Therapy (2009) 16, 521–532 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt ORIGINAL ARTICLE Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

M Hellstro¨m1, MJ Ruitenberg1, MA Pollett1, EME Ehlert2, J Twisk3, J Verhaagen2 and AR Harvey1 1School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia; 2Netherlands Institute for Neuroscience, Amsterdam, The Netherlands and 3Amsterdam Molecular Therapeutics, Amsterdam, The Netherlands

Recombinant adeno-associated virus (rAAV) vectors are cell phenotype was based on cell morphology and immuno- increasingly being used as tools for gene therapy, and histochemistry. rAAV2/2 and rAAV2/6 transduced the great- clinical trials have begun in patients with genetically linked est number of cells, whereas rAAV2/5 and rAAV2/8 were retinal disorders. Intravitreal injection is optimal for the least efficient. Most vectors primarily transduced RGCs; transduction of retinal cells (RGCs), although however, rAAV2/6 had a more diverse tropism profile, with complete selectivity has not been achieved. There may also 46% identified as amacrine or bipolar cells, 23% as RGCs be advantages in using intravitreal approaches for the and 22% as Mu¨ller cells. Mu¨ller cells were also frequently transduction of photoreceptors. Here we compared the transduced by rAAV2/4. The highest photoreceptor transduc- cellular tropism and transduction efficiency of rAAV2/1, tion was seen after intravitreal rAAV2/3 injection. These data -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after facilitate the design and selection of rAAV vectors to target intravitreal injection. Each vector encoded green fluorescent specific retinal cells, potentially leading to an improved gene protein (GFP), and the number, laminar distribution and therapy for various human retinal pathologies. morphology of transduced GFP+ cells were determined Gene Therapy (2009) 16, 521–532; doi:10.1038/gt.2008.178; using fluorescent microscopy. Assessment of transduced published online 18 December 2008

