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Neuroscience Letters 576 (2014) 73–78

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Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection Jesse R. McLean 1 , Gaynor A. Smith 1 , Emily M. Rocha, Melissa A. Hayes, Jonathan A. Beagan, Penelope J. Hallett, Ole Isacson ∗ Neuroregeneration Institute, McLean Hospital/Harvard Medical School, Belmont, MA, USA

h i g h l i g h t s • • • • •

Injection of rAAV9 in neonatal mice exhibits high transduction patterns in the CNS. Ubiquitous promoter usage and varying tropism limit cell-type-specific targeting. We use the hSYN1 promoter to drive neuron-specific GFP expression. We identify GFP expression in CNS and PNS neurons after neonatal i.c.v. injection. High transduction is also observed in neuronal populations vulnerable to disease.

a r t i c l e

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Article history: Received 24 March 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 29 May 2014 Keywords: AAV9 Neurodegeneration Gene delivery ALS Parkinson’s disease Alzheimer’s disease

a b s t r a c t Adeno-associated viral (AAV) gene transfer holds great promise for treating a wide-range of neurodegenerative disorders. The AAV9 serotype crosses the blood–brain barrier and shows enhanced transduction efficiency compared to other serotypes, thus offering advantageous targeting when global transgene expression is required. Neonatal intravenous or intracerebroventricular (i.c.v.) delivery of recombinant AAV9 (rAAV9) have recently proven effective for modeling and treating several rodent models of neurodegenerative disease, however, the technique is associated with variable cellular tropism, making tailored gene transfer a challenge. In the current study, we employ the human synapsin 1 (hSYN1) gene promoter to drive neuron-specific expression of green fluorescent protein (GFP) after neonatal i.c.v. injection of rAAV9 in mice. We observed widespread GFP expression in neurons throughout the brain, spinal cord, and peripheral nerves and ganglia at 6 weeks-of-age. Region-specific quantification of GFP expression showed high neuronal transduction rates in substantia nigra pars reticulata (43.9 ± 5.4%), motor cortex (43.5 ± 3.3%), hippocampus (43.1 ± 2.7%), cerebellum (29.6 ± 2.3%), cervical spinal cord (24.9 ± 3.9%), and ventromedial striatum (16.9 ± 4.3%), among others. We found that 14.6 ± 2.2% of neuromuscular junctions innervating the gastrocnemius muscle displayed GFP immunoreactivity. GFP expression was identified in several neuronal sub-types, including nigral tyrosine hydroxylase (TH)-positive dopaminergic cells, striatal dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)-positive neurons, and choline acetyltransferase (ChAT)-positive motor neurons. These results build on contemporary gene transfer techniques, demonstrating that the hSYN1 promoter can be used with rAAV9 to drive robust neuron-specific transgene expression throughout the nervous system. © 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

∗

Corresponding author at: Neuroregeneration Institute, McLean Hospital/Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA. Tel.: +1 617 855 3283; fax: +1 617 855 3284. E-mail addresses: [email protected], [email protected] (O. Isacson). 1 These authors contributed equally to this work.

Adeno-associated viral (AAV) gene delivery holds great promise for treating a wide-range of neurodegenerative disorders [1]. Recombinant AAV (rAAV) vectors may be tailored for long-term, cell-type-specific transgene expression or gene knockdown with minimal cytotoxic effects [2]. AAV vectors pseudotyped with natural or engineered capsid serotypes have been used to facilitate

