Subretinal Delivery of Recombinant AAV Serotype 8 Vector in Dogs Results in Gene Transfer to Neurons in the Brain

original article

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Subretinal Delivery of Recombinant AAV Serotype 8 Vector in Dogs Results in Gene Transfer to Neurons in the Brain Knut Stieger1, Marie-Anne Colle2, Laurence Dubreil2, Alexandra Mendes-Madeira1, Michel Weber3, Guylène Le Meur3, Jack Yves Deschamps4, Nathalie Provost1, Delphine Nivard1, Yan Cherel2, Philippe Moullier1 and Fabienne Rolling1 INSERM U649, CHU Hotel-Dieu, Nantes, France; 2INRA UMR 703, Ecole Nationale Vétérinaire de Nantes, Nantes, France; 3CHU Hotel-Dieu, Service d’Ophtalmologie, Nantes, France; 4Ecole Nationale Vétérinaire de Nantes, Service d’Urgences, Nantes, France 1

Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere. Received 18 October 2007; accepted 14 February 2008; published online 11 March 2008. doi:10.1038/mt.2008.41

Introduction Recombinant adeno-associated virus 2 (rAAV2) vectors are among the most efficient gene delivery vehicles for the treatment of retinal diseases. Since the discovery of AAV2, over 100 AAV isolates have been described and >10 of them have been cloned for development as gene therapy vectors.1,2 Because different rAAV vectors exhibit distinct tissue tropism and interact with different cellular receptors,3–6 we investigated the behavior of different rAAV vectors in ophthalmological application. We as well as others have studied rAAV chimeric serotypes in which the vector is flanked by AAV2 inverted terminal repeats but encapsidated in an AAV1, -2, -3, -4, or -5 shell, generating rAAV2/1, -2/2, -2/3, -2/4, and -2/5, respectively. Subretinal injection of rAAV2/1, -2/2, -2/3, -2/4, and -2/5 in rats results in a hierarchy in the levels of transgene expression, with rAAV2/4 and -2/5 capsids being the most efficient.7 Interestingly, subretinal injection of rAAV2/2 and rAAV2/5 results in the transduction of retinal pigment epithelium

(RPE) and photoreceptor cells, whereas subretinal injection of rAAV2/4 and rAAV2/1 results in transgene expression in the RPE only.8–13 Intravitreal injection of rAAV2 leads to efficient ganglion cell transduction,14–17 whereas intravitreal injections of rAAV2/1, rAAV2/4, and rAAV2/5 result in no detectable transduction. Recently, a number of new rAAV serotype vectors, including rAAV8, have become available. Vectors derived from AAV8 are of particular interest because of their exceptionally high gene transfer efficiency in vivo. When this vector is delivered by intravascular infusion into rodents, nearly complete transduction of multiple organs is seen.18 Moreover, after administration of vector pseudotyped with the AAV8 capsid, gene expression in the liver is higher (1–2 logs) than when the more established rAAV2 serotype vector is used.19,20 The serotype 8 vectors achieve a much more efficient gene transfer to the brain than the rAAV2 vector does.21,22 Very recently, it was reported that rAAV8 efficiently transduces murine photoreceptors.23,24 In this study, we investigated the behavior of vectors pseudotyped with the AAV8 serotype capsid with regard to intraocular delivery in rats and dogs. Our results indicate that, after subretinal delivery, the serotype 8 capsid allows transgene expression in all layers of the retina and, most surprisingly, in distal neuronal cell bodies in the brain. Moreover, the recombinant genome was found to be widely distributed in the brain and in peripheral organs.

Results Subretinal delivery of AAV2/8.gfp in rats and dogs results in efficient transduction of the neuroretina including the ganglion cells We administered AAV2/8.gfp through subretinal injections into 12 Wistar rats and 2 Beagle dogs (D1 and D2). As described earlier, diluted fluorescein was added to the vector suspension in order to monitor, by fluorescence fundus photography, the accuracy and the precise localization of the injection site (bleb) in the rats (Figure 1a and e). In dogs, standard fundus photography is sufficient to localize the targeted area of the retina (Figure 2a). Fluorescent imaging in live animals displayed, as early as 4 days

The first two authors contributed equally to this work. Correspondence: Fabienne Rolling, Laboratoire de Thérapie Génique, INSERM U649, CHU-Hotel Dieu, Bât. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, Nantes, France. E-mail: [email protected]

