Efficient Gene Delivery and Selective Transduction of Glial Cells in the Mammalian Brain by AAV Serotypes Isolated From Nonhuman Primates

original article

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Efficient Gene Delivery and Selective Transduction of Glial Cells in the Mammalian Brain by AAV Serotypes Isolated From Nonhuman Primates Patricia A Lawlor1, Ross J Bland2, Alexandre Mouravlev1, Deborah Young1,3 and Matthew J During1 1 Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand; 2Animal Health Section, AgResearch Limited, Hopkirk Research Institute, Grasslands Research Centre, Hamilton, New Zealand; 3Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Auckland, New Zealand

Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1–9, with efficient gene transfer to neurons only—selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the ­constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated—widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology. Received 17 December 2008; accepted 24 June 2009; published online 28 July 2009. doi:10.1038/mt.2009.170

Introduction Recombinant adeno-associated viral (AAV) vectors are derived from nonpathogenic, replication-deficient members of the Parvovirus family and are efficient at transducing the nondividing cells of the central nervous system (CNS). Following infusion of AAV into the brain, stable, long-lasting neuronal transgene expression is observed, with no apparent toxicity.1–3 AAV vectors are versatile tools, allowing upregulation or knockdown of gene expression in specific brain regions, and can be used for in vivo

functional genomics studies,4–6 to create animal models of neurodegenerative disease,7–9 and as vehicles for gene therapy of these disorders.10–13 Treatment of neurodegenerative diseases by gene therapy may require AAV vectors capable of transducing large volumes of brain tissue from a single injection site. Additionally, the ability to target transgene expression to non-neuronal cell populations would be useful. Astrocytes have traditionally been considered as merely neuronal support cells; however, it is becoming evident that they contribute to the pathogenesis of neurodegenerative disorders.14–16 Given that both neuronal loss and astroglial ­proliferation are common characteristics of neurological diseases, the ability to target transgene expression to astrocytes may be useful when designing gene therapy treatments. Likewise, vectors that preferentially transduce oligodendrocytes, the cells responsible for CNS myelination, could be used for gene therapy of demyelination disorders such as Canavan disease and multiple sclerosis. To date, attempts to target transgenes to glial cell populations by  varying the cellular promoter have resulted in limited transduction only.17,18 Preclinical data on AAV-mediated gene transfer in the CNS has largely been based on the use of AAV2, a serotype that transduces neurons efficiently in the immediate vicinity of the injection site but requires multiple injections or addition of agents such as mannitol or heparin to transduce larger volumes of brain.19–21 AAV2 does not transduce all neurons with equal effectiveness, e.g., neurons of the substantia nigra (SN) are readily transduced, but specific hippocampal neuronal subpopulations are refractory to AAV2 transduction.22,23 Clinical application of AAV2 may also be limited by preexisting immunity to AAV2 in most humans.24,25 These limitations of AAV2 have necessitated the evaluation of alternative AAV serotypes with broader cellular targets, capable of evading preexisting immunity to AAV2. Delivery of AAV1, 5, 7, 8, and 9 (refs. 26–29) into adult rodent brain has been shown to result more widespread transgene expression than achieved with AAV2. However, transduction with these serotypes is still overwhelmingly neuronal—targeted and widespread transduction of glial cell populations is still not possible. PCR-based screens have led to the isolation of many additional endogenous AAVs from a variety of human and nonhuman

Correspondence: Patricia A Lawlor, Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand. E-mail: [email protected]

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Figure 1  Enhanced green fluorescent protein (EGFP) expression following intrastriatal, intrahippocampal, and intranigral infusion of vectors. Rats injected with adeno-associated viral (AAV) vectors were killed 3 weeks following infusion and brains processed immunohistochemically for detection of EGFP. (a) Transduction volume (mm3) following vector infusion. Every 12th section was used to measure the transduction volume according to the Cavalieri estimator. Overall, transduction volumes observed following infusion of cy5, rh20, and rh39 were greater than that achieved with AAV8 in all structures (striatum: ANOVA P < 0.001; LSD post hoc cy5 > AAV8, P = 0.002; rh20 > AAV8, P < 0.001; rh39 > AAV8, P = 0.026; hippocampus: ANOVA P = 0.006; LSD post hoc cy5 >AAV8, P = 0.028; rh39 > AAV8, P = 0.017; SN: ANOVA P = 0.021; LSD post hoc rh20 > AAV8, P = 0.03; rh39 > AAV8, P = 0.05). Bars represent mean + SEM, n = 3–5 per treatment. (b) The number of sections containing EGFPimmunoreactive staining was used to estimate the rostrocaudal spread (mm) of each vector. This was significantly different between the serotypes only in the striatum (striatum: ANOVA P = 0.003; cy5 > AAV8, P = 0.009; rh20 > AAV8, P = 0.001; rh39 > AAV8, P = 0.009; hippocampus: ANOVA P = 0.571; SN: ANOVA P = 0.977). Bars represent mean + SEM, n = 3–5 per treatment. (c) Low magnification views of the striatum show the extent of transduction obtained by infusing 4.5 × 109 vg of each serotype. (d) The extent of transduction obtained by infusing 4.5 × 109 vg of each serotype into the hippocampus. (e) Transduction of the SNpc following infusion of 4.5 × 109 vg of each serotype. ANOVA, analysis of variance; LSD, least significant difference; SNpc, substantia nigra pars compacta; vg, viral genomes. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