http://dx.doi.org/10.1016/j.neulet.2014.05.044 0304-3940/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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varied transduction efficiencies and tropism throughout the nervous system [3,4]. In recent years, AAV-mediated gene transfer has been successfully used to assess or treat preclinical models of neurodegenerative disease [5] and human clinical efficacy trials have been demonstrated for Parkinson’s disease (PD) [6,7] and Alzheimer’s disease [8]. While several AAV serotypes efficiently cross the blood–brain barrier [9], transduction efficiency and cellular tropism is varied, making gene transfer to the nervous system a challenge. Historically, intraparenchymal injections have been the most widely used delivery routes, allowing for direct targeting of a circumscribed population of cells. This technique is particularly useful for assessing region-specific effects in adult animals, but relies on invasive, stereotaxic manipulation and is associated with limited AAV diffusion from the injection site. Recent studies have utilized the trans blood–brain barrier capacity and high transduction rate of the AAV9 serotype to facilitate widespread transgene expression throughout the nervous system by intravenous (i.v.) or intracerebroventricular (i.c.v.) injection in neonatal rodents [10–13]. This combinatorial approach is increasingly used to model various neurobiological processes and as a therapeutic tool for treating neurodegeneration [14,15], most notably in mouse models of amyotrophic lateral sclerosis (ALS) [16] and spinal muscular atrophy (SMA) [17–22]. Neonatal i.v. or i.c.v. injection studies have, to date, used AAV9 vectors containing strong, ubiquitous promoters, such as cytomegalovirus (CMV), CMV/chicken ␤-actin (CMV/␤-actin), or phosphoglycerate kinase, to drive robust transgene expression. These promoters are strongly active in both neurons and nonneuronal cells, which may be advantageous for targeting non-cell autonomous toxicity [23]. However, as the utility of this technique grows, cell-type-specific transgene expression would offer a more tailored approach for modeling and targeting neurodegenerative processes. Interestingly, these promoters have helped identify an age-dependent range of AAV9 cellular tropism in neonatal mice [11,13]; here, reductions in neurotropism are observed in favor of astroglial transduction as early as 48 h after birth. While the reason(s) for these age-dependent variations in AAV9 tropism remain unknown, they present a challenge for cell-type-specific transduction, and, thus may limit the effectiveness of targeted therapeutics. In the present study, we use the human synapsin 1 (hSYN1) promoter to drive widespread, neuron-specific expression of green fluorescent protein (GFP) throughout the nervous system after rAAV9 neonatal i.c.v. injection. 2. Materials and methods 2.1. AAV9 production and preparation Single-stranded rAAV9 viruses expressing GFP under the hSYN1 promoter (∼495 bp) and human growth hormone first (hGH) intron enhancer (AAV9–hSYN1–GFP) were produced by Virovek as previously described [24]. For neonatal injections, AAV9–hSYN1–GFP was diluted to ∼2 × 1012 vg/mL in filtered (0.22 ␮M) Phosphate Buffered Saline (PBS) with magnesium chloride and calcium chloride containing 0.01% FastGreen dye (Sigma–Aldrich). 2.2. Neonatal i.c.v. delivery All animal procedures were approved by McLean Hospital’s Institutional Animal Care and Use Committee. Neonatal injections were performed under cryoanesthesia as previously described [25,26]. Pups were injected with 3.5 ␮L AAV9–hSYN1–GFP into the lateral ventricle of each hemisphere, located 1 mm lateral to the superior sagittal sinus and 2 mm rostral to the transverse sinus,