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Figure 1 Subretinal injection of AAV2/8.gfp vector into rats results in green fluorescent protein (GFP) expression outside of the injected area, in contrast to the results when AAV2/5.gfp is used. Intravitreal injection of AAV2/8.gfp does not result in any transduction of retinal cells, in contrast to the results when AAV2/2.gfp is used. Live fluorescent retinal images. (a,b) Subretinal injection of AAV2/8.gfp. (c,d) Subretinal injection of AAV2/5.gfp. (e,f) intravitreal injection of AAV2/8.gfp. (g,h) Intravitreal injection of AAV2/2.gfp. Animals were injected with the vector solution containing 0.1% fluorescein in order to visualize the injected area (a,c,e,g). This fluorescent signal disappeared within 24 hours after injection. The GFP expression in the injected animals was recorded at 2 months after the injection (b,d,f,h). AAV, adeno-associated virus.

after the injection, a strong green fluorescent protein (GFP) signal in rats (Figure 1b) as well as in dogs (Figure 2b and c). Surprisingly, in both species, the GFP signal was detected not only within the targeted part of the retina but in a much larger area. In rats, this characteristic of rAAV2/8-mediated gene transfer is immediately apparent when compared with an rAAV2/5-transduced retina, in which the GFP-expressing area of the retina perfectly matches the targeted area (Figure 1c and d). In dogs, it was striking to observe the increase in the GFP-expressing area of the retina between days 4 and 13 after the injection (Figure 2b and c). In the rats as well as in the dogs, GFP expression in neurons can be seen outside the targeted area. In rat and dog retina flatmounts, with the use of a fluorescence inverted microscope, rAAV2/8-mediated gene expression Molecular Therapy vol. 16 no. 5 may 2008

Figure 2 Subretinal injection of AAV2/8.gfp vector into dogs results in green fluorescent protein (GFP) expression in an area exceeding the injected area in all cell layers of the retina. (a–c) Live fluorescent retinal images, (d–f) retinal flatmounts, and (g,h) flatmount sections of dog D1 injected subretinally with AAV2/8.CMV.gfp. (a) Retinal detachment created by the subretinal injection 30 minutes after ­ injection. (b) GFP signal at 4 days after injection. (c) GFP signal at 13 days after injection. (d) Choroid/RPE flatmount displayed fluorescent retinal pigment epithelium (RPE) cells. (e,f) Neuroretina flatmount displayed fluorescent cells and axons of ganglion cells forming the optic nerve. (g) Choroid/RPE section. (h) Neuroretina section. Arrows in h indicate ganglion cells and cells in the inner nuclear layer. Bar = 100 μm. AAV, adeno-associated virus; CMV, cytomegalovirus.

was detected within the choroid/RPE layer (Figures 2d and 3a–c) and within the neuroretina (Figures 2e and f and 3e, g and i). Flatmount sections of rat and dog retinas clearly showed transgene expression in the RPE within the choroid/RPE layer (Figures 2g and 3d), and in the photoreceptors, the cells from the inner nuclear layer, and the ganglion cells within the neuroretina (Figures 2h and 3f and h). Interestingly, it was observed in the rat retina flatmount, that RPE cells expressing GFP were present not only in the bleb area but also over the entire retina (Figure 3a and b). In contrast to the results obtained using the AAV2/2.gfp vector (Figure 1g and h), intravitreal injections of AAV2/8.gfp resulted in no detectable transduction (Figure 1e and f). Only a few cells displayed GFP expression at the needle insertion site.

Subretinal delivery of AAV2/8.gfp in rats results in a high level of GFP signal along the visual pathway before the first synapse In order to evaluate the pattern of GFP expression in the brains of rats after subretinal administration of AAV2/8.gfp into both eyes, six rats were killed 2 months after injection. The brains were cut into coronal slices and examined for GFP expression using laser scanning confocal microscopy. A strong GFP signal was observed along the visual pathway in the optic nerve, the optic chiasm, the suprachiasmatic nucleus, the optic tract, the ventral and dorsal lateral geniculate nucleus, and the superior colliculus (Figures 3j and 4). No GFP signal was detected outside the visual pathway. In the lateral geniculate nucleus, the GFP signal did not colocalize with cellular bodies of neurons, astrocytes, or oligodendrocytes, 917

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AAV8-mediated Gene Transfer in the Retina of Dogs