primate tissues30,31 that could be developed as human gene therapy vehicles.27,32 In this study, transgene expression obtained following injection of nonhuman primate AAV serotypes bb2, cy5, rh20, rh39, and rh43 directly into brain tissue was compared to transduction obtained with AAV8—a nonhuman primate– derived AAV that has previously been found to perform well in mammalian brain.26–29 The transduction volume and phenotype of transduced cells following infusion of AAV vectors into the adult rat striatum, hippocampus, and SN were compared. The results show widespread neuronal transduction following infusion of Molecular Therapy vol. 17 no. 10 oct. 2009

cy5, rh20, and rh39, to a level greater than that observed with AAV8, with limited transduction following infusion of bb2 or rh43. However, preferential astrocytic transduction was observed following infusion of rh43. This tropism for glial cells was further optimized by use of cell-specific, rather than constitutively active, promoters. The results show marked alterations in rh43and AAV8-induced expression patterns following use of the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, allowing widespread transduction of selected glial cell populations. 1693

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Vector stocks encoding the enhanced green fluorescent protein (EGFP) reporter under control of the chicken β-actin/cytomegalovirus (CMV) hybrid (CAG) promoter were titer matched and 3 µl [total of 4.5 × 109 viral genomes (vg)] injected unilaterally into the striatum, hippocampus, or SN. Brain tissue was harvested 3 weeks postinfusion and examined for visible EGFP fluorescence and EGFP-immunoreactivity. The volume of EGFP-immunoreactivity within the target structure was quantified using stereological methods. Overall, infusion of new serotypes cy5, rh20, and rh39 resulted in measurably greater transduction than obtained with AAV8. Use of AAV8 resulted in transduction of ~10 mm3 within the striatum; two- to threefold higher transduction was observed following infusion of cy5, rh20, or rh39 (Figure  1a). However, transgene expression with these serotypes was not confined to the striatum, extending into the cortex, medial forebrain bundle, and septum (Figure  1c). AAV8 EGFP-immunoreactivity extended for 2.2 mm around the injection site, with cy5, rh20, and rh39 EGFP-immunoreactivity extending significantly further than this (Figure 1b). Although infusion of bb2 resulted in no visible EGFP fluorescence, immunohistochemistry for EGFP showed that bb2mediated transduction extended over a greater portion of the striatum (~15.5 mm3) than observed with AAV8; however, transduction was confined to a sparse subpopulation of medium spiny neuron (Figures 2a and 3e). Intrastriatal infusion of rh43 resulted in the least transgene expression of all serotypes (Figure 1a–c). EGFP-positive fibers were observed in striatal projection areas (globus pallidus and SN pars reticulata) following AAV8, cy5, rh20, and rh39 infusion. Retrograde transport of vector to the SN was also observed following intrastriatal infusion of AAV8, cy5, rh20, and rh39 with EGFP-immunoreactive cell bodies observed in SN pars compacta. In contrast, intrastriatal infusion of bb2 resulted in few positive fibers within the globus pallidus, and no observed transduction of SN pars compacta, consistent with the sparse transduction of neurons observed within the striatum; infusion of rh43 did not result in transgene expression in striatal projection areas. As has been shown previously, AAV8 transduced cells in all principal layers of the hippocampus—dentate gyrus, hilus, CA1, CA2, and CA4—with EGFP-immunoreactive fibers and cell bodies also observed in the contralateral hippocampus. Overall, cy5 and rh39 transduced a significantly larger portion of the hippocampus than AAV8 (Figure 1a), with transduced neurons present in all principal layers of the hippocampus (Figure 1d). Intense staining of fibers was observed on the contralateral side, along with immunoreactive neuronal cell bodies. With these serotypes, there was also a large amount of transduction outside the target area, extending into the cortex and thalamus of the injected side. Similar to observations made in the striatum, infusion of bb2 into the hippocampus resulted in transduction of only a small subpopulation of neurons, and infusion of rh43 resulted in minimal transduction (<5 cells transduced throughout the hippocampus) with no volume measurement possible. There was no difference in rostrocaudal spread between serotypes (Figure 1b). Projection areas for the hippocampus include the nucleus accumbens and septum. EGFP-immunoreactivity was not detected in the nucleus

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Figure 2 Density of transduction following intrastriatal infusion of new adeno-associated viral (AAV) serotypes. (a) Higher magnification views of the striatum show the density of transduction obtained following infusion of 4.5 × 109 viral genomes of each serotype. (b) The total number of transduced cells within the striatum was counted using unbiased stereological techniques. Only infusion of rh20 resulted in transduction of significantly more cells than transduced with AAV8 (ANOVA P = 0.021; LSD post hoc rh20 > AAV8, P = 0.027). (c) The number of cells transduced per mm3 of striatum was calculated, with neither cy5, rh20 or rh39 resulting in a significantly different density of transduction than AAV8 (although both bb2 and rh43 had a significantly lower density of transduced cells than AAV8—ANOVA P = 0.019; post hoc LSD bb2 < AAV8, P = 0.006; rh43 < AAV8, P = 0.004). ANOVA, analysis of variance; LSD, least significant difference. *P ≤ 0.05.