to a depth of 2 mm (Fig. 1A). Injections were performed with a 33-gauge, 30◦ beveled needle (point style 4) and gastight syringe retrofit with a Neuros adaptor and blind stop (Hamilton). Injection efficiency was monitored by dissociation of FastGreen dye throughout the lateral ventricles. 2.3. Immunofluorescence Injected mice were terminally anaesthetized at 6 weeks-of-age with sodium pentobarbital (130 mg/kg, i.p.) and transcardially perfused with ice-cold 0.9% heparinized saline, followed by a second perfusion with 4% paraformaldehyde (PFA) in PBS. Tissues were dissected, post-fixed in 4% PFA in PBS for 6 h, and cryoprotected in 30% sucrose in PBS for storage at 4 ◦ C. Serial sections were cut at 40 ␮m thickness on a freezing sledge microtome or cryostat and stored in antifreeze (30% glycerol, 30% ethoxyethanol, and 40% PBS) at −20 ◦ C. Free-floating sections were washed in PBS, blocked in 10% normal donkey serum in PBS for 1 hr at room temperature, and incubated in primary antibodies [anti-GFP (Aves Labs, 1:1000); anti-GFAP (Chemicon, 1:1000); anti-Iba-1 (Wako, 1:1000); anti-NeuN (Millipore, 1:1000); anti-peripherin (Millipore, 1:1000); anti-TH (Millipore, 1:500); anti-DARPP-32 (Cell Signaling, 1:1000); anti-ChAT (Millipore, 1:500); and anti-NF-H (Millipore, 1:500)] in blocking solution overnight at 4 ◦ C. Sections were washed in PBS and incubated with Alexa Fluor secondary antibodies (Life Technologies, 1:300) in PBS and applied for 2 h at room temperature. For detection of gastrocnemius neuromuscular junctions (NMJs), ␣-bungarotoxin conjugated to tetramethylrhodamine (Life Technologies, 1:500) in PBS was applied at this step. Sections were washed in PBS and coverslipped on slides with aqueous mounting media. High magnification fluorescent images are presented as 10–15 ␮M z-stacks taken with a Zeiss LSM 510 Confocal Microscope (Carl Zeiss), while whole brain and spinal cord images were captured at 4× magnification and stitched together with a Keyence BZ-9000 Fluorescence Microscope (Keyence). 2.4. Immunoblotting Injected mice were terminally anaesthetized at 6 weeks-of-age with sodium pentobarbital (130 mg/kg, i.p.) and transcardially perfused with ice-cold 0.9% heparinized saline. Select brain sections were immediately dissected using a McIlwain Tissue Chopper (Ted Pella), while the spinal cord, DRG, and gastrocnemius muscle were removed intact. Tissues were homogenized in 2% sodium dodecyl sulfate (SDS) in PBS containing 1× HALT Protease Inhibitors (Pierce Biotechnology) and boiled for 5 min. Protein samples (10–15 ␮g) were separated on Criterion 4–15% SDS polyacrylamide gels (BioRad) and transferred to polyvinyldifluoride membrane. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.2% Tween-20 for 1 h at room temperature, then incubated in primary antibodies [anti-GFP (Aves Labs, 1:2500) and anti-NSE (Abcam, 1:5000)] in blocking solution overnight at 4 ◦ C. Antibody binding was revealed using horseradish peroxidase conjugated IgG and visualized by chemiluminescence using the ChemiDoc XRS+ (Bio-Rad). 2.5. Quantification Viral transduction was quantified from brain regions corresponding to anterior–posterior coordinates relative to Bregma [27]: motor and somatosensory cortex (layer V) and dorsolateral and ventromedial striatum, 0.9–0.7 mm; hippocampus (CA1), −2.2 to −2.4 mm; substantia nigra pars compacta and reticulata, −3.0 to −3.3 mm; cerebellum (central or ansiform lobes), −6.1 to −6.5 mm. For spinal cord, the ventral horns of well-transduced cervical and lumbar sections were quantified. Anti-NeuN was used to

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Fig. 1. Neonatal i.c.v. injection of AA9-hSYN1-GFP shows widespread transgene expression in 6-week-old mouse brain and spinal cord. P2 neonatal pups were injected with AAV9–hSYN1–GFP into each lateral cerebral ventricle (A). Schematic of the rAAV vector shows GFP under the control of the hSYN1 promoter and hGH first intron enhancer (B). Low magnification immunofluorescent images show GFP expression (green) throughout most brain regions and in the spinal cord. A negative control (FCF dye only) is the last section in the series (C). High magnification images of select brain regions using anti-NeuN (red) and anti-GFP (green) antibodies show strong, neuron-specific expression of GFP in motor cortex (D/D ), hippocampus (E/E ), striatum (F/F ), substantia nigra (G/G ), cerebellum (H/H ), and lumbar spinal cord (I/I ). Mice injected with FastGreen dye only did not show GFP expression (J/J ; motor cortex shown). Panels indicated by  are higher magnification insets of the dashed boxes above. Abbreviations: V, cortical layer 5; C, pars compacta; CA-1, cornu ammonis-1; R, pars reticulata; VL, ventrolateral; VM, ventromedial; VR, ventral root. Staining with anti-GFAP (K; red, arrowheads) and anti-Iba-1 (L; red, arrowheads) antibodies show that astrocytes and microglia did not express GFP (green), respectively. Scale bars are 200 ␮M. Neuronal transduction efficiency was quantified by co-localization of NeuN (or NF-H for cerebellum, not shown) with GFP (n = 6–13; M). Western blots (N) and corresponding densometric quantification (n = 6; O) shows GFP expression differs among neuronal regions when normalized to NSE. Data presented as mean ± SEM.