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Figure 3 Subretinal injection of AAV2/8.gfp vector into rats results in green fluorescent protein (GFP) expression in all cell layers of the retina. (a–c,e,g,i) Retinal flatmounts and (d,f,h,j) corresponding sections of rats subretinally injected with AAV2/8.gfp. (a–c) Sclera/choroid/RPE flatmount and (d) section of the sclera/choroid/RPE flatmount showing fluorescent retinal pigment epithelium (RPE) cells. The spotted line in a outlines the injected area, and the asterisk (∗) marks the injection point. Arrows in a highlight GFP-expressing RPE cells outside the injected area. The square in a defines the area displayed with higher magnification in b. GFP-expressing RPE cells in c and d are located within the injected area. (e,f) Neuroretina flatmount and corresponding section displaying fluorescent photoreceptor cells. Arrows in f indicate photoreceptor nuclei. (g,h) Neuroretina flatmount and corresponding section showing fluorescent ganglion cells and cells of the inner nuclear layer. Arrows in h indicate ganglion cells and cells of the inner nuclear layer. (i) Neuroretina flatmount displaying fluorescent axons of the ganglion cells forming the optic nerve. (j) Transverse section through the optic nerve showing GFP signal in axons. Bar = 100 μm. AAV, adenoassociated virus.

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Figure 4  In rats, the green fluorescent protein (GFP) signal can be detected along the visual pathway, reaching the first synapse within the lateral geniculate nucleus. The GFP signal in the brains of AAV2/ 8.gfp–injected rats. The animals were killed at 2 months after the injection, and the brains were removed and cut into 100 μm coronal slices and examined for GFP expression using laser scanning confocal microscopy. Images shown in a–d correspond to coronal slices at the indicated location on the brain image. c-1 and c-2 correspond to two locations on the same slice c. Bar = 200 μm. dLGN, dorsal lateral geniculate nucleus; OC, optic chiasm; OT, optic tract; SC, superior colliculus; SCN, suprachiasmatic nucleus; vLGN, ventral lateral geniculate nucleus. AAV, adenoassociated virus.

as confirmed by immunofluorescent staining using the specific markers, NeuN, GFAP, and Olig2, respectively (Supplementary Figure S1). This result suggests that the GFP signal was present only in nerve fibers that are the axonal projections of ganglion cells in the retina.

Subretinal delivery of AAV2/8.gfp in dogs results in a high level of GFP signal along the visual pathway before and after the first synapse, and in transfer of vector DNA and GFP mRNA to the contralateral lateral geniculate nucleus In order to evaluate the pattern of GFP expression in the brains of dogs after subretinal injection of AAV2/8.gfp, dogs D1 and D2 were killed at 4 weeks after injection of the vector into the right eye. The brains of the dogs were cut into coronal slices (Supplementary Figure S2) and analyzed for GFP expression using laser scanning confocal microscopy. The GFP signal was detected in nerve fibers in the optic nerve, in the optic chiasm, within the contralateral hemisphere in the optic tract, in the nerve fibers and cellular ­bodies www.moleculartherapy.org vol. 16 no. 5 may 2008

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Figure 5  In dogs, the green fluorescent protein (GFP) signal can be detected along the visual pathway reaching the optic radiation, passing the first synapse in the lateral geniculate nucleus. The GFP signal in the brain of AAV2/8.gfp vector–injected dog D1. Inserts show the slab in which the GFP signal was detected. Circles within the inserts indicate the location of the GFP signal. LGN, lateral geniculate nucleus; OC, optic chiasm; ON, optic nerve; OR, optic radiation; OT, optic tract; SC, superior colliculus. Bar = 50 μm. AAV, adeno-associated virus.

in the lateral geniculate nucleus, and in the superior colliculus (Figure 5, Table 1, and Supplementary Table S1). No GFP signal was observed in the contralateral visual cortex. Interestingly, the GFP signal in cellular bodies within the lateral geniculate nucleus colocalized with the NeuN marker, a specific marker for neurons (Figure 6a–c), thereby demonstrating that the GFP signal is present beyond the first synapse. This conclusion is supported and confirmed by the detection of a GFP signal in nerve fibers of the optic radiation (Figure 5e). The GFP signal did not colocalize with cellular bodies from astrocytes or oligodendrocytes, as confirmed by immunofluorescent staining with the markers GFAP and Olig2, respectively (Figure 6d–i). The location of GFP-positive neurons was restricted to the A layer of the left lateral geniculate nucleus, which receives its projections from ganglion cells of the contralateral right retina (data not shown). Vector DNA was detected in the left lateral geniculate nucleus by PCR analysis of frozen tissue from dog D2, thereby indicating that the vector traveled from the right retina along the visual pathway into the contralateral hemisphere of the brain (Figure 7a). Furthermore, reverse transcription-PCR analysis of RNA extracted from both the lateral geniculate nuclei of D2 clearly demonstrated Molecular Therapy vol. 16 no. 5 may 2008