accumbens with any serotype; EGFP-immunoreactive fibers were observed in both ipsilateral and contralateral septum following AAV8, cy5, rh20, and rh39 infusion, but not in bb2- and rh43injected brains. www.moleculartherapy.org vol. 17 no. 10 oct. 2009

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extending into the thalamus of the injected hemisphere (Figure 1a). There was no difference in rostrocaudal spread between serotypes (Figure 1b). Use of bb2 resulted in transduction of a small number of neurons within the SN, detectable only by immunostaining. Given the minimal immunostaining observed with rh43 in the SN (<5 cells throughout the SN), this serotype was excluded from quantitative analysis of transduction volume.

The density of transduction following infusion of new serotypes Although infusion of cy5, rh20, and rh39 resulted in more widespread transduction than observed with AAV8, we sought to determine whether there was a difference in the total number of cells transduced or the density of transduction (cells transduced per mm3). As all serotypes transduced the striatum, a large region with a homogeneous cell population, this structure was selected for further characterization. The total number of transduced cells within the striatum was counted (i.e., transduced areas outside the striatum were not included) using unbiased stereological techniques. Infusion of rh20 resulted in transduction of the greatest number of cells (~400,000) of all serotypes tested (Figure 2b), and although more widespread transduction was seen with cy5 or rh39 than with AAV8, neither serotype transduced significantly more cells than AAV8 (~160,000). This could be attributed to a lower transduction density with these serotypes—rh20 and AAV8 transduced ~14,000–16,000 cells per mm3, respectively, whereas cy5 and rh39 transduced ~8,000– 10,000 cells per mm3. Examples of striatal transduction density are shown in Figure 2a.

Figure 3  Bb2, cy5, rh20, and rh39 transduced neurons, whereas infusion of rh43 resulted in transduction of astrocytes. Neurons within the (a) striatum, (b) substantia nigra, and (c,d) hippocampus were transduced following infusion of cy5, rh20, and rh39. Transduction following bb2 infusion was detectable only by use of immunohistochemistry using (e) anti-GFP. Infusion of 4.5 × 109 vg of rh43 into the (f) striatum resulted in low-level astrocytic transduction. Infusion of 3 × 1010 vg of rh43 into the (g) striatum and (h) hippocampus resulted in increased numbers of transduced astrocytes, along with transduction of neurons. (i) Infusion of 4.5 × 109 vg AAV8-CAG-EGFP resulted in transduction of a small number of astrocytes, as indicated by arrowhead. These were not visible under fluorescence, detectable only by immunohistochemistry. Bars = (a–f) 50 µm; (g–i) 100 µm. AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; vg, viral genomes.

Infusion of AAV8 resulted in extensive transgene expression in the SN, extending into the thalamus around the needle tract. Infusion of cy5, rh20, and rh39 resulted in more widespread transgene expression than obtained with AAV8 (Figure 1e), with EGFP-positive immunoreactivity evident throughout the SN and Molecular Therapy vol. 17 no. 10 oct. 2009

Infusion of bb2, cy5, rh20, and rh39 resulted in neuronal transduction With the exception of rh43, infusion of these serotypes resulted in neuronal transduction—examination of both EGFP fluorescence and immunostaining showed that transduced cells were morphologically consistent with a neuronal phenotype (Figure  3a–d). The presence of fiber staining in the contralateral hippocampus after hippocampal infusion (Figure  1d) and striatal EGFP expression in SN-injected brains further confirmed the neuronal phenotype of transduced cells (data not shown). Notably, bb2 transduced only a subpopulation of medium spiny neuron within the striatum. Transduction of astrocytes following rh43 infusion Quantifiable transgene expression following infusion of rh43 was observed only in the striatum. Notably, however, rh43 transduced only astrocytes at this titer (Figure 3f)—these cells were morphologically distinct from neurons, with large cell bodies and multiple, highly ramified processes. The observation that intrastriatal infusion of rh43 resulted in exclusive, but limited, astrocytic transduction led us to investigate whether injecting an increased number of vector genomes would result in more widespread astrocytic transduction. Additional animals were injected with 3 × 1010 vg of rh43-CAG-EGFP. However, although more widespread astrocytic transduction was observed at this titer, this was accompanied by significant neuronal transduction in both the striatum (Figure 3g) and hippocampus (Figure 3h). 1695

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Altered tropism of AAV8 and rh43 following use of cell-specific promoters The observed propensity of rh43 to transduce astrocytes, combined with the observation that AAV8-derived expression cassettes under the control of the CAG promoter resulted in transduction of a small number of astrocytes (maximum of five per section, not visible under fluorescence but detectable by immunohistochemistry, Figure  3i) led us to investigate the effect of varying the promoter on transgene expression in these two serotypes. In order to optimize targeted glial cell transduction, we generated further AAV8 and rh43 vectors driven by the cell-specific promoters GFAP and MBP, and compared this to transduction obtained using the constitutive promoters, CAG and CMV. Each vector (3 × 1010 vg) was infused into the striatum or hippocampus, and brain tissue examined immunohistochemically for transgene expression 3 weeks postinfusion.

