WO2010071454A1 - Adeno-associated viral vectors and uses thereof - Google Patents

Abstract

The present invention relates to viral vectors and in particular to adeno-associated viral vectors with enhanced gene transfer capabilities into cells of the central nervous system (CNS). The invention has been developed primarily for gene transfer in glial cells and further for the treatment of diseases associated with glial cell pathology.

Description

Adeno-associated viral vectors and uses thereof

TECHNICAL FIELD

The present invention relates to viral vectors and in particular to adeno- associated viral vectors with enhanced gene transfer capabilities into cells of the central nervous system (CNS).

The invention has been developed primarily for gene transfer in glial cells and further for the treatment of diseases associated with glial cell pathology. However, it will be appreciated that the invention is not limited to this particular field of use.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Recombinant adeno-associated viral (AAV) vectors are derived from nonpathogenic, replication-deficient members of the Parvovirus family and are efficient at transducing the non-dividing cells of the central nervous system. Following infusion of AAV into the brain, stable, long-lasting neuronal transgene expression can be achieved, with no apparent toxicity [1-3]. AAV vectors are versatile tools, allowing up-regulation or knock-down 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 ultimately as vehicles for gene therapy treatment of these disorders [10-13]. Treatment of neurodegenerative diseases by gene therapy may require AAV vectors capable of transducing large brain structures from a single injection site. Additionally, the ability to target transgene expression to non-neuronal cell populations would be useful. For example, transduction of the entire striatum, hippocampus or substantia nigra (SN) would be advantageous for developing therapies for Huntington's,

Alzheimer's or Parkinson's disease. Astrocytes have traditionally been considered as merely neuronal support cells, however it is becoming evident that astrocytes contribute to the pathogenesis of neurodegenerative disorders [14-16]. Given that both neuronal loss and astroglial proliferation are common characteristics of these neurodegenerative disorders, the ability to target transgene expression to astrocytes may be useful when considering cell targets in design of 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 AAV to glial cells by varying the cellular promoter has resulted in limited transduction only [17, 18]. Chen et al., investigated the feasibility of cell type specific gene expression in oligodendrocytes as a possible therapeutic approach for demyelinating diseases. This was achieved using recombinant adeno associated viral vectors (rAAV) carrying the green fluorescent protein (GFP) gene driven either by the myelin basic protein (MBP) promoter or the cytomegalovirus (CMV) promoter. Overall the study showed that transduction with the CMV promoter was inefficient, even when the vector was injected into white matter. Cells transduced by rAAV-CMV-GFP appeared to be primarily neuronal. This in vivo study also revealed that using rAAV-MBP-GFP, GFP expression in mouse brains was in the corpus callosom in cells typically resembling oligodendrocytes. This expression was very limited (Le. not many cells and not widespread) and occurred in cells arranged in rows along nerve fibres. There was no detectable expression in astrocytes. The authors make the point that they only see GFP expression in white matter but see no expression in oligodendrocytes in gray matter. It was concluded by the authors that it would be useful to have a vector having the ability to show widespread gene and cell specific expression. In another study, Feng et al investigated the biological and pathological function of ApoE in Alzheimer's Disease. The comparison was made between recombinant Adenovirus (rAd) and recombinant adeno associated virus (rAAV) with respect to time course, expression, immune activity and cell type specificity of vectors. rAAV was found to be less immunogenic than rAD. The authors were able to show cell type specific expression of ApoE in astrocytes when driven by the GFAP promoter. In this transgenic mouse model for Alzheimer's Disease, expression of ApoE using an AAV vector was shown to be long term and stable, more so than expression of ApoE when using an Adenoviral vector. The authors proposed that such a vector expression system could provide a helpful tool for understanding the function of ApoE expression in Alzheimer's Disease. Comments made above in relation to Chen et al are also relevant to this reference. Some transgene expression was achieved but there is no indication on how far it spread.

Pre-clinical data on AAV-mediated gene transfer in the CNS has largely been based on 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 are easily transduced, but some hippocampal neurons are refractory to AAV2 transduction [22, 23]. Infusion of AAV2 driven by the GFAP promoter did not appreciably alter that serotype's neuronal tropism in favour of astrocytic transduction [18]. In other studies, attempts to restrict transgene expression to oligodendrocytes by manipulation of the promoter has been undertaken with limited success [17, 38]. Use of AAV2 driven by the MBP promoter resulted in oligodendroglial transduction, however this was not widespread.