Figure 6   In dogs, green fluorescent protein (GFP) is expressed in neurons within the lateral geniculate nucleus, but not in astrocytes and oligodendrocytes. Immunofluorescent staining of coronal slices containing the lateral geniculate nucleus (slices of slab LH 9 in Supplementary Figure S2). NeuN, specific marker for neurons; GFAP, specific marker for astrocytes; Olig2, specific marker for oligodendrocytes. Arrows indicate colocalization of GFP and NeuN-positive cells. Bar = 50 μm.

the presence of GFP transcripts in the lateral geniculate nucleus contralateral to the injected eye (Figure 7b). These results confirm that gene transfer occurred in the neurons of the contralateral lateral geniculate nucleus.

Subretinal delivery of AAV2/8.gfp in rats and dogs results in a wide biodistribution of the recombinant genome in the brain and in peripheral organs Vector distribution was studied in brain tissue of unilaterally injected rats and dogs, using total DNA extracted from the 100-μm slices that had been earlier examined for GFP expression. The peripheral organs were also assayed. Analysis by PCR was performed on total DNA extracts, using primers that are localized within the gfp gene. In the rats, the vector genome was detected in DNA extracts from slices comprising the frontal lobe, the optic nerve, the dorsal and ventral lateral geniculate nucleus, the superior colliculus, the optic radiation, and some (but not all) slices that contained the visual cortex, the cerebellum, and the brain stem (Supplementary Figure S3). In contrast, the vector genome was not found in DNA extractions from slices that contained the optic chiasm, suprachiasmatic nuclei, or optic tract. The vector genome was found in the injected right retina but not in the contralateral noninjected left retina. The vector genome was also found in the spinal cord, liver, and heart of two of the four animals tested (data not shown). In dogs, the vector genome was detected using PCR in DNA extracts almost exclusively from the left hemisphere (Table 1 and Figure 7a). Positive results were obtained from slices located within the frontal lobe (LH 4A and 5A), slices containing the optic chiasm and the optic tract (LH 5B and 6B), and slices containing the lateral geniculate nucleus, the optic radiation, and the visual cortex (LH 8AB continuously to LH 15, with the exception of LH 919

RH 1

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LH 4 B

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RH 4 B

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RH 5 A

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LH 5 B

++ f

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RH 5 B

+++ f

LH 6 A

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+f

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RH 7 B

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LH 8 A

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LGN

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−

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+f

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LH 11 B

++ f

OR

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RH 11 B

+f

LH 12 A

++ f

OR

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RH 12 B

+f

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Abbreviations: AAV, adeno-associated virus; c, cellular bodies; f, nerve fibers; GFP, green fluorescent protein; LGN, lateral geniculate nucleus; LH, left hemisphere; OC, optic chiasm; OR, optic radiation; OT, optic tract; RH, right hemisphere.

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Retina L

424 bp –

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GFP GFP positive signal structures

350 bp –

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GFP GFP positive signal structures

Vector genome

Transgene

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Vector genome

Transgene

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Table 1  Detection of gfp signal and vector DNA in both hemispheres of AAV2/8.gfp–injected dogs

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AAV8-mediated Gene Transfer in the Retina of Dogs

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Figure 7  In dogs, vector DNA and green fluorescent protein (GFP) messenger RNA are detected within the contralateral lateral geniculate nucleus. (a) PCR analysis of frozen tissue from dog D2. PCR was carried out using primers that amplified a 424-bp region within the GFP sequence. As control, for an equal amount of DNA as that used in the PCR, each sample was also used for amplifying a 350-bp fragment of the cytochrome β gene. Reaction products were separated on an agarose gel (top) and then transferred to a nylon membrane and hybridized to a gfp probe (bottom). A PCR sensitivity assay for the detection of gfp sequences is shown at the bottom of a. (b) Reverse transcriptionPCR (RT-PCR) of RNA extracted from the left and right retina and lateral geniculate nucleus (LGN), respectively, of dog D2. PCR was performed in an identical manner as in a. As control, for an amount equal to the RT mix used in the PCR, each sample was used for the amplification of a 650-bp fragment of the β-actin gene. As a control for DNA contamination, each sample was processed identically without adding the reverse transcriptase to the RT mix (RT−). A PCR sensitivity assay for the ­detection of gfp sequences is shown at the bottom.