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The phenotype of transduced cells varied markedly depending on the promoter used. Overall, transduction with AAV8 resulted in widespread visible EGFP fluorescence, whereas transduction with rh43 resulted in weak EGFP fluorescence, and the full extent of transduction was detectable only by immunohistochemistry for EGFP—colabeling results presented in Figure  4 are from AAV8injected brains. It has been noted previously that immunohistochemical detection of GFP is more sensitive than quantification of visible EGFP fluorescence, so although the images of visible EGFP fluorescence in Figure  4 depict the predominant cell type transduced with each promoter, results presented in Figure 5 show additional transduction of other cell populations detectable only after immunohistochemistry with anti-GFP. Striatal or hippocampal infusion of 3 × 1010 vg AAV8-CAGEGFP (Figure  4a–f) resulted in widespread neuronal EGFP expression (Figure  4a,d), confirmed by colocalization of EGFP with the neuronal marker NeuN (Figure 4b,e), and lack of EGFP/ GFAP colocalization (Figure  4c,f). Within the hippocampus, ­neuronal transduction was additionally confirmed by the presence of widespread contralateral fiber staining (Figure  5g). As shown previously, at this titer, infusion of rh43-CAG-EGFP resulted in widespread transduction of both neurons and astrocytes (Figure  3g,h). Following infusion of AAV8-CMV-EGFP (Figure  4g–l), visible EGFP fluorescence was observed predominantly in neurons, although immunohistochemical analysis showed some glial cells were also transduced. Infusion of rh43CMV-EGFP into the striatum or hippocampus resulted in EGFP transgene expression in both neurons and astrocytes, restricted to the immediate vicinity of the injection site.

Astrocytic transgene expression driven by the GFAP promoter Use of the GFAP promoter resulted in widespread astrocytic transduction with both serotypes. Following AAV8-GFAP-EGFP ­infusion (Figure  4m–r), EGFP fluorescence was observed in ­astrocytes only (Figure  4m,p), confirmed by colocalization of EGFP with GFAP (Figure  4o,r), and the lack of EGFP/NeuN colabeling (Figure 4n,q). The lack of contralateral fiber staining following intrahippocampal infusion (Figure  5) and the lack of EGFP-positive fibers in striatal projection areas further confirm Figure 4  Varying the promoter altered transgene expression following infusion of AAV8 and rh43 in the striatum and hippocampus. Examples shown are AAV8 (3 × 1010 viral genomes). (a–f) Use of the CAG promoter resulted in widespread neuronal EGFP expression with transduced cells morphologically consistent with neurons (a,d). This was confirmed by colocalization of EGFP transgene and the neuronal marker NeuN within cells (b,e; colabeled cells appear yellow), and lack of colocalization between EGFP and the astrocytic marker GFAP (c,f). (g–l) Use of AAV8 with the CMV promoter resulted in predominantly (g,h,j,k) neuronal transduction with minimal (i,l) astrocytic transduction. (m–r) Use of the GFAP promoter resulted in widespread (m,p) astrocytic transduction, confirmed by the lack of EGFP/NeuN colocalization in (n,q) and the large number of GFP/ GFAP colabeled cells, appearing yellow (o,r). (s–x) Use of the MBP promoter resulted in oligodendroglial transduction within the (s,v) striatum and hippocampus. Transduced cells colocalize with the oligodendroglial marker (u,x) CAII, rather than with (t,w) NeuN. Bar = (v) 20 µm (applies to a,d,g,j,m,p,s). Bar = (x) 50 µm (applies to b,c,e,f,h,i,k,l,n,o,q,r,t,u,w). AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; MBP, myelin basic protein.

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transgene expression driven by the GFAP promoter as predominantly astrocytic. However, immunohistochemical detection of EGFP-immunoreactive fibers in AAV8-GFAP-EGFP injected tissue indicates that some neurons were also transduced, detectable only by immunohistochemistry. Following rh43-GFAP-EGFP infusion, visible fluorescence was less widespread than with AAV8;

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however, immunohistochemical results (Figure  5) show that transduction was almost exclusively astrocytic, confirmed by the lack of EGFP-immunoreactive fibers.