Keywords: AAV; photoreceptors; retinal ganglion cells; CNS regeneration

Introduction capsid surface.23 Variation in capsid structure affects viral tropism and expression kinetics in terms of the Recombinant, replication-deficient adeno-associated onset and expression levels of therapeutic , and the virus (rAAV) vectors are increasingly being used in gene use of different serotypes may allow for a more cell- therapy trials in the central nervous system.1 rAAV specific gene transfer.3,6,8,12,24,25 vectors transduce a variety of cell types while inducing Robust photoreceptor transduction has been minimal immune responses in the host.2 In the eye, reported after subretinal injection of many vector types; rAAV has been used in experimental studies aimed at however, photoreceptors are only infrequently trans- treating retinal degenerative diseases or for cell rescue duced after intravitreal injection of the serotypes tested following trauma.3–14 On the basis of the success of some to date.3,4,6–8,11,12,26 Intravitreal vector delivery is a less of these animal studies, clinical trials with AAV vectors invasive technique compared with subretinal delivery. A have recently been initiated in humans with genetically larger vector volume can be delivered and this approach linked ophthalmic diseases.15,16 also allows repeated injections, and can thus be Human AAV serotype 2 was originally found as a combined with the delivery of other pharmacotherapeu- contaminant in adenovirus preparations17 and has not tic agents.9,10,27–31 The ability to target photoreceptor cells been associated with any human pathogenicity. Since in the outer retina through intravitreal injections may then, several other AAV serotypes have been identified therefore be of clinical benefit. with slight variations in amino-acid capsid identity. AAV Retinal ganglion cells (RGCs) are targeted directly by serotypes 1–8 share about 60% amino-acid capsid intravitreal injections and gene therapy may be an homology,18–22 the largest amino-acid variations being effective method for rescuing these in degen- concentrated to the hypervariable regions exposed on the erative conditions such as glaucoma, ischaemia, optic neuritis and optic neuropathy.28 rAAV2 vectors trans- duce RGCs8–12 but not with absolute selectivity,11 which Correspondence: Professor AR Harvey, School of Anatomy and may complicate gene therapy treatment of ophthalmic Human Biology (M309), The University of Western Australia, disease. It is, therefore, important to evaluate the tropism 35 Stirling Highway, Crawley, Western Australia 6009, Australia. E-mail: [email protected] and transduction efficiency of other AAV serotypes to Received 9 September 2008; revised 3 November 2008; accepted determine if there are even more efficient and specific 5 November 2008; published online 18 December 2008 vectors that target RGCs. In this study, we assessed the Transduction properties of AAV vectors in retina M Hellstro¨m et al 522 cellular tropism and transduction efficiencies of titre- matched rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 vectors in adult rat retina after a single intravitreal injection. All vectors encoded the gene for green fluorescent protein (GFP) and possessed an AAV2 backbone, cross-packed into capsids from another serotype, hence the pseudoserotype nomenclature AAV2/capsid serotype. Retinas were analyzed 10 weeks post-injection, a time point at which all vector types should maintain a strong gene expression.6,8,12,24 For each serotype, the number, morphology and laminar distribu- tion of GFP-positive (+) cells were quantified using Figure 1 Transduction efficiency of the seven rAAV serotypes after confocal and fluorescence microscopy. Phenotype was an intravitreal, titre-matched (2.6  109 genomic copies) injection in also assessed by double immunolabelling with various adult Wistar rats, assessed 10 weeks later. Note that, for each + markers for retinal cell types.11 animal, transduced GFP cells were counted in retinal sections sampled from across the entire retina. The average number of transduced cells per mm retina was then determined (mean± s.e.m.). GFP, green fluorescent protein; rAAV, recombinant adeno- Results associated virus. Intensity measurements of retinal GFP expression Cellular tropism of rAAV serotypes in eyecups Transduced retinal cells were identified on the basis