Discussion 11A and 12A). In the right hemisphere, the vector genome was found only in DNA extracts from slices containing the optic tract and visual cortex (RH 6A, 13A, and 15). In samples of the brain stem, DNA extracts from slices containing the superior colliculus (B 1), and caudal parts of the brain stem (B 4A and 6B) tested positive for the presence of the vector genome (Supplementary Table S1). In both the dogs, the vector genome was detected in the injected right retina and in the right optic nerve, but not in the contralateral, noninjected control retina or in the left optic nerve (Supplementary Table S2 and Figure 7a). The medulla oblongata, heart, liver, and gonads tested positive for the presence of vector genome in both the dogs (Supplementary Table S2). The spinal cord and cerebrospinal fluid were negative for the vector genome (Supplementary Table S2). 920

This study shows that, after subretinal delivery of AAV2/8.gfp, the type 8 AAV capsid results in efficient transgene expression in the RPE, the photoreceptors and, more surprisingly, in the cells from the inner nuclear layer and in the ganglion cells. Most important is the finding of GFP in the brain along the visual pathway, with transgene expression occurring in the lateral geniculate nucleus. As regards the distribution of vector expression, vector sequences were found in many parts of the brain including some areas of the brain stem, in the medulla oblongata, and in peripheral organs such as the heart, liver, and gonads. The detection of a GFP signal in all cell types within the inner nuclear layer, as well as in ganglion cells, was unexpected. It is known that, after subretinal administration in mice, rats, and dogs, rAAV2 and rAAV5 vectors transduce both RPE and www.moleculartherapy.org vol. 16 no. 5 may 2008

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­ hotoreceptors cells, while rAAV4 transduces only RPE cells.25 p None of these vectors demonstrated transduction of ganglion cells when administered through this route. However, in contrast to the results with rAAV2, intravitreal administration of rAAV8 does not allow transduction of ganglion cells, thereby suggesting that transduction of ganglion cells after subretinal delivery is caused by the transit of the rAAV vector genomes from the photoreceptors to the ganglion cells through the inner nuclear layer. This hypothesis of the passage of the recombinant vector from one cell to another is confirmed by the fact that, in contrast to results from the use of other rAAV serotypes or lentiviral vectors,13,26 the GFP signal was detected not only within the targeted area but also in a much larger area of the retina. This spreading of the GFP signal outside the original bleb is observed for the cells within the neuroretina and most surprisingly for the RPE cells. None of the other rAAV serotypes subretinally delivered in the eye has provided such high levels of transgene expression in the brain nor shown such spread of vector sequences in the brain and in peripheral organs. After subretinal delivery of rAAV type 8 in rats and dogs, the GFP signal was detected in the brain tissue along the visual pathway but not in other parts of the brain. This has never been observed after subretinal injection of rAAV types 2, 4, and 5.27 Although intravitreal injection of rAAV type 2 vector leads to GFP expression in ganglion cells, no GFP signal could be detected in the brain, and only vector sequences were detected by PCR.27 Overall, these results suggest the capacity of the rAAV8 pseudotyped vector to transit along the different neurons of the visual pathway leading to transgene expression at different locations. Interestingly, we observed a difference in the patterns of GFP expression in the brains of the rats when compared to those of the dogs. In the rats, GFP was detected in the optic chiasm, the suprachiasmatic nucleus, the optic tract, the superior colliculus, and the lateral geniculate nucleus. Within the lateral geniculate nucleus, axonal projections from the ganglion cells were GFP positive but not the neuronal cell bodies, thereby suggesting that GFP expression does not extend beyond the first synapse. In contrast, in the dogs, the GFP signal was detected both before and beyond the first synapse, as confirmed by the GFP signal being detected in the lateral geniculate nucleus and also in the optic radiation. Moreover, in the dogs, GFP transcripts were detected in the lateral geniculate nucleus, thereby confirming that gene transfer occurred in the neuronal cells from this part of the brain. The transgene expression observed was not caused by undesirable AAV replication in distal tissues, because AAV vectors cannot replicate in dogs in the absence of helper virus and virus rep/cap proteins that can propagate AAV2 inverted terminal repeats. Using a replication center assay, we confirmed that the batch of AAV2/8.gfp used in this study was not contaminated with adenovirus nor rep-positive AAV.28 In view of the fact that the same vector titer was used for the rats as well as for the dogs, and that the GFP label is stronger in the rat optic tract and in the rat lateral geniculate nucleus fibers than in the corresponding structures in the dog, there is a strong possibility that the absence of GFP label in the cellular bodies in the rat lateral geniculate nucleus is not because of a lack of sensitivity, but is truly linked to the species difference. This last result shows that, in contrast to the retina where the same tropism for rAAV type 8 was found between the two species Molecular Therapy vol. 16 no. 5 may 2008