Use of the MBP promoter results in oligodendroglial transduction Following infusion of AAV8-MBP-EGFP (Figure  4s–x), widespread EGFP fluorescence was observed in oligodendrocytes within the striatum, along with EGFP-positive fibers within the striosomes (Figure  4s). The small size and morphology of these transduced cells were consistent with an oligodendroglial phenotype, confirmed by colocalization of EGFP with the oligodendroglial marker CAII (Figure 4u) and lack of colabeling with NeuN (Figure 4t). Similarly, intrahippocampal infusion of AAV8-MBPEGFP resulted in transduction of oligodendrocytes within the hilus (Figure  4v–x). Transgene expression observed following rh43MBP-EGFP infusion was less  widespread than seen with AAV8, and was not solely ­oligodendroglial—following intrastriatal infusion, EGFP fluorescence was observed in oligodendrocytes only in the immediate vicinity of the injection site, although immunohistochemistry showed that astrocytes within the striatum were also transduced (Figure 5c). Intrahippocampal infusion of rh43-MBPEGFP resulted in no visible EGFP fluorescence and immunostaining detected transgene expression only within a subpopulation of neurons within the dentate gyrus (Figure 5d). Detection of EGFP transgene expression in the corpus callosum, a region rich in glia but devoid of neurons, further demonstrated that AAV8- and rh43derived expression cassettes under the control of the MBP promoter result in transduction of oligodendrocytes (Figure 5e,f). Results presented in Figures 6 and 7 show the extent of transduction obtained with each vector following intrastriatal infusion. The volume of EGFP-immunoreactivity and the number of transduced neurons, astrocytes, and oligodendrocytes within the striatum were quantified using stereological methods. Infusion Figure 5  Immunohistochemical detection of EGFP transgene in the hippocampus and striatum following infusion of AAV8 and rh43 vectors driven by CAG, CMV, GFAP, and MBP promoters. Images have been taken on the periphery of the transduced area (rather than the area of maximal transduction) in order to observe the morphology of transgene-expressing cells. Use of rh43-GFAP-EGFP (3 × 1010 viral genomes) resulted in widespread astrocytic transduction in both the (a) hippocampus and (b) striatum, and extending into (b) corpus callosum (CC). Within the striatum, infusion of rh43-MBP-EGFP resulted in transduction of oligodendrocytes, although some (c) astrocytes were also transduced. When infused into the hippocampus, a population of hippocampal neurons adjacent to the (d) granule cell (GC) layer was transduced. Examination of transgene expression in the corpus callosum further showed that use of the MBP promoter resulted in oligodendroglial transgene expression with both (e) rh43 and (f) AAV8. Bar = (a,c-f) 50 µm; (b) 100 µm. (g) Low magnification views of both ipsilateral and contralateral EGFP expression in hippocampus of AAV8and rh43-injected brains. Upper panel: use of AAV8-CAG resulted in extensive ipsilateral neuronal transduction, confirmed by the detection of significant contralateral fiber staining. In contrast, EGFP transgene expression in contralateral fibers was negligible in AAV8-GFAP-injected tissue, despite extensive ipsilateral transgene expression, confirming further that the majority of transduced cells are astrocytes. Similar observations were made with rh43-CAG and rh43-GFAP injected tissue (lower panel). Bar = 200 µm. AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; str, striatum.

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Figure 6 The effect of promoter on the amount of EGFP transgene expression with AAV8 and rh43. EGFP under control of the CAG, CMV, GFAP, or MBP promoters was packaged into AAV8 and rh43, titer matched and injected into the striatum or hippocampus. Low magnification views of the striatum or hippocampus show the extent of transduction obtained with (a) AAV8 and (b) rh43 driven by the various promoters at a dose of 3 × 1010 vg. When injected at a lower dose (4.5 × 109 vg), the extent of transgene expression with (c) MBP and GFAP promoters was reduced. (d) Transduction volume (mm3) in the striatum following infusion of 3 × 1010 vg AAV8 and rh43 vectors driven the CAG, CMV, GFAP, and MBP promoters. Every 12th section was used to measure the transduction volume according to the Cavalieri estimator. Transduction volumes did not vary between promoters or between serotypes. Bars represent mean + SEM, n = 3 per treatment. AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; vg, viral genomes.

of 3 × 1010 vg of AAV8 resulted in widespread striatal transduction regardless of the promoter used (Figure 6a,d). The volume of transduction obtained following injection of an equivalent dose of rh43 was not significantly less than observed with AAV8 (Figure 6b,d); however, the total number of transduced cells and density of transduction obtained with rh43 were less than observed with AAV8 for both the GFAP and MBP promoters (Figure 7a–c). For each serotype and promoter used, the proportion of neurons, astrocytes, and oligodendrocytes was calculated as a percentage of the total number of cells transduced. These results (Figure 7d) confirm that use of the GFAP promoter resulted in predominantly astrocytic transduction (AAV8-GFAP: 88% of total transduced 1698

cells were astrocytes; rh43-GFAP: 93%, astrocytes), whereas use of the MBP promoter resulted in predominantly oligodendroglial transduction (AAV8-MBP: 91% of transduced cells were oligodendrocytes; rh43-MBP: 82%, oligodendrocytes). Infusion of lower titers (4.5 × 109 vg, as used in the previous study) driven by the GFAP and MBP promoters resulted in more localized transduction (Figure 6c) with both serotypes.