of m rAAV titres were matched for each serotype group (4 l their laminar location, morphology and confirmed using  9 eye injections, 2.6 10 genomic counts per injection). In cell-specific immunomarkers (Figures 2 and 3). With the each group, there was a sustained GFP expression in the exception of rAAV2/6, all rAAV serotypes preferentially retinas at 10 weeks post-injection. Examination of the transduced cells located within the RGC layer (Figures 2a whole retina before sectioning revealed that GFP and 4). Calculated as a proportion of the total number of fluorescence was not uniform, typically being much GFP+ cells, between 57 and 71% were located in the RGC brighter in one quarter to one half of the retina, in the layer; however for rAAV2/6, only about 24% of vicinity of the initial lower-temporal vector injection. transduced cells were found in this layer. Multiple group Native GFP fluorescence levels revealed detectable comparisons revealed that this value was significantly variation between groups (Supplementary Figure 1), less than that found for rAAV2/2 and rAAV2/8 and although these data provided no information about serotypes. GFP+ cells in rAAV2/6-injected animals were laminar or cellular location, overall, the comparative mainly found in the inner nuclear layer (61%) (Figures 2e levels of GFP transduction for each serotype were and 4g). Note that, even after intravitreal injection, consistent with subsequent quantitative histological rAAV2/3 and rAAV2/5 showed some tropism towards analyses of retinal sections. cells in the outer nuclear layer (Figures 2d, 4d and f). A further characterization of transduced (GFP+) retinal cell types was based on cell morphology supported by Transduction efficiencies of rAAV vectors: histological immunostaining with various cell-specific markers11 analysis (Figures 2, 3 and 5). No allocation to a specific cell type Two to five slides (10–12 retinal sections per slide) per was made unless clearly defined cell bodies and animal in each rAAV serotype group (n ¼ 3–6 per group) processes were distinguished; overall, 56% of GFP+ cells were quantified. For all serotypes, there was some were phenotyped. Of these characterized cells, RGCs variation in the intensity of native GFP immunofluores- constituted the highest proportion of transduced cells in cence in individual cells, and GFP immunohistochem- six serotype groups (49–64%; Figure 5), but in the istry was carried out on retinal sections from each group. rAAV2/6 group, only 23% of transduced cells were Within each serotype group there was no significant RGCs (Figure 5f). As transduction efficiency was difference in cell counts between immunostained and highest in the rAAV2/2 group, many GFP+ axons were non-immunostained sections and data were therefore seen in the optic nerves of these animals (Figure 2c). pooled. Consistent with the initial whole retina analysis, Note here that the tropism results for rAAV2/2 were the extent of AAV transduction seen in retinal sections remarkably similar to data obtained in an earlier study varied across the retina, with an average of 50–60% of using the same rAAV2/2 vector construct.11 GFP+ axons retinal sections containing at least some GFP+ cells. were also seen in the optic nerve of rAAV2/6-injected Retinas from rAAV2/2- and rAAV2/6-injected eyes animals (Figure 3g) even though this vector displayed a contained the greatest number of transduced cells (that much lower tropism for RGCs. In both vector groups, is, highest transduction efficiency), with an average almost all GFP+ axons exited from the temporal side of of 1.9 and 1.7 cells per mm retinal section, respectively the retina, confirming more frequent transduction of (Figure 1). Transduction was lowest in rAAV2/5- and RGCs in the vicinity of the initial intravitreal injection. rAAV2/8-injected eyes. Statistical analysis revealed no rAAV2/8 displayed the highest degree of tropism for significant difference between rAAV2/2 and rAAV2/6; RGCs (Figure 5g), but because of very low transduction however, rAAV2/2 GFP transduction was significantly efficiency in these eyes (Figure 1), the total number of higher than rAAV2/4 and rAAV2/8. rAAV2/6 transduc- transduced cells in inner retina in this group was low tion was also significantly greater than that found using and far fewer than that seen using the rAAV2/2 vector rAAV2/8 vectors. serotype.