Clinical application of AAV2 may also be limited by pre-existing immunity to AAV2 in most humans [24] [25]. Delivery of AAVl, 5, 7, 8 and 9 [26-29] into adult rodent brain has been shown to result in greater numbers of transduced neurons and more wide-spread transgene expression than achieved with AAV2. However transduction with these serotypes is still overwhelmingly neuronal - targeting and widespread transduction of glial cell populations is still not possible.

AA V4 preferentially targets a specific population of astrocytes, those of the sub- ventricular zone destined for the rostral migratory stream, [39] but not a broader population of glial cells. AAVl 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 astrocytic transduction with AAV5-CAG-GFP has been observed (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 [28].

Some newer serotypes (hu32, pi2, hu48R3 and rh8) have recently been reported to transduce both astrocytes and oligodendrocytes [41] of the corpus callosum and external capsule, when under the control of the CMV promoter. However, it was noted that 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.

AAV9 has also recently been reported to result in widespread transduction of astrocytes in brain and spinal cord following systemic (intravascular) delivery. Although this non-invasive 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 can be expected following infusion via the tail vein, is undesirable. Thus there is a need for alternative AAV vectors and techniques that enable broader cellular targets, capable of evading pre-existing immunity to AAV2.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a purified adeno- associated viral (AAV) vector stock comprising a vector having a cell specific promoter, wherein the vector preferably transduces non-neuronal brain tissue.

According to a second aspect, the present invention provides a purified AAV vector comprising a promoter that enhances transduction of non-neuronal brain tissue.

According to a third aspect, the present invention provides use of a vector according to the first or second aspect for gene therapy.

According to a fourth aspect, the present invention provides a method of preparing a purified AAV vector stock for preferential non-neuronal brain tissue transduction comprising purifying a vector having a cell specific promoter, such that transduction is preferably of non-neuronal cells.

According to a fifth aspect, the present invention provides a method of preparing an AAV vector stock capable of preferential non-neuronal brain cell transduction, comprising the steps: (a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.

According to a sixth aspect, the present invention provides a AAV vector stock prepared by the method of the fourth or fifth aspects. Preferably, the non-neuronal brain tissue comprises glial cells such as astrocytes and/or oligodendrocytes.

The promoter is preferably a cell specific promoter, selected from the group consisting of GFAP, MBP , adenosine kinase, aspartoacylase promoters, JC virus early promoter, SlOOB, vimentin, CAR2, CD44, GLUL, PDGFRA, RLBPl, SLCl A3 or parts thereof, for any gene which is highly or relatively specifically expressed within glial or subglial populations.

Preferably the adeno-associated viral vector may be derived from AAV serotypes including cy5, rh20, rh39, rh43 and AAV8. A vector such as AAV9 may also be used in the methods of the present invention, as described herein.

Preferably, transduction of brain tissue occurs in vivo.

The adeno-associated viral vector is preferably able to evade pre-existing immunity.

Preferably, the adeno-associated viral vector is recombinant. Preferably, the adeno-associated viral vector is non-human.

More preferably, the adeno-associated viral vector is of primate origin.

Preferably, the brain tissue is human brain tissue.

According to a seventh aspect there is provided a purified AAV vector according to the first or second aspect, further comprises a therapeutic gene or a sequence which reduces expression of a specific target gene by use of RNA interference (short hairpin RNA, micro RNA), antisense or ribozyme sequences.

Preferably, the gene is selected from the group consisting of, but not limited to neuropeptide Y (NPY), excitatory amino acid transporter 2 (EAAT2) and glutamine synthetase. Target genes where RNA interference, antisense or ribozyme sequences would be used to reduce gene expression would include adenosine kinase, ion channels (potassium and calcium), water channels (AQP4), glutamate receptors, inflammatory genes (e.g. cytokines like interleukin or interferon), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), aspartoacylase (ASPA) or any gene functioning abnormally in an astrocyte and/or an oligodendrocyte in diseased brain. According to an eighth aspect the invention provides use of a purified AAV vector according to the first or second aspects for the preparation of a medicament for therapeutic or prophylactic treatment of a neurological disorder and/or a neurodegenerative disease by gene therapy.