identify neurons, except for the cerebellum and muscle, where NF-H and ␣-bungarotoxin were used, respectively. We performed simple immunofluorescent counts using StereoInvestigator software (MBF Bioscience). Neuronal transduction rates (presented as

mean ± S.E.M; n = 6–13) were calculated by dividing the number of NeuN- or NF-H-positive neurons expressing GFP by the total number of NeuN- or NF-H-positive neurons from z-stack projections of the aforementioned brain and spinal cord regions. Similar counts

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Fig. 2. Neonatal i.c.v. injection of AAV9–hSYN1–GFP transduces neurons in 6-weekold mouse PNS. Immunofluorescent images show strong GFP expression (green) in PNS neurons, including DRG, indicated by NeuN (red; A/A ), sciatic nerve, indicated by peripherin (red, B/B ), and gastrocnemius muscle NMJs (arrowheads) and their axons (arrows), indicated by ␣-bungarotoxin (red) and peripherin (blue), respectively (C/C ). Panels indicated by  are higher magnification insets of the dashed boxes above. Scale bars are 200 ␮M (A/A –B/B ) and 40 ␮M (C).

were performed to evaluate transduction rates in DARPP-32-, TH-, or ChAT-positive cells (n = 3). Western blot images were analyzed by ImageJ (NIH) using the integrated density function after background subtraction (n = 6); here, the values representing each GFP band were divided by the values of the corresponding NSE loading control to obtain relative protein expression. 3. Results The lateral ventricles of P2 neonatal mice were injected with 3.5 ␮L of ∼2 × 1012 vg/mL AAV9–hSYN1–GFP per hemisphere (Fig. 1A). The rAAV9 vector contains the hSYN1 promoter and hGH first intron enhancer upstream of the GFP open reading frame (Fig. 1B). We previously used the hSYN1 promoter to drive neuronspecific transgene expression in the substantia nigra of adult rats after intraparenchymal injection with rAAV2 [28]. In the current study, widespread GFP expression was observed throughout the CNS, including the forebrain, midbrain, hindbrain, and spinal cord 6 weeks after injection (Fig. 1C). At higher magnification, neuronspecific GFP expression was identified from its co-localization with NeuN in the motor cortex (Fig. 1D/D ), hippocampus (Fig. 1E/E ), striatum (Fig. 1F/F ), substantia nigra (Fig. 1G/G ), cerebellum (Fig. 1H/H ), and spinal cord (Fig. 1I/I ). NF-H was used as the neuronal marker in the cerebellum (not shown), where the anti-NeuN antibody does not recognize some neurons. GFP immunoreactivity was not observed in the cortex when injected with FastGreen dye only (Fig. 1J/J and C, back image). GFP expression was observed only in NeuN- or NF-H-positive cells and not in astrocytes or microglia, which were identified by anti-GFAP (Fig. 1K, arrowheads) and anti-Iba-1 (Fig. 1L, arrowheads) antibodies, respectively. Neuronal transduction rates (mean ± S.E.M; n = 6–13), quantified from specific regions, were as follows: 43.5 ± 3.3% and 32.4 ± 3.7% in the motor and somatosensory cortices, respectively; 43.1 ± 2.7% in the hippocampus; 16.8 ± 4.5% and 16.9 ± 4.3% in the dorsolateral and ventromedial striatum, respectively; 22.5 ± 4.2% and 45.9 ± 5.4% in the substantia nigra pars compacta and reticulata, respectively; 29.6 ± 2.3% in the cerebellum; and 24.9 ± 3.9% and 20.9 ± 2.6% in the ventral horns of the cervical and lumbar spinal cord, respectively (Fig. 1M). Region-specific transgene expression was further analyzed by immunoblotting (Fig. 1N). Densometric analysis of GFP expression, relative to the neuron-specific reference protein, NSE, revealed differences among the regions examined (n = 6). The