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 523

Figure 2 Confocal images of immunostained retinal sections from AAV2/2- and AAV2/3-injected eyes. (a) GFP-expressing cells in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) in an AAV2/2-injected eye. Closed arrows in the GCL present double-labelled GFP+ and bIII-tubulin+ retinal ganglion cells; open arrows show bipolar-like GFP+ cells. (b)TransducedGFP+ cells double labelled with RGC-specific protein bIII-tubulin (closed arrows). (c)GFP+ axons (arrows) in the optic nerve from an eye that had been injected with AAV2/2; note the localization of GFP+ axons originating from temporally located RGCs. The open arrow points towards the retina. T, temporal side of nerve; N, nasal side; on, optic nerve. (d) Retinal section from an AAV2/3-injected eye. Note transduced photoreceptors (closed arrows) in the outer nuclear layer (ONL); a Mu¨ller glial cell is also labelled (open arrow). (e) Retinal section from an AAV2/6-injected eye showing a GFP+ RGC immunostained for bIII-tubulin (closed arrow) and two GFP+ cells with amacrine-like morphology (open arrows) located at the inner edge of the inner nuclear layer (INL). The latter cells were not bIII-tubulin positive. A bipolar-like cell is marked by the thin arrow. Green ¼ GFP transduced cells; red ¼ bIII-tubulin; blue ¼ Hoechst 33342 nuclear staining. Scale bars: 20 mm(a); 20 mm(b); 100 mm(c); 30 mm(d); 40 mm(e). AAV, adeno-associated virus; GFP, green fluorescent protein; RGCs, retinal ganglion cells. See online html-version for colour figure.

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 524

Figure 3 Confocal images of retinal sections containing transduced cells from AAV2/6-injected eyes. (a)TransducedbIII-tubulin double-labelled RGCs (closed arrows) and a cell with bipolar morphology (open arrow). (b)GFP+ bipolar cell immunolabelled for PKCa (see inset). (c–e)Two transduced Mu¨ller glial cells double stained with glutamine synthetase; arrows pointing towards cell soma and processes. (f)GFP+ cell with bipolar-like morphology adjacent to a transduced horizontal cell immunopositive for (arrow; see inset). (g)GFP+ axons in the optic nerve (on) from an eye injected with AAV2/6; open arrow points towards the retina. T, temporal side of nerve; N, nasal side; on, optic nerve. Green ¼ GFP-labelled cells (transduced cells); red ¼ bIII-tubulin (a), PKCa (b), glutamine synthetase (d and f)andcalbindin(f); blue ¼ Hoechst nuclear staining. Scale bars: 20 mm (a, b and f); 40 mm(c–e); 100 mm(g). AAV, adeno-associated virus; GFP, green fluorescent protein; PKCa,proteinkinaseC-a; RGCs, retinal ganglion cells.