Discussion Nonhuman primate–derived AAVs are attractive candidates for use as human gene therapy vehicles because they can potentially overcome the problem of preexisting immunity33 against human www.moleculartherapy.org vol. 17 no. 10 oct. 2009

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AAV serotypes. In this study, the transgene expression obtained following injection of new nonhuman primate AAV serotypes bb2, cy5, rh20, rh39, and rh43 directly into brain tissue was compared to that obtained with AAV8—a nonhuman primate–derived AAV that has previously been found to perform well in mammalian brain.27,34 The vector dose (4.5 × 109 vg per injection) used in the current study was selected because pilot experiments with AAV8

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at this titer showed widespread striatal and hippocampal transduction, whereas use of a higher titer (3 × 1010 vg) resulted in significant spread outside the target area (Figure 1c,d compared with Figure  6a). Additionally, it was important to observe transduction with the new serotypes at low titer and exclude the possibility that differences between the serotypes would be indistinguishable because of saturating conditions. The amount of transgene expression observed following infusion of AAV8-CAG-EGFP into adult rodent brain was comparable to that observed previously using this serotype and promoter,26,28,35 and was predominantly neuronal, although differences in titer and vector purification method32 must also be taken into account. Infusion of an equivalent dose of cy5, rh20, and rh39 driven by the CAG promoter resulted in more widespread transduction than obtained with AAV8, and the phenotype of cells transduced by these serotypes was neuronal. Despite cy5, rh20, and rh39 transducing a significantly larger striatal volume than AAV8, only use of rh20 resulted in a greater number of transduced cells within the striatum suggesting that although cy5 and rh39 spread further than AAV8, the density of transduction obtained may be lower than AAV8. A recent report suggesting increased immune reaction following use of higher titer viruses35 highlights the benefit of attaining maximal transgene expression with the lowest vector titer possible—our results suggest that cy5, rh20, and rh39 could be useful as human gene therapy vehicles in disorders where widespread neuronal transgene expression is advantageous. Notably, at this titer, infusion of rh43-CAG-EGFP resulted in a minimal amount of transduction, in accordance with the only previous report of rh43 infusion into the adult rat brain.32 However, where detectable, transgene expression was exclusively astrocytic. The observed propensity of rh43-CAG-EGFP to transduce astrocytes, along with the observation that AAV8-CAGEGFP transduced a small number of astrocytes (as also previously observed in refs. 28, 29), led us to investigate whether the use of cell-specific promoters in these two serotypes would enhance transgene expression in glial cells. The results show that it is possible to transduce specific glial cell populations by varying the promoter in these serotypes—use of the GFAP promoter resulted in widespread astrocytic transduction; use of the MBP promoter resulted in oligodendroglial transduction, although this was more readily observed following intrastriatal infusion of AAV8 where

CAG CMV GFAP MBP

AAV8

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rh43

Figure 7 Density of transduction following intrastriatal infusion of AAV8 and rh43. (a) Higher magnification views of the striatum show the density of transduction obtained following infusion of 3 × 109 viral genomes of AAV8 and rh43 driven by the CAG, CMV, GFAP, and MBP promoters. (b) The total number of transduced cells within the striatum was counted using unbiased stereological techniques. Use of rh43 driven by the GFAP and MBP promoters resulted in transduction of fewer cells than obtained with AAV8 vectors driven by the same promoters (GFAP rh43 < AAV8, P = 0.041; MBP rh43 < AAV8, P = 0.046). (c) The number of cells transduced per mm3 of striatum with each promoter was calculated—use of rh43 resulted in a lower density of transduction than AAV8 when the GFAP or MBP promoters were used (GFAP rh43 < AAV8, P = 0.03; MBP rh43 < AAV8, P = 0.008). (d) For each serotype and promoter used, the proportion of neurons, astrocytes, and oligodendrocytes was calculated as a percentage of the total number of cells transduced. Bars represent mean ± SEM, n = 3 per treatment. AAV, adeno-associated virus; CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein. *P ≤ 0.05.