order of GFP expression among these regions, from highest to lowest, were as follows: hippocampus > cortex > striatum > ventral midbrain > cerebellum > lumbar spinal cord > cervical spinal cord (Fig. 1O). We identified that AAV9–hSYN1–GFP neonatal i.c.v. injections showed neuron-specific GFP expression in the PNS. GFP expression was observed in DRG neurons (Fig. 2A/A ) and in nerve bundles of the sciatic nerve (Fig. 2B/B ). GFP immunoreactivity was further observed in 14.6 ± 2.2% of NMJs in the gastrocnemius muscle, which were identified by staining with ␣-bungarotoxin (Fig. 2C, arrowheads), and along the length of their innervating axons (Fig. 2C, arrows). The surrounding muscle cells did not show GFP immunoreactivity. Given the widespread neuron-specific transduction pattern associated with AAV9–hSYN1–GFP neonatal i.c.v. injection, we looked at whether GFP was expressed in neuronal sub-types relevant to some neurodegenerative diseases, including Huntington’s disease (HD), PD, and ALS. GFP expression co-localized with DARPP32-positive medium spiny neurons in the ventromedial striatum (Fig. 3A, upper panel, arrowheads), TH-positive dopaminergic neurons in the substantia nigra pars compacta (Fig. 3A, middle panel, arrowheads), and ChAT-positive motor neurons in the lumbar spinal cord (Fig. 3A, lower panel, arrowheads). Here, transduction rates (n = 3) were as follows: 11 ± 2.1% of DARPP-32 neurons; 15.9 ± 3% of TH neurons; and 40.8 ± 7.3% of ChAT neurons (Fig. 3B).

4. Discussion Neonatal i.v. or i.c.v. gene transfer using AAV9 vectors is growing in popularity due to the unprecedented rates of transgene expression obtained in the adult nervous system. This is largely due to AAV9’s higher transduction rates and more widespread biodistribution [2,29,30] when compared to other serotypes that also cross the blood–brain barrier [9,13]. An interesting finding to emerge from AAV9 neonatal injections was that neurons, astrocytes, and vascular endothelia are transduced at varying degrees throughout the nervous system [11]. The viral-receptor mechanisms underlying these transduction patterns remain unknown, but, in general, greater neurotropism is observed for injection at P0-1, while enhanced astrocytic tropism is observed for injections at P2 and older [13]. Other explanations may also account for these age-dependent changes, including route of administration, viral titer and purification, and AAV diffusion, but because these studies employed the use of general promoters, such as CMV and CMV/chicken-␤-actin, transgene expression may occur in a noncell-specific manner [31]. We previously used the hSYN1 promoter to drive rAAV2-mediated expression of human mutant ␣-synuclein into the substantia nigra of rats in a model of ␣-synucleinopathy in PD [28]. In that study, the hSYN1 promoter offered strong, protracted neuron-specific activity up to 17 weeks, whereupon the rats were sacrificed due to significant dopaminergic neuron loss associated with disease. Others have reported hSYN1 promoter-driven expression after 2.5 and 9 months in vivo after intraparenchymal injection using lentiviral or adenoviral vectors, respectively [32,33]. In the current study, we show that the hSYN1 promoter can be used with rAAV9 to achieve high neuron-specific transduction rates in the CNS and in sciatic nerve, DRG, and NMJs after neonatal injection at P2, an age associated with preferential astrocytic tropism [13]. While neonatal i.c.v. injections with rAAV9 using general promoters are associated with varied cellular tropism, and hence some neuronal expression is expected, we did not observe GFP expression in astrocytes or microglia, thus demonstrating that the hSYN1 promoter confers neuronal specificity. The transduction rates reported here are appreciable when compared to other AAV9 studies, but since the methodological approach to gene