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 525

Figure 4 The proportion of transduced cells in each retinal layer for each AAV serotype. (a) Overall transduction efficiency for the tested vectors (same as Figure 2). (b–h) Data for each serotype were derived from the location of GFP+ cell nuclei within the different retinal layers. The inner nuclear layer (INL) was further divided into two halves: RGC layer side and photoreceptor (PR) side. Mu¨ ller cells were placed in a separate category. AAV, adeno-associated virus; GFP, green fluorescent protein; RGCs, retinal ganglion cells.

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 526

Figure 5 (a–g) The proportions of GFP+ cells transduced for each serotype that could be identified on the basis of nuclear location and morphology, and characterization using immunomarkers. GFP, green fluorescent protein.

In both rAAV2/3- and rAAV2/5-injected eyes, over than that for rAAV2/5; thus the greatest number of GFP+ 20% of transduced cells were identified as photorecep- photoreceptors was seen in the rAAV2/3-injected group. tors (Figures 2d, 5c and e). However, overall rAAV2/3 rAAV2/4 transduced relatively larger numbers of ama- transduction efficiency was about three times greater crine cells and Mu¨ ller within the inner nuclear layer

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 527 (Figure 5d). rAAV2/6 had the most diverse tropism the retina, but GFP+ cells were usually most frequently profile among the tested vectors (Figure 5f); the majority encountered in ventral and temporal retina, in the of the transduced cells were amacrine cells (25%) and vicinity of the original intravitreal injection. In this bipolar cells (21%; Figure 3b), but a large number were regard, note that the transduction efficiency data also classified as Mu¨ ller glia (22%; Figures 3c–e). Mu¨ ller presented here were obtained from sections sampled glia were only infrequently transduced by rAAV2/8 and across the entire retina, contributing to the apparently rAAV2/5, at levels that were significantly lower than low average number of GFP+ cells per mm of retinal those seen in retinas from rAAV2/4- and rAAV2/6- section. Nonetheless, for rAAV2/2 at least, when encod- injected eyes. Horizontal cells were only rarely transduced ing the appropriate transgene, this level of transduction by any of the seven rAAV vectors tested in this study is sufficient to significantly enhance the survival and (Figures 3f and 5). For rAAV2/1–5 vectors, between 15 regeneration of injured RGCs.9 The localized nature of and 25% of the GFP+ cells found in the RGC layer were rAAV retinal transduction after a single 4 ml intravitreal not obviously immunoreactive for bIII-tubulin, and injection suggests that a more homogeneous transduc- where a clear profile was seen these were counted as tion (and perhaps even better functional outcomes) may displaced amacrine cells.28 Interestingly, these trans- be achieved by using multiple injections and larger duced bIII-tubulinÀ cells in the RGC layer were more injection volumes, perhaps carried out sequentially over frequent after rAAV2/6 or rAAV2/8 injection (43 and a period of days. In this context, vector transfer in 45%, respectively), which at least for AAV2/6 was combination with specific epidermal growth factor consistent with this vector’s overall lack of transduction receptor protein tyrosine inhibitors or with specificity (Figure 5f). proteasome inhibitors may also result in higher trans- duction efficiency in ocular gene therapy.32,33 Of course, increased transduction and stronger transgene expres- Discussion sion from gene therapy applications may not necessarily lead to better outcomes, and there is likely to be a fine This study compared the transduction efficiency of balance between therapeutic and detrimental expression seven pseudoserotyped rAAV vectors after a single levels, especially for genes that encode secretable intravitreal injection into adult Wistar rat eyes. Vectors proteins.34–36 This should also be taken into account consisted of the AAV2 backbone cross-packed into the when choosing promoters that may increase the speci- capsids from AAV serotype 1–6 or AAV8. Earlier ficity of transgene expression37–39 and when designing investigations that compared the transduction efficiency vector systems that allow the temporal regulation of of rAAV serotypes in the eye used in vivo fluorescent transgene expression.40–43 imaging, fundus luminescence quantification, and in Although comparable in terms of overall transduction some studies, quantitative PCR.3,4,6–8,12 Quantitative efficiency, the tropism profile between rAAV2/2 and analysis of immunostained retinal sections has rAAV2/6 was considerably different. AAV2 and AAV6 been carried out after intravitreal rAAV2/2 injections,11 share 82% homology and differ by 53 amino acids in the but to our knowledge, this type of analysis has not been variable region of the Cap gene.21 AAV2 is known to used to compare the transduction efficiency and tropism enter cells through the heparin sulphate proteoglycan of different rAAV serotypes after injection into the receptor, fibroblast growth factor receptor 1 and the vitreous. integrin receptors aVb1 and aVb5.44–47 A laminin When analyzed 10 weeks after a single titre-matched receptor has also been implicated for AAV2 cell entry.48 intravitreal injection, we here confirm earlier tropism and AAV6 can also bind to heparin sulphate proteoglycan transduction findings for rAAV2/211 and report new receptors,49,50 but unlike AAV2, AAV6 is not inhibited by data on six additional rAAV vectors. Measurement of soluble heparin.51 Instead, AAV6 has been shown to be native GFP expression in whole retina before sectioning dependent on cell-bound sialic compounds presented on revealed between-group differences in fluorescence N-linked glycoproteins.52 These different vector–cell levels that were largely consistent with subsequent interaction characteristics may cause the profile diver- GFP+ cell counts in cryosectioned material. rAAV2/2 gence seen between rAAV2/2 and rAAV2/6 in this and rAAV2/6 displayed the highest transduction effi- study. Interestingly, AAV6 and AAV1 share 99% se- ciency, followed by rAAV2/3. rAAV2/5 and rAAV2/8 quence similarity in the VP1 capsid genome.19 None- were the least efficient. rAAV2/6 had the most diverse theless, the vector carrying the rAAV1 capsid transduced tropism profile, transducing many neuronal cell types as very few cells in this experiment. Cell entry is not well as Mu¨ ller glia. These retinal glia were also relatively the only obstacle in vector transduction, the intracellular more frequently transduced by rAAV2/4. Apart from trafficking is also known to be a limiting factor53 rAAV2/6, all other vectors transduced RGCs more and may explain the poor outcomes seen here with frequently than other cells, but because of greater rAAV2/1. Interpretation is also complicated by the fact transduction efficiency, the highest number of GFP+ that the process of injecting rAAV into the vitreous will RGCs and axons was seen after rAAV2/2 injections. rapidly alter the expression of growth factors and some With this vector, about two-thirds of all transduced cells receptors such as fibroblast growth factor receptor 1,54 were RGCs. Following rAAV2/3 or rAAV2/5 injection, potentially influencing the types of cell transduced by 20–25% of GFP+ cells were photoreceptors; however, the rAAV vectors. total number of these cells was greatest in the rAAV2/3 rAAV2/3 was the third most efficient vector tested in group, again due to relatively greater transduction this experiment and over 20% of transduced cells were efficiency. photoreceptors. Other serotypes delivered intravitreally For all vector groups, there was some interanimal were less effective in transducing these outer retinal variability in the spatial extent of GFP expression across neurons. This tropism was unexpected due to generally