Preferably the neurological disorder and/or neurodegenerative disease is associated with glial cell pathology. The neurological disorder and/or a neurodegenerative disease may be selected from, but not limited to, Alzheimer's disease, Huntington's disease,

Parkinson's disease, Canavan disease, amyotrophic lateral sclerosis, spinal cord disease or injury, spinal muscular atrophy, epilepsy, multiple sclerosis and leukodystrophies. Preferably, the brain cells are glial cells such as astrocytes and/or oligodendrocytes. Preferably, the brain cells are human brain cells.

According to a ninth aspect the present invention provides a method of therapeutic or prophylactic treatment of a neurological disorder and/or a neurodegenerative disease by administering to a subject in need thereof an AAV vector according to the first or second aspects, wherein the neurological disorder and/or a neurodegenerative disease is selected from spinal muscle atrophy, Alzheimer's disease, Huntington's disease, Parkinson's disease, epilepsy, Canavan disease, amyotrophic lateral sclerosis, spinal cord disease or injury, multiple sclerosis and leukodystrophies. Preferably the neurological disorder is epilepsy and/or depression however it will be understood that the present invention is not limited to these disorders.

Preferably the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis but it will be understood that the present invention is not limited to these disorders. According to a tenth aspect the present invention provides a method for selecting an AAV vector that preferentially transduces non-neuronal brain cells, comprising the steps of:

(a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.

According to an eleventh aspect the present invention provides a method for selecting an AAV vector that transduces s desired brain cell type, comprising the steps of:

(a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that transduces the desired brain cell type.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions.

In this document, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: EGFP transgene expression following intrastriatal, intrahippocampal and intranigral infusion of vectors. Rats injected with AAV vectors were killed 3 weeks following infusion and brains processed immunohistochemically for detection of EGFP. (A) Transduction volume (mm³) following vector infusion. Every 12^th section was used to measure the transduction volume according to the Cavalieri estimator [44, 45, 46]. Overall, transduction volumes observed following infusion of cy5, rh20 and rh39 were greater than that achieved with AAV8 in all structures (striatum: ANOVA PO.001; LSD post-hoc cy5>AAV8, p=0.002; rh20>AAV8, pO.OOl, 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). Use of bb2 resulted in a larger striatal transduction volume than that observed with AAV8 but this transduction was restricted to one neuronal sub-type. Transduction with bb2 in the hippocampus and SN was more restricted than that seen with AAV8. Use of rh43 resulted in restricted astrocytic expression in the striatum and no measurable transduction in the hippocampus or SN at this titer. Bars represent mean + SEM, n=3-5 per treatment. (B) The number of sections containing EGFP immunoreactive staining was used to estimate the rostro-caudal spread (mm) of each vector. This was significantly different between the serotypes only in the striatum (ANOVA P=O.003; cy5>AAV8 p= 0.009; rh20>AAV8 p=0.001 ; rh39>AAV8 p=0.009). 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 x 10⁹ vg of each serotype. Transduction obtained with AAV8 was wide-spread throughout the striatum. More wide-spread transduction was observed following infusion of cy5, rh20 or rh39, but transduction was not confined to the target area, extending into the cortex. Minimal transduction of striatum was obtained with bb2 or rh43. (D) The extent of transduction obtained by infusing 4.5 x 10⁹ vg of each serotype into the hippocampus. Following infusion into the hippocampus, transduction with AAV8 was wide-spread throughout the principal cell layers of the ipsilateral hippocampus, with some fibre staining evident in the contralateral hippocampus. More wide-spread ipsilateral transduction was obtained with cy5, rh20 and rh39, with intense fibre staining and some cell bodies in the contralateral hemisphere. (E) Transduction of the SNpc following infusion of 4.5 x 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. Wide-spread transduction of neurons of the SNpc was obtained following infusion of AAV8. Greater transduction of SNpc neurons was observed with cy5, rh20 and rh39 although transduction was not confined to target area, extending upwards to the thalamus. Minimal transduction was seen with bb2 and rh43.

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 ^χ 10⁹ viral genomes of each serotype. (B) The total number of transduced cells within the striatum was counted using unbiased stereo logical 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.

Figure 3: Infusion of new serotypes cy5, rh20 and rh39 resulted in transduction of neurons within the striatum (A), hippocampus (C, D) and SN (B). Transduction following bb2 infusion was detectable only by use of immuno histochemistry using anti- GFP (E). Infusion of 4.5 x 10⁹ vg of rh43 into the striatum (F) resulted in low-level astrocytic transduction. Infusion of 3 x 10¹⁰ vg of rh43 into the striatum (G) and hippocampus (H) resulted in increased numbers of transduced astrocytes, along with transduction of neurons. (I) Infusion of 4.5 x 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.