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Fig. 3. Multiple neuronal sub-types are transduced by neonatal i.c.v. injection of AAV9–hSYN1–GFP. Immunofluorescent images show GFP expression in DARRP-32-positive medium spiny neurons of the striatum (upper panels, arrowheads), in TH-positive dopaminergic neurons of the substantia nigra pars compacta (middle panels, arrowheads), and in ChAT-positive motor neurons of the lumbar spinal cord (lower panels, arrowheads). The arrows in the upper panels show GFP expression in traversing nerve fibers. Abbreviations: C, pars compacta; R, pars reticulata; VM, ventromedial; VR, ventral root. Scale bar is 200 ␮M. Neuronal transduction efficiency was quantified by co-localization of DARPP-32, TH, or ChAT with GFP (n = 3; B). Data presented as mean ± SEM.

transfer often differs (serotype, promoter, injection route, etc.), direct comparisons among these studies are difficult. Despite this, a similar approach to the current study was recently reported by Dirren and colleagues, who used the minimal glial fibrillary acidic protein promoter (gfaABC1D) to restrict expression to astrocytes [34], thus highlighting the growing interest in cell-type specific expression after neonatal injection. Neonatal i.v. and i.c.v. injections using rAAV9 are increasingly used to model or treat neurodegenerative disease in rodents, notably for the motor neuron diseases, ALS and SMA. A rat model of ALS was recently created by i.v. delivery of rAAV9 expressing human TDP-43 and a pathological fragment, TDP-25, emulating the phenotype observed in TDP-43 transgenic models [35,36]. In the mutant (G93A) human superoxide dismutase (SOD1) mouse model of ALS, increased survival and reduced disease

progression were observed after neonatal i.v. injection of rAAV9 encoding shRNA targeting human SOD1 [16]. In the SMN7 and SMN2 mouse models of SMA, dramatic extensions in survival and motor function were demonstrated after neonatal i.v. or i.c.v. delivery of exogenous survival motor neuron protein (SMN) [17–22]. Interestingly, gene therapy in SMA mice was associated with survival variability when the rAAV9 was injected at different postnatal ages [18,21]. Given that age-dependent differences in cellular tropism are associated with AAV9 neonatal injection, this suggests that cell-type-specific targeting may be important for effective gene therapy during narrow therapeutic windows. Another point to emerge from rAAV9-mediated gene therapy in SMA mice was that i.c.v. injections were associated with longer survival than i.v. injections [19], suggesting that, in some models, restriction of rAAV9 within the nervous system may be beneficial. Although the use of