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spread of vector to the corpus callosum resulted in widespread EGFP expression in this oligodendrocyte-rich region. However, obtaining widespread glial cell transduction required the infusion of a high dose of vector (3 × 1010 vg). Brains injected with lower titers (4.5 × 109 vg) showed less widespread glial transduction (Figure 6c,d). Importantly, use of these promoters resulted in only low-level neuronal transgene expression accompanying the observed glial transduction. Although the ability to transduce glial cells with AAV in vitro has been shown previously,36,37 this has not translated into reliable, widespread transduction of these cell types in vivo. Infusion of AAV2 driven by the GFAP promoter did not appreciably alter that serotype’s neuronal tropism in favor of astrocytic transduction.18 Likewise, previous attempts to restrict AAV2-mediated transgene expression to oligodendrocytes have been undertaken with limited success—AAV2 driven by the MBP promoter resulted in oligodendroglial transduction; however, this was not widespread.17,38 AAV4 preferentially targets a specific subpopulation of subventricular zone astrocytes.39 AAV1 and AAV8 driven by the CAG or CMV promoters have been observed to transduce a small number of astrocytes,28,29,40 as has AAV5.28 Although marked astrocytic transduction was observed with AAV5-CAG-GFP (22% of transduced cells were astrocytes), this transduction was not limited to astrocytes (58% of transduced cells were still neurons), and the overall amount of transduction with AAV5 was less than seen with AAV8 (ref. 28). Some newer serotypes (hu32, pi2, hu48R3, and rh8) have recently been reported to transduce both astrocytes and oligodendrocytes41 of the corpus callosum and external capsule, when under the control of the CMV promoter. However, outside these areas, transduced cells were predominantly neuronal. Additionally, although this study measured spread of vector genomes by in situ hybridization, EGFP transgene expression itself was not quantified, so it is difficult to compare the level of glial transgene expression obtained with that of our own study. AAV9 has also recently been reported to result in widespread transduction of astrocytes in brain and spinal cord following systemic (intravascular) delivery.42 Although this noninvasive method could be used to obtain widespread astrocytic transduction, focal gene delivery, as can be achieved by intraparenchymal injection of AAV8 or rh43, is not possible. Also, transduction of organs outside the CNS, as may be expected following infusion via the tail vein, was not discussed. Another factor that may influence transduction (in addition to serotype and promoter) is purity of the vector stock. As was recently demonstrated,32 vector stocks prepared by CsCl density gradients (as used in refs. 28, 41) may contain significant levels of contaminating proteins resulting in altered tropism profiles when compared to iodixanol density gradient–purified stocks. Of note, stocks in the current study were all purified concurrently by iodixanol density gradient and were free from visible protein contamination as visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue detection (Supplementary Figure S1). This suggests that the variations in tropism result from differences in promoter rather than purification method and contaminant levels. It is becoming evident that astrocytes contribute to the pathogenesis of neurodegenerative disorders14–16 and may be an ideal cellular target for therapeutic genes. The ability to efficiently transduce oligodendrocytes will be useful in developing treatments for 1700

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disorders such as Canavan disease—this has been limited by the fact that AAV preferentially targets neurons.43 In conclusion, this study is the first report of infusion of bb2, cy5, rh20, and rh39 into adult rat brain —the observation that these serotypes transduce a larger volume of brain tissue than AAV8 confirms their potential as gene therapy agents, especially as widespread transduction was obtained with a relatively low vector dose. The ability to alter the tropism of both rh43 and AAV8 by varying the cellular promoter means that reliable widespread transduction of glial cell populations is possible and expands the potential utility of AAV to treatment of diseases with glial pathology.

Materials and Methods Vector production. EGFP was cloned into an AAV expression plasmid under the control of the CAG (hybrid CMV-chicken β-actin) promoter and containing woodchuck hepatitis virus post-transcriptional regulatory element, and bovine growth hormone polyadenylation signal flanked by AAV2 inverted terminal repeats. Human embryonic kidney 293 cells were co-transfected with three plasmids—AAV plasmid, appropriate helper plasmid encoding rep and cap genes, and adenoviral helper pF Δ6—using standard CaPO4 transfection. Cells were harvested 60 hours following transfection, cell pellets lysed with sodium deoxycholate and AAV vectors purified from the cell lysate by ultracentrifugation through an iodixanol density gradient, then concentrated and dialyzed against phosphate-buffered saline (PBS), as previously described.8 Vectors were titered using real-time PCR (ABI Prism 7700; Applied Biosystems, Foster City, CA), and purity of vector stocks was confirmed by running a 10 µl sample on sodium dodecyl sulfate polyacrylamide gel electrophoresis and staining with Coomassie blue. For the initial study comparing transduction volume obtained with each serotype, all vector stocks were diluted to 1.5 × 1012 vg/ml and 3 µl injected into each site (total of 4.5 × 109 vg per injection). For the promoter analysis study using AAV8 and rh43, constructs and vectors were made as above, with the CAG promoter replaced by either the 560 bp human CMV (immediate-early promoter), 2,200 bp human GFAP (generously provided by M. Brenner; GenBank accession number M67446) or 1,350 bp mouse MBP (GenBank accession number M24410) promoter. Vectors were titer matched to 1 × 1013 and 1.5 × 1012 vg/ml, and 3 µl injected into the striatum or hippocampus (giving 3 × 1010 and 4.5 × 109 vg per injection, respectively). Infusion of vectors. Animal studies were approved by the University of