Figure 4: Altering the promoter changed the tropism of AAV 8 and rh43 in the striatum and hippocampus. Examples shown are AAV8 (3 x 10¹⁰ vg). Use of the CAG promoter (A-F) resulted in wide-spread neuronal EGFP expression with transduced cells morphologically consistent with a neuronal phenotype - cells had a rounded soma with multi-polar axonal and dendritic projections (A, D). This was confirmed by co- localisation of EGFP transgene and the neuronal marker NeuN within cells (B, E; co- labelled cells appear yellow), and lack of co-localisation between EGFP and the astrocytic marker GFAP, (C, F). Use of AAV 8 with the CMV promoter resulted in predominantly neuronal transduction (G, H, J, K) with minimal astrocytic transduction (I, L). Use of the GFAP promoter resulted in wide-spread astrocytic transduction (M, P) - these cells had large soma surrounded by multiple, highly-branched processes, however these processes were shorter than those observed on neurons. These cells were confirmed to be astrocytes by the lack of EGFP/NeuN co-localisation in (N, Q) and the large number of GFP/GFAP co-labelled cells, appearing yellow (O, R). Use of the MBP promoter resulted in oligodendroglial transduction within the striatum and hippocampus (S, V). Transduced cells had smaller cell bodies than either neurons or astrocytes with no cellular processes, and EGFP expression co-localised with the oligodendroglial marker CAII (U, X), rather than with NeuN (T, W). Scale bar on v = 20μm (applies to A, D, G, J, M, P, S). Scale bar on x = 50μm (applies to B, C, E, F, H, I, K, L, N, O, Q, R, T, U, W).

Figure 5: Immunohistochemical detection of EGFP transgene in the hippocampus and striatum following infusion of AAV8 and rh43 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 x 10¹⁰ vg) resulted in wide-spread astrocytic transduction in both the hippocampus (A) and striatum (B), and extending into corpus callosum (CC) (B). Within the striatum, infusion of rh43 -MBP-EGFP resulted in oligodendroglial transduction, although some astrocytes were also transduced (C). When infused into the hippocampus, a population of hippocampal neurons adjacent to the granule cell (GC) layer were transduced. Examination of transgene expression in the corpus callosum (an area rich in oligodendrocytes) farther showed that use of the MBP promoter resulted in oligodendroglial transgene expression with both rh43 (E) and AAV8 (F). Scale bars: A, C-F: 50μm; B lOOμm.

(G) Low magnification views of both ipsi- and contra-lateral EGFP expression in hippocampus of AAV8- and rh43-injected brains. Upper panel - use of AAV8-CAG resulted in extensive ipsilateral neuronal transduction, confirmed by the detection of significant contralateral fibre staining. In contrast EGFP transgene expression in contralateral fibres 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). Scale bar = 200μm. AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; str, striatum.

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 AAV8 (A) and rh43 (B) driven by the various promoters at a dose of 3 x 10¹⁰ vg. When injected at a lower dose (4.5 x 10⁹ vg), the extent of transgene expression with the MBP and GFAP promoters was reduced, (C). Transduction volume (mm³) in the striatum following infusion of 3 x 10¹⁰ vg AAV8 and rh43 vectors driven the CAG, CMV, GFAP, and MBP promoters (D). 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. Figure 7: Density of transduction following intrastriatal infusion of AAV 8 and rh43. (A) Higher magnification views of the striatum show the density of transduction obtained following infusion of 3 x 10⁹ viral genomes (vg) of AAV 8 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 stereo logical 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.

Figure 8: Immunohistochemical detection of EGFP transgene in the striatum and hippocampus following infusion of AAV9 vectors driven by CAG and GFAP and promoters. (A) Infusion of AAV9-CB A-GFP resulted in widespread (low mag) neuronal transduction (high mag) in the striatum, whilst infusion of AAV9-GF AP-GFP resulted in widespread astrocytic transgene expression. 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. (B) Low magnification views of both ipsi- and contra-lateral EGFP expression in hippocampus of AAV9-injected brains. Upper panel - use of AAV9-CAG resulted in extensive ipsilateral neuronal transduction, confirmed by the detection of significant contralateral fibre staining. In contrast EGFP transgene expression in contralateral fibres was negligible in AAV9-GFAP-injected tissue, despite extensive ipsilateral transgene expression, confirming further that the majority of transduced cells are astrocytes. DESCRIPTION OF THE PREFERRED EMBODIMENT