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general promoters may be helpful for global gene targeting, celltype-specific promoters may help tailor this expression to augment therapeutic potential. In the current study, we show GFP expression in DARPP-32-positive medium spiny neurons of the striatum, THpositive neurons of the substantia nigra, and ChAT-positive motor neurons of the spinal cord, which are vulnerable neuronal populations in HD, PD, and ALS, respectively. Future studies will warrant the examination of cell-type specific promoters with different serotypes and delivery routes. For example, Levites and colleagues utilized the strong transduction capacity of AAV1-CMV/␤-actin to deliver recombinant anti-A␤ single-chain variable fragments to P0 CRND8 transgenic mice in an anti-A␤ immunotherapy model of Alzheimer’s disease [37]. In addition, the development of neuron-specific shRNA promoters, for example, a hybrid between hSYN1 and polymerase III U6/H1 promoters, would significantly aid current gene therapy studies using RNAi. Because these diseases are associated with non-cell autonomous toxicity to some degree [23], the use of cell-type-specific promoters to overexpress or knockdown genes-of-interest will help identify disease-related processes and/or refine prospective therapies. Acknowledgements This work was supported by a Department of Defense grant, W81XWH-11-1-0708 (O.I.). We thank Professors Claudio Hetz (University of Chile) and Nicholas Mazarakis (Imperial College London) for helpful discussions. References [1] F. Mingozzi, K.A. High, Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges, Nat. Rev. Genet. 12 (5) (2011) 341–355. [2] C. Zincarelli, S. Soltys, G. Rengo, J.E. Rabinowitz, Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection, Mol. Ther. 16 (6) (2008) 1073–1080. [3] D.F. Aschauer, S. Kreuz, S. Rumpel, Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain, PLoS ONE 8 (9) (2013) e76310. [4] C. Burger, O.S. Gorbatyuk, M.J. Velardo, C.S. Peden, P. Williams, S. Zolotukhin, P.J. Reier, R.J. Mandel, N. Muzyczka, Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system, Mol. Ther. 10 (2) (2004) 302–317. [5] T.J. McCown, Adeno-associated virus (AAV) vectors in the CNS, Curr. Gene Ther. 11 (3) (2011) 181–188. [6] R.T. Bartus, T.L. Baumann, J. Siffert, C.D. Herzog, R. Alterman, N. Boulis, D.A. Turner, M. Stacy, A.E. Lang, A.M. Lozano, C.W. Olanow, Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients, Neurology 80 (18) (2013) 1698–1701. [7] G. Mittermeyer, C.W. Christine, K.H. Rosenbluth, S.L. Baker, P. Starr, P. Larson, P.L. Kaplan, J. Forsayeth, M.J. Aminoff, K.S. Bankiewicz, Long-term evaluation of a phase 1 study of AADC gene therapy for Parkinson’s disease, Hum. Gene Ther. 23 (4) (2012) 377–381. [8] M.S. Rafii, T.L. Baumann, R.A. Bakay, J.M. Ostrove, J. Siffert, A.S. Fleisher, C.D. Herzog, D. Barba, M. Pay, D.P. Salmon, Y. Chu, J.H. Kordower, K. Bishop, D. Keator, S. Potkin, R.T. Bartus, A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer’s disease, Alzheimers Dement. (2014). [9] H. Zhang, B. Yang, X. Mu, S.S. Ahmed, Q. Su, R. He, H. Wang, C. Mueller, M. Sena-Esteves, R. Brown, Z. Xu, G. Gao, Several rAAV vectors efficiently cross the blood–brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system, Mol. Ther. 19 (8) (2011) 1440–1448. [10] S. Duque, B. Joussemet, C. Riviere, T. Marais, L. Dubreil, A.M. Douar, J. Fyfe, P. Moullier, M.A. Colle, M. Barkats, Intravenous administration of selfcomplementary AAV9 enables transgene delivery to adult motor neurons, Mol. Ther. 17 (7) (2009) 1187–1196. [11] K.D. Foust, E. Nurre, C.L. Montgomery, A. Hernandez, C.M. Chan, B.K. Kaspar, Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes, Nat. Biotechnol. 27 (1) (2009) 59–65. [12] M.R. Haddad, A. Donsante, P. Zerfas, S.G. Kaler, Fetal brain-directed AAV gene therapy results in rapid, robust, and persistent transduction of mouse choroid plexus epithelia, Mol. Ther. Nucleic Acids 2 (2013) e101. [13] P. Chakrabarty, A. Rosario, P. Cruz, Z. Siemienski, C. Ceballos-Diaz, K. Crosby, K. Jansen, D.R. Borchelt, J.Y. Kim, J.L. Jankowsky, T.E. Golde, Y. Levites, Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain, PLoS ONE 8 (6) (2013) e67680.

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