Auckland Animal Ethics Committee. 250–300 g male Sprague–Dawley rats were used for all studies. Animals were anesthetized with sodium pentobarbital (75 mg/kg, intraperitoneal) and placed in a Kopf stereotaxic frame. AAV vectors were infused unilaterally into the brain using the following stereotaxic coordinates: hippocampus: flat skull—anteriorposterior 4.0 mm, medial-lateral 2.1 mm, and dorsal-ventral 4.3 mm from skull surface, bregma = zero; striatum: anterior-posterior +1.4 mm, medial-lateral 2.5 mm, dorsal-ventral 5.5 mm; SN: anterior-posterior −5.2 mm, medial-lateral 2.4 mm, dorsal-ventral 8.1 mm. Three microliters of vector was infused at 70 nl/minute. Following infusion, the needle was left in place for 5 minutes prior to being slowly retracted from the brain. Animals were killed 3 weeks after AAV infusion. Three to five animals were injected per vector per injection site. Immunohistochemistry. Rats were euthanized with pentobarbital and perfused transcardially with 60 ml saline followed by 60 ml 4% paraformaldehyde in 0.1 mol/l phosphate buffer. Brain tissue was post-fixed for 24 hours in 4% paraformaldehyde, cryoprotected in increasing concentrations (10, 20, and 30%) of sucrose in PBS and cut into 40 µm free-floating sections using a cryostat. Alternate sections were selected for immunohistochemistry as described below, or mounted for examination of native GFP fluorescence. www.moleculartherapy.org vol. 17 no. 10 oct. 2009

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Immunostaining for EGFP was done according to the following protocol. Sections were washed in 1× PBS containing 0.2% Triton (PBS-T), and incubated in 1% H2O2 in 50% methanol for 30 minutes to bind endogenous peroxidase present in the tissue. Sections were washed extensively in 1× PBS-T. Two hundred microliters of primary antibody (anti-GFP, ab290; Abcam, Cambridge, UK) diluted 1:20,000 in immunobuffer (1× PBS-T containing 1% normal goat serum, 0.4 mg/ml Thimerosal) was applied overnight at room temperature on a rocking table. The following day, sections were washed in 1× PBS-T and incubated in 200 µl of biotinylated anti-rabbit (diluted 1:250 in immunobuffer; Sigma) for 3 hours at room temperature. Following further washes in 1× PBS-T, sections were incubated in 200 µl ExtrAvidin Peroxidase (diluted 1:250 in immunobuffer; Sigma) for 2 hours at room temperature. Sections were washed in PBS-T and antibody binding was visualized using either 3′, 3-diaminobenzidine (0.5 mg/ml in 0.1 mol/l phosphate buffer with 0.01% H2O2; Sigma, St Louis, MO) or Vector VIP substrate kit (as per manufacturer’s instructions; Vector Labs, Burlingame, CA). In sections used for stereological cell counting in the promoter comparison study, the biotin/avidin amplification step was omitted to reduce the density of immunostaining and enable individual cells to be distinguished. Following incubation with anti-GFP, these sections were instead incubated in horseradish peroxidase–conjugated anti-rabbit (1:250; Chemicon, Temecula, CA) for 3 hours at room temperature and antibody binding visualized using Vector VIP substrate kit. Fluorescent immunolabeling: Once the area of maximal EGFP transgene expression had been identified, sections were selected for immunostaining with antibodies to the following phenotypic markers: anti-NeuN (to detect neurons; 1:2000; Chemicon), anti-GFAP (astrocytes; 1:2000; Sigma), CAII (oligodendrocytes; 1:1000; S Ghandour). Sections were hydrogen peroxidetreated and primary antibodies applied overnight as detailed above. Sections were then washed extensively in PBS-T and the appropriate fluorescent Cy3conjugated secondary antibody (Jackson Labs, West Grove, PA) applied at 1:250 in immunobuffer for 3 hours at room temperature. Sections were again extensively washed in PBS-T prior to mounting onto slides. Stereology. The volume of brain tissue transduced was quantified ste-

reologically using the Cavalieri estimator in Stereo Investigator 7 (MBF Bioscience, Willeston, VT). The area within the target structure containing EGFP-positive immunoreactivity was outlined and markers placed at a grid size of 100 µm to estimate the area of transduction within each section. The area in every 12th 40 µm section was measured (4–11 sections per brain measured, depending on brain structure and vector), then averaged and multiplied by the rostrocaudal distance between the first and last sections to give an estimate of transduction volume. The number of cells transduced within the striatum was quantified for each serotype using unbiased stereological techniques. The number of immunoreactive cell bodies within the transduced area of the striatum was determined for every 12th 40 µm section (4–9 sections per brain) using a ×40 objective and 100 µm counting frame, and the total number of transduced cells within the striatum calculated using the Optical Fractionator probe in Stereo Investigator. For each brain, the total number of cells transduced was divided by the total transduction volume to determine the mean number of cells transduced per mm3 of striatal tissue.

Statistics. Results were analyzed by analysis of variance with Fisher’s

least significant difference used for post hoc comparisons (SPSS; SPSS, Chicago, IL), with significance set at P < 0.05.

Supplementary Material Figure S1.  SDS-PAGE analysis of AAV8 and rh43 vectors.

Acknowledgments We thank Guang-Ping Gao and the Gene Therapy Program Vector Core, Department of Medicine, University of Pennsylvania for providing adeno-

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associated virus packaging plasmids. We thank Dahna Fong (Department of Molecular Medicine and Pathology, the University of Auckland) and Henry Waldvogel (Department of Anatomy with Radiology, the University of Auckland) for assistance. This work was funded by the NZ Foundation for Research Science and Technology, the Gus Fisher Charitable Trust, and the US National Institutes of Health (NS44576).

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