AAV8-mediated Gene Transfer in the Retina of Dogs

tested, the pattern of GFP expression in the brain was different between rats and dogs, after the subretinal injection. It was previously reported that, in the retina, gene transfer using rAAV2, 4, and 5 always resulted in the same pattern of transduction in species such as rats, dogs, and primates.13 In contrast, earlier studies have described discrepancies between species with respect to transduction in the brain. For example, injection of rAAV2/5 did not result in detectable transduction in the brains of cats, whereas the same batch of rAAV2/5 resulted in transduction of the choroid plexus and ependymal cells in the brains of mice.29 In this study, we have demonstrated that subretinal delivery of rAAV8 vector in dogs results in gene transfer to the lateral geniculate nucleus. It has been shown earlier that intracranial injections of rAAV8 vectors in rodents results in efficient neuronal gene transfer.21,22,30,31 Moreover, the rAAV type 8 vector, when injected into the lateral ventricles, has been shown to allow widespread gene delivery to the brain in neonatal mice, and proved to be more efficient than either rAAV1 or rAAV2 (ref. 32). PCR analysis of DNA extracts from multiple brain samples of subretinally injected rats and dogs showed broad dispersion of rAAV8 vector genomes within the brain. In contrast to rAAV types 2, 4, and 5, for which vector sequences were found only in the optic nerve after subretinal injection in rats and dogs,27 subretinal injection of rAAV type 8 leads to the presence of vector sequences in many parts of the brain. Interestingly, in the brain of the dog, where it was possible to separate the two hemispheres, vector genome was detected almost exclusively in the left hemisphere (65% of vectorpositive sections in the left hemisphere versus 11% in the right hemisphere). Given that only the right eye was injected, this last observation suggests the transport of the vector genomes through the partial decussation of the optic nerve and their trans-synaptic passage from neuron to neuron along the visual pathway. The fact that no GFP signal was detected in the frontal lobe and in the brain stem (where vector genomes were detected), suggests that the vector sequences were present only at a low level and/or were not transcriptionally active in these two parts of the brain. In summary, this study showed that subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer to the RPE, photoreceptors, cells from the inner nuclear layer, and ganglion cells in the retina, and to distal neurons of the contralateral lateral geniculate nucleus. From the standpoint of therapeutic application, this characteristic of rAAV2/8 may be of distinct benefit in conditions where widespread or global expression is an objective. However, from the standpoint of safety, widespread dissemination of the vector genome in the brain and transgene expression in the lateral geniculate nucleus following subretinal injection of rAAV8 serotype is a serious concern when discrete retinal gene therapy is the objective.

Materials and methods Production of AAV2/8.gfp vector. Plasmid SSV9.CMV.gfp, used for the

production of AAV2/8.gfp, consists of the AAV2 inverted terminal repeats, between which is the coding sequence of the GFP complementary DNA flanked by the cytomegalovirus promoter and the simian virus 40 polyadenylation signal. The AAV2/8.gfp vector was produced as described earlier7 in the Vector Core at the University Hospital of Nantes (http://www.vectors. nantes.inserm.fr). The titer was determined using dot blot and is expressed as vector genomes/ml (vg/ml).28 The vector titer was 1 × 1012 vg/ml. The

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vector batch was characterized using a modified replication center assay.28 No infectious adenovirus or rep-positive AAV was detected. Subretinal delivery. The two Beagle dogs (D1 and D2) used in this study were