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 528 poor diffusion of rAAV vectors across the retina after regulatory element. The transgene expression cassette is intravitreal delivery and because rAAV2/3 has not been flanked by the AAV2 inverted terminal repeats. shown to be efficient when applied subretinally.6 The Helper plasmids used for cross-packaging were majority of cells in the retina are photoreceptors and generously provided by Dr Kleinsmidt (University of desired vectors for the treatment of photoreceptor- Heidelberg, Germany) and used as described earlier.65 associated disorders will need high transduction effi- Briefly, pTRCGW (50 mg per roller bottle) and the helper ciencies. However, the levels of transduction seen for plasmids (ratio 1:3) were co-transfected into 293T cells rAAV2/3 may be sufficient if the vector encodes cultured in roller bottles (850 cm2 surface, Greiner, secretable factors. Intravitreal injections for the treatment Alphen aan Den Rijn, The Netherlands) using calcium of photoreceptor dysfunction may have advantages, phosphate. Each roller bottle contained 2.5 Â 107 cells. including capacity for larger, as well as multiple, vector Cells were grown in 50 ml of Iscove’s modified injections and possible combinations with other pharma- Eagle’s medium without phenol red and pyruvate, and cotherapeutic agents.9,10,27–31 supplemented with 10% fetal calf serum and pen/strep In our hands, rAAV2/1, rAAV2/4, rAAV2/5 and (Invitrogen, Breda, The Netherlands). Medium was rAAV2/8 were the least efficient among the tested refreshed 2 h before and on the day of transfection. vectors. Earlier studies based on fluorescent intensity Recombinant AAV was harvested 66 h post-transfection measurements have also shown low transduction effi- by removing the medium and adding 5 ml of lysis ciencies for these vector types;4,7,8,12 however, when buffer (50 mM Tris (pH 8.5), 2 mM MgCl, 150 mM injected subretinally, others have reported that rAAV2/ NaCl and 0.1 % Triton X-100) per roller bottle. The lysis 8 is efficient, even for transducing RGCs.4 The authors buffer from roller bottles was pooled and incubated speculated that, in contrast to the other vectors tested, for 1 h with 10 mgmlÀ1 DNAse. This harvest was spun the rAAV2/8 vector may have been transited from down (30 min, 4000 g) and filtered through a 0.45-mm2 transduced photoreceptor cells to other neurons in the mesh filter. visual pathway because they also reported poor RGC AAV serotypes 1, 2, 3 and 5 were purified using transduction after an intravitreal injection.4 Interestingly, affinity chromatography with AVB Sepharose High for rAAV2/2 at least, although this vector remains the Performance (GE Healthcare Bio-Sciences AB, Brussels, most effective for targeting RGCs after intravitreal Belgium). After loading the column with crude lysate, injection into adult eyes, the transduction profile varies the column was rinsed with 10 column volumes of 0.1 M significantly depending on the age of the injected animal. Tris-HCl, pH 8.5, and eluted in DPBS (Invitrogen), pH Thus, after intravitreal rAAV2/2 injection into neonatal 3.5. For neutralization to pH 7.4, the eluate was collected eyes, about two-thirds of the GFP+ cells are found in the in 10% v/v 0.1 M Tris, pH 8.5. A buffer change was photoreceptor layer and a more adult-like pattern is performed in Amicon Ultra-15 (Millipore, Billerica, MA, established by 2 weeks after birth.11 USA) concentrators to DPBS (pH 7.4) supplemented with It is important to continuously evaluate novel rAAV 5% sucrose, and the rAAV vector was concentrated to the vectors for human gene therapy as a large proportion of appropriate volume. the human population carry sera with neutralizing AAV serotypes 4, 6 and 8 were isolated on an antibodies against many different AAV serotypes.25,55 iodixanol density gradient. Crude lysate (18.5 ml) was Our quantitative serotype data in adult eyes provide new underlayered with 9 ml 15%, 5 ml 25%, 5 ml 40% and information on transduction and cellular tropism char- 3 ml 60% iodixanol (Sigma, St Louis, MO, USA) in acteristics of different rAAV vectors that may aid in the phosphate-buffered saline (PBS), pH 7.4, supplemented

