunohistochemical staining of brain sections for detection of GFP and CD68

Immunohistochemical staining of brain sections was used to detect GFP expression. Endogenous peroxidase activity was depleted by incubating sections in 1% H202 in TBS solution for 30min and after rinsing three times in TBS, sections were then incubated for 30min in a solution of 15% normal goat serum (NGS; Vector Laboratories Inc., Burlingame, CA, USA) in TBS-T (TBS solution containing 0.3% Triton X-100) for 30min to block non-specific immunoglobulin binding. Subsequently sections were incubated at 4°C overnight in rabbit anti-GFP antibodies (1:10000; Abcam, Cambridge, UK) in TBS-T/10% NGS. After rinsing in TBS and incubation for 2h in goat anti-rabbit IgG (Vector Laboratories Inc.) diluted in TBS-T/10% NGS (1:1000), the sections were rinsed in TBS and incubated for 2h in a 1:1000 solution of Vectastain avidin–biotin solution (ABC; Vector Labs, Peterborough, UK) in TBS and prepared 30min before use. To visualise immunoreactivity sections were rinsed and incubated in 0.05% solution of DAB containing 0.01% H202. The staining reaction was stopped by rinsing sections in ice cold TBS and sections were mounted onto chrome-gelatine-coated Superfrost-plus slides (VWR, Poole, UK) and left to dry overnight before dehydrating in a series of industrial methylated spirits, cleared in xylene for 20min before being coverslipped using DPX mounting medium (VWR).

Immunohistochemical detection of activated microglia was performed via a similar protocol to reveal CD68 immunoreactivity, using normal rabbit serum for blocking and incubation in rat anti-mouse CD68 antibodies (1:100; AbD Serotec, Kidlington, UK) followed by rabbit anti-rat IgG diluted in TBS-T/10% normal rabbit serum (1:1000). These sections were rinsed, incubated in avidin–biotin solution, washed in TBS, visualised with DAB and mounted on slides, as described above.

All DAB-stained sections were viewed under an Axioskop 2 Mot microscope (Carl Zeiss Ltd., Hertfordshire, UK) to examine and compare staining patterns. Representative images were captured using an Axiocam HR camera and Axiovision 4.2 software (Carl Zeiss Ltd).

Quantitative analysis of immunohistochemical staining

The expression of GFP and CD68 was measured by quantitative thresholding image analysis as previously described13, 48 with each antigen analysed blind to the type of vector injected. Briefly, 40 non-overlapping RGB images were captured across four consecutive sections through the CA1 subfield of the hippocampus, caudate putamen, motor and piriform cortex, medial septum, VPM/VPL region of the thalamus and the cerebellar nodule. All the images were captured using a live video camera (JVC, 3CCD, KY-F55B) mounted onto a Zeiss Axioplan microscope using a × 40 objective. During the capture phase, light intensity, microscope calibration and video camera settings were kept constant. Images were analysed for optimal segmentation of immunoreactive profiles, which were determined using Image-Pro Plus (Media Cybernetics, Bathesda, MD, USA). Foreground immunostaining was accurately defined according to averaging of the highest and lowest immunoreactivities within the sample population for a given immunohistochemical marker (per colour/filter channel selected) and measured on a scale from 0 (100% transmitted light) to 255 (0% transmitted light) for each pixel. This threshold setting was then applied as a constant to all subsequent images analysed for the vector injected or the antigen used. Immunoreactive profiles were discriminated in this manner to determine the specific immunoreactive area (the mean grey value obtained by subtracting the total mean grey value from non-immunoreacted value per defined field). Macros were recorded to transfer the data to a spreadsheet for subsequent statistical analysis. Data were separately plotted graphically as the mean percentage area of immunoreactivity per field±s.e.m. for each region.

Phenotypic analysis of transduced cells

Viral vector-transduced cells were assessed for phenotype via double labelling with neuronal and astrocytic markers. Sections were blocked in 15% NGS in TBS-T and incubated overnight in rabbit anti-GFP (1:4000; Abcam) and either mouse anti-NeuN (1:500; Millipore, Watford, UK) or mouse anti-S100β (1:500; Dako, Ely, UK) diluted in 10% NGS in TBS-T. The following day, sections were rinsed in TBS and incubated for 2h with goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 546 (1:1000; Invitrogen, Paisley UK). After rinsing, sections were counterstained with Topro-3 (1:1000; Invitrogen), mounted onto chrome-gelatin-coated slides and coverslipped with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Slides were visualised with a laser scanning confocal microscope (Leica SP5, Leica Microsystems, Milton Keynes, UK). Confocal z-stacks were captured in the cortex to determine the phonotypic identity of transduced cells.

IVIS imaging

Mice were anaesthetised with isofluorane (Abbot Laboratories, Abbot Park, IL, USA) and injected with D-luciferin (15mgml−1; Gold Biotechnology, St Louis, MO, USA). The mice were imaged with a cooled charge-coupled device (CCCD) camera (IVIS; Caliper Life Sciences) 5min later. Following acquisition of a grey-scale image, a bioluminescence image was captured with a 12-cm field of view, a binning resolution factor of 8 and a 1/f stop and open filter. Signal intensities were calculated using Living Image software (Caliper Life Sciences) and expressed as photons per second per cm2.

SPR-biosensor immunoassay

Samples were analysed using a Biacore T-100 instrument (GE Healthcare, Little Chalfont, UK). GFP (10179 RU) was immobilised on flow cell 2 of a CM5 chip by amine coupling according to the manufacturer's instructions. Subtracted sensorgrams were generated by subtracting the signal from flow cell 1 whose surface was subjected to a blank amine immobilisation. Sensorgrams were generated at a flow rate of 10μlmin−1 using 10mM Hepes pH7.4, 150mM NaCl, 3mM EDTA, 0.05% tween 20 as running buffer. All serum samples were diluted 1:10 in sample diluent 25mM TRIS pH9, 250mM NaCl, 3mM EDTA, 0.05% tween 20, 2mgml−1 dextran. Each sample was injected for 2min and sample binding was recorded 60s after the end of the injection. Immediately after completion of the sample injection, the goat anti-mouse (IgA+IgG+IgM; Thermo Scientific, Loughborough, UK) was injected at a concentration of 50μgml−1 for 2min and the binding response was recorded 30s after the injection. Finally, the sensor chip surface was regenerated between injections with a 30s injection of glycine pH1.5. This was followed by a 2min stabilisation period before another sample was injected. A mouse monoclonal anti-GFP antibody (632375; Clontech, Mountain View, CA, USA) was used as a positive control at a dilution of 1:100 in sample diluent.


Conflict of interest

The authors declare no conflict of interest.



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We thank Gennadij Raivich, University College London (UCL) for useful discussion. The UCL-based laboratory and AHB were supported by the Biotechnology and Biological Sciences Research Council. AAR and SNW had also received support from the UK Gauchers Association. The King's College London-based laboratory was supported by the Wellcome Trust (GR079491MA), Batten Disease Family Association and the Batten Disease Support and Research Association.

Supplementary Information accompanies the paper on Gene Therapy website