purchased from the Centre d’Elevage du Domaine des Souches (Mezilles, France). Twelve Wistar rats were acquired from Charles River laboratory (Domaine des oncins, France). The animals were cared for in accordance with the Association for Research in Vision and Ophthalmology guidelines for the use of animals in ophthalmic and vision research. Injection protocols were approved by the Institutional Animal Care and Use Committee of the University of Nantes. In the rats, subretinal injection was performed either in both eyes (n = 6) or unilaterally in the right eye (n = 6). Anesthesia, surgical procedures and post surgical care were performed as described earlier.26 Briefly, a transscleral/transchoroidal approach was used by first puncturing the sclera and the choroid. A 33-gauge needle was then inserted into the globe through the sclerotomy in a tangential direction under an operating microscope. The vector concentration was 1 × 1012 vg/ml and the total vector dose was 2.5 × 109 vg/injection. Fluorescein was added to the viral solution at a 1/1,000 final dilution, and the injection mixture was delivered into the subretinal space. The accuracy of the injections was monitored by fluorescence fundus photography immediately after the injection procedure. In dogs, the surgical procedure was conducted by a transvitreal approach in the right eye as described earlier by Weber et al.13 Briefly, a 41-gauge cannula connected to a viscous fluid injection system was used for delivering a controlled, automated injection. The cannula was inserted into the eye through a sclerotomy, advanced through the vitreous, and viral solution was injected into the subretinal space underlying the tapetal central retina with the guidance of a microscope. The vector concentration was 1 × 1012 vg/ml and the total vector dose was 5 × 1010 vg/injection. Postsurgical care was provided as described earlier.13 In vivo GFP fluorescence imaging, retina flatmounting, and tissue sections of the retina. GFP expression in rats and dogs was monitored at

weekly intervals by fluorescence retinal imaging using a Canon UVI retinal camera connected to a digital imaging system (Lhedioph Win Software, Lheritier SA, Saint-Ouen-l’Aumône, France). The preparation of retinal flatmounts was performed identically in rats and dogs on 4% paraformaldehyde (PFA)-fixed enucleated eyes as described earlier.13,26 Fixed flatmounts were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA) and snap frozen in liquid nitrogen. Cryosections were cut (7 μm) and observed using epifluorescence microscopy (Nikon, Champigny-sur-Marne, France).

Brain tissue processing For confocal microscopy and immunocytochemistry: Two months after the

subretinal injection, six of the rats were killed by overdose of anesthesia, and the brains were immediately removed and fixed in 4% PFA for 24 hours at 4 °C. Each of the brains was cut into four blocks and again fixed for 1 hour in 4% PFA at 4 °C followed by a 48-hour cryopreservation step using a 30% sucrose solution at 4 °C. The blocks were embedded and frozen as described for the flatmounts. Coronal slices were cut in intervals alternating between 1 × 100 and 3 × 10 μm. The 100-μm sections were used in examining for GFP signals using confocal microscopy, and the 10-μm sections were used for immunocytochemistry. Four weeks after the injection, the two dogs (D1 and D2) were anesthetized by induction with ketamin (Imalgène; Mérial, Lyon, France)/xylazin (Rompun; Bayer, Puteaux, France), and maintained under isofluoran gas anesthesia. Perfusion was accomplished through the transcarotid route using 150 ml phosphate-buffered saline (PBS) followed by 600 ml of 4% PFA diluted in PBS. The brains were removed and fixed in 4% PFA for 24 hours at 4 °C. The hemispheres were separated from

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the brain stem and the two parts of the brain were cut into 4-mm slabs followed and fixed again in 4% PFA for 24 hours at 4 °C. These slabs were then cryopreserved in a 30% sucrose solution for 48 hours at 4 °C, and the hemispheres were cut into four pieces each, while the brain stem was cut into two pieces (Supplementary Figure S2). These tissue blocks were then embedded as described earlier and frozen in isopentan at −45 to −60 °C. The canine coronal slices were sectioned for analysis in a manner identical to those of the rats. For PCR analysis: The 100-μm coronal sections used for the detection of GFP signals were removed from the slide and total DNA was purified using a phenol/chloroform extraction protocol. Peripheral tissue samples were removed from the animals and immediately frozen in liquid nitrogen, and total DNA was purified, again using a phenol/chloroform extraction. In order to avoid cross-contamination between tissues, each sample or slice was collected using separate disposable material and supplies. We also processed the samples from each individual animal at different time intervals in a BL3 environment. GFP expression and immunocytochemistry, and analysis using laser scanning confocal microscopy. The 100-μm sections were mounted on