development of improved protocols for the treatment of with 10 mM MgCl2 and 25 mM KCl. Centrifugation was many types of retinal disease. RGC loss is a feature of performed for 1 h 10 min in a 70Ti Beckman rotor at many degenerative ophthalmic conditions,28 and there is 69 000 r.p.m. at 16 1C. The viral vector will concentrate also ongoing, often aberrant, remodelling of intraretinal between the 40 and 60% iodixanol bands. Approximately circuitry as a consequence of photoreceptor loss.56,57 Use 5 ml was collected by puncturing the centrifuge tube of appropriate AAV vectors may prevent or reduce such with a syringe. Before concentrating in an Amicon Ultra- anomalous reorganizations within the inner nuclear 15 concentrator, rAAV was diluted 1:1 in DPBS to reduce layer, potentially enhancing the effectiveness of other the viscosity of the iodixanol solution. At least three therapies such as cellular or bionic implants.57 Finally, volumes of DPBS were spun through the filter device new vector constructs, different modes of delivery,24,25 before the vector stock was spun down with two and the isolation and modification of different sero- volumes of DPBS supplemented with 5% sucrose. All types7,58–62 will lead to novel vectors that have greater vector stocks were kept at À80 1C until use. packaging capacity33,63 and cell specificity, and may even Characterization of purified rAAV stocks included further minimize immunological sequelae following quantitative PCR analysis to determine the number of therapeutic application. genomic copies (GCs), SDS-polyacrylamide gel electro- phoresis analysis for visualization of the three capsid proteins65 and an infection assay of 293T cells to verify Materials and methods the infectivity. The infection assay was performed using serial dilutions of the vector, by adding the viral vector to Vector production and purification camptothecin-treated (4.8 mgmlÀ1 for 4 h) 293T cells. The AAV vectors were generated using the plasmid presence of transduced cells was determined at 3 days pTRCGW.64 This plasmid harbours a transgene expres- post-infection under a fluorescence microscope. GC titres sion cassette that contains the human cytomegalovirus were in the range of 6.6 Â 1011 to 1.5 Â 1012 GC mlÀ1. promoter, a cDNA encoding GFP, a polyadenylation Before intravitreal injection, all AAV vector stocks were (polyA) signal and the woodchuck post-transcriptional titre matched at 6.6 Â 1011 GC mlÀ1.

Gene Therapy Transduction properties of AAV vectors in retina M Hellstro¨m et al 529 Animals anti-PKCa (bipolar cells;67,68 1:500; Transduction Labs, All animals used for the experiment were 10-week-old Sydney, Australia), rabbit anti-GFAP (Mu¨ ller glia and outbred female Wistar rats purchased from the Animal astrocytes;69 1:150; Dako, Sydney, Australia), mouse Resources Centre (Perth, Australia). Experiments were anti-glutamine synthetase (GS; Mu¨ ller glia;70,71 1:1000; approved by The University of Western Australia (UWA) Chemicon), mouse anti-ED1 (macrophages;72 1:200; AbD- Animal Ethics Committee and conformed to NHMRC Serotec, Raleigh, NC, USA), mouse anti-calbindin guidelines. Animals were maintained on a 12 h day/ (horizontal73 cells and some amacrine cells; 1:2000; Sigma), night cycle and supplied with food and water ad libitum. rabbit anti-PV-28 (amacrine cells;74 1:5000; Swant, Bellinzona, Switzerland), overnight at +4 1C, followed Eye injections by 3  5 min washes in PBS. Sections were then incu- Intravitreal eye injections were carried out as described bated for 1 h with an anti-mouse or anti-rabbit earlier.9–11 In anaesthetized rats (1–1.5 ml kgÀ1 i.p. of a 1:1 Cy3-conjugated secondary antibody (1:300; Jackson, West mixture of ketamine (100 mg mlÀ1) and xylazine Grove, PA, USA), rinsed for 3  5 min in PBS before (20 mg mlÀ1)), rAAV was injected into the vitreous being coverslipped with Dako fluorescent mounting through a glass micropipette inserted into peripheral media containing Hoechst 33342 (Sigma) nuclear dye. lower temporal retina. Each animal (n ¼ 6 per rAAV serotype) received a total injection volume of 4 ml titre- Quantification methods and phenotype-identification matched rAAV vector equivalent to 2.6  109 GC per procedures injection. Rats were killed 10 weeks post-injection. After examination of sectioned material, four retinas were excluded from further analysis because it was clear Tissue processing and native GFP fluorescence that the initial injection procedure had damaged poster- measurements ior retina and that some vector had been delivered directly into retinal tissue. In the remaining 38 animals, Animals were overdosed with an i.p. injection of all retinal sections on selected slides were examined and Lethabarb (sodium pentobarbitone) and were perfused GFP+ cells were counted. Quantification, localization and with PBS (pH 7.4) containing 0.1% heparin followed by phenotype analysis were performed at  400 using a 4% paraformaldehyde in 0.1 M phosphate buffer (pH fluorescent microscope (Nikon). Sections were also 7.4). Both eyes were removed, the cornea and lens cut photographed at  20 and the length of each section away and retinas were post-fixed in the eyecups for 1 h. measured along the inner retinal surface (Image-Pro Before cryosectioning, native GFP fluorescence was Express software; Media Cybernetics). The number of visualized in the eyecup from most of the rAAV-injected transduced cells was divided by the total retinal length animals (Kodak imager 2000MM; excitation, 465 nm; per animal and averaged for each serotype tested. GFP+ emission, 535 nm; 1 min exposure). On the retrieved cells were counted and documented according to the image, a circle was drawn (circle area ¼ 2044 pixels) and retinal layer in which they were found. Characterization placed in the centre of each eyecup. Intensity levels were of transduced (GFP+) retinal cell types was based on recorded with Adobe Photoshop (CS2) histogram func- laminar location and morphology, and was confirmed tion. For each animal, the fluorescence of the non-injected using various cell-specific immunomarkers.11 No counts eye was also recorded to establish a baseline fluorescence were made unless a clearly defined cell body could be level. Eyecups and optic nerves were then cryoprotected distinguished. GFP+ non-neuronal Mu¨ ller cells were in Jung freezing medium (Leica, Sydney, Australia) and included in a separate group. frozen at À25 1C and put in À80 1C until further processing. Cryostat sections (16 mm) were cut horizon- tally through the eyecup from ventral to dorsal side so Statistical analysis that each section contained a complete temporal-to-nasal For each rAAV serotype, the number of identified cells cross-section of the retina. Every third section was and the total length of analyzed retina were determined, collected in a series of 10 slides with 10–12 sections per allowing a calculation of the number of transduced cells slide, thus each slide sampled sections from across the per mm retina per animal. The mean and standard error entire retina. Slides were stored at À20 1C. of the mean (s.e.m., error bars in all graphs) were calculated and significance tested using one-way analy- sis of variance and post hoc Bonferroni and/or Kruskall– Immunohistochemistry Wallis tests (significance level Po0.05). After removal from the freezer, slides were air-dried for 30 min at room temperature and rehydrated for 5 min in PBS before being blocked for 60 min with PBS buffer containing either 0.2% Triton X-100 and 10% normal goat Acknowledgements serum or 5% horse serum plus 2% bovine serum This study was supported by NHMRC and WA albumin. Depending on the source of the secondary Neurotrauma research grants to ARH, an ARC grant to antibody, antibodies were diluted in one or the other of MJR and a grant from SenterNovem to Sander van these blocking solutions. Selected slides were stained Devender (AMT) and JV. We thank Dr Jenny Rodger for with a primary rabbit or mouse anti-GFP antibody (1:100; the gift of the glutamine synthetase antibody. Millipore, Sydney, Australia), followed by an appropriate fluorescein isothiocyanate-conjugated secondary anti- body (1:100; Sigma), to assure that a weak native GFP References fluorescence was not overlooked in the analysis. Anti- bodies used for GFP+ cell characterization were mouse 1 McCown TJ. Adeno-associated virus (AAV) vectors in the CNS. anti-bIII-tubulin (RGCs;9,66 TUJ1; 1:2000; Sigma), mouse Curr Gene Ther 2005; 5: 333–338.

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