slides with a mounting medium (Mowiol medium; Calbiochem, San Diego, CA) and examined for GFP expression using a laser scanning confocal microscope (Nikon C1, Champigny-sur-Marne, France), equipped with a blue argon ion laser emitting at 488 nm. The 10-μm sections for immunocytochemical analysis were thawed and rehydrated with PBS (pH 7.4). Nonspecific antigen binding was blocked by incubating sections for 20 minutes at room temperature in blocking buffer (0.1% PBS/Tween, 5% goat serum) followed by over night incubation at 4 °C in the blocking buffer with the following primary antibodies at the indicated dilution mouse monoclonal anti-NeuN anti­ body at 1:800 (MAB377; Chemicon, Euromedex, Mundolsheim, France); rabbit polyclonal anti-GFAP antibody at 1:4,000 (Z-0334; DakoCyto­ mation, France SAS, Trappes, France); and mouse monoclonal antiOlig2 anti­body at 1:100 (AB9610; Chemicon, Euromedex, Mundolsheim, France). After being washed with PBS, the sections were incubated for 2 hours at room temperature with Alexa fluor 546 conjugated goat antirabbit immunoglobulin G at 1:300 (A11010; Invitrogen Molecular Probes, Eugene, OR) or goat anti-mouse immunoglobulin G at 1:100 (A11030; Invitrogen Molecular Probes, Eugene, OR) secondary antibodies diluted in blocking solution for 2 hours at room temperature. After being washed with PBS, the sections were mounted with an antifade medium (Mowiol; Calbiochem, San Diego, CA). The immunolabeled sections were scanned serially using the argon ion laser (488 nm) to observe GFP signals, and with a helium neon laser (543 nm) to observe Alexa fluor 546 signals. Each image was recorded in a separated channel (channel green for GFP and channel red for Alexa fluor 546) and overlayed to allow detection of colocalized fluorescent signals. PCR analysis. For PCR analysis, DNA was extracted either from 100-μm

sections or from stored, unfixed tissue. The amount of DNA used in the PCR was 750 ng. The 5′ primer (5′-AAGTTCATCTGCACCACCG-3′) and the 3′ primer (5′-TGTTCTGCTGGTAGTGGTCG-3′) are both located within the gfp DNA sequence. The PCR-amplified vector sequence yielded a 424-base pair (bp) fragment. The PCR employed Taq DNA polymerase (Taq Gold; Roche, Neuilly-sur-Seine, France) and a Perkin-Elmer thermocycler (Groton, CT) and the reaction profile was as follows; an initial denaturation step at 95 °C for 5 minutes, followed by 40 cycles at 94 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, and a final incubation step at 72 °C for 10 minutes. The amplified products were analyzed by agarose gel electrophoresis followed by transfer under alkaline conditions to a positive TM membrane (MP Biomedicals Europe, Illkirch, France). The membrane was hybridized to a 608-bp PvuII–AvaI fluorescein-labeled gfp www.moleculartherapy.org vol. 16 no. 5 may 2008

© The American Society of Gene Therapy

probe (Amersham, Otelfingen, Switzerland; Gene Images random prime labeling module). The sensitivity of the gfp PCR assay was evaluated as described earlier33 and showed that a threshold of 10−1 vg can be detected. Reverse transcription-PCR analysis. RNA was extracted from frozen tissue

as described earlier.34 Contaminating DNA was removed by DNAse digestion for 1 hour at room temperature (DNase I; Roche, Neuilly-sur-Seine, France). Reverse transcription was performed on 1 μg of RNA using a random oligo dT primer in 25 μl (M-MLV; Invitrogen, Cergy-Pontoise, France), followed by an inactivation step at 65 °C for 30 minutes. The volume was brought up to 50 μl with water, and 2.5 μl was used as a template for the PCR which was conducted in accordance with the protocol described earlier.

Acknowledgments This work was supported by INSERM, the Association Française contre les Myopathies, the Fondation pour la Thérapie Génique en Pays de la Loire, and the Lions Clubs International foundation. We thank the Vector Core (http://www.vectors.nantes.inserm.fr) at the University Hospital of Nantes. We also thank Matthew Ellinwood (Department of Animal Science, Iowa State University, Ames, IA) for his critical reading and editing of the article.

Supplementary Material Figure S1. GFP is only expressed in axonal projections but not in cellular bodies within the lateral geniculate nucleus of rats. Figure S2. Procedure to obtain coronal serial slabs of dog brain. Table S1. Detection of gfp signal and vector DNA in the brain stem of AAV2/8.gfp–injected dogs. Figure S3. Vector genome can be found in the optic nerve and in the brain of rats, but does not necessarily match the presence of GFP signal. Table S2. Detection of gfp signal and vector DNA in the retina, optic nerve and peripheral organs of AAV2/8.gfp–injected dogs.

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