The present inventors have for the first time demonstrated the influence of promoters on tropism of AAV. This was in part enabled by novel purification techniques in the preparation of the AAV stocks. The present inventors have also for the first time demonstrated the infusion of cy5, rh20 and rh39 into the adult rodent brain and observed that these serotypes transduce a larger volume of brain tissue than AAV8, which is currently considered the best serotype for widespread transduction of brain parenchyma. Furthermore, the present inventors have demonstrated that AAV having cell specific, rather than constitutively active promoters, specifically target non-neuronal brain cells i.e. glial cells. These findings confirm the potential of these serotypes as both therapeutic agents and for disease modelling and functional genomics studies.

The ability of the AAV vectors of the present invention to transduce a broader range of cell targets within brain tissue is in part based on the AAV vector stock purification and packaging adopted herein. Whereas prior art techniques use live adenovirus for AAV production (and thus may result in AAV vector stocks contaminated with residual adenovirus), the methods of the present invention make use of a helper virus-free production method. Further, the prior art methods make use of CsCl density ultracentrifugation to purify the AAV vector stocks, which results in vector stocks heavily contaminated with proteins other than AAV particles and hence high potential for creating artefacts. The problem with CsCl (and sucrose gradients) is that they are hyperosmotic at the densities used to band viruses, and need to be diluted out of the vector before use in an animal. A great deal of work is required to remove contaminating particles from the final vector stock, with loss of vector particles with every additional purification step undertaken. Additionally, use of CsCl tends to result in viral vectors with reduced infectivity. The methods of the present invention make use of iodixanol density gradient-purified stocks. Iodixanol is less toxic and is easier to remove from the final vector stock than CsCl or sucrose.

Non-human primate derived AAVs are attractive candidates for use as human gene therapy vehicles because they can potentially overcome the problem of pre-existing immunity against human AAV serotypes.

In this study, transgene expression obtained following injection of recently isolated non-human primate AAV serotypes bb2, cy5, rh20, rh39 and rh43 directly into brain tissue was compared to that obtained with AAV8 - a non-human primate-derived AAV, that has previously been found to perform well in mammalian brain [27,34].

Titer-matched vector stocks encoding the EGFP reporter driven by the constitutive CAG promoter were injected into the hippocampus, striatum, or SN of adult rats. The vector dose (4.5 x 10⁹ vg per injection) used for comparative purposes in the current study, although lower than that used in some previous reports, was selected because the pilot experiments with AAV8 at this titer showed wide-spread striatal and hippocampal transduction, whereas use of a higher titer (3 xlO¹⁰ vg) resulted in significant spread outside the target area (Fig. 1C, D compared with Fig. 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 results show wide-spread neuronal transduction following infusion of 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 enhanced for both rh43 and AAV8 by use of cell-specific, rather than constitutively active, promoters. The results show marked alterations in AAV8 and rh43 tropism following use of the glial flbrilliary acidic protein (GFAP) and myelin basic protein (MBP) promoters, allowing targeted and wide-spread transduction of selected glial cell populations.

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 predominatly neuronal, although differences in injection titer and vector purification method [32] must also be taken into account.

Infusion of an equivalent dose of cy5, rh20 and rh39 driven by the CAG promoter resulted in more wide-spread transduction than obtained with AAV8, and the phenotype of cells transduced by these serotypes was neuronal. The titer used for comparative purposes in the current study was relatively low compared to some previous reports - pilot experiments with AAV8 at this titer showed wide-spread striatal and hippocampal transduction, whereas use of a higher titer (3 xlO¹⁰ vg) resulted in significant spread outside the target area (compare Fig. 1C with Fig. 3A). 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. Thus there is a benefit of attaining maximal transgene expression with the lowest vector titer possible - the use of novel serotypes with higher transduction efficiency addresses this issue by potentially allowing the lowering of viral vector load without detrimental effects on transgene expression. AAV8 driven by the CAG promoter results in predominantly neuronal transduction in the striatum, hippocampus and substantia nigra. The phenotype of cells transduced by cy5, rh20 and rh39 was exclusively neuronal. Notably at this titer, infusion of rh43- CAG-EGFP resulted in a minimal amount of transduction and where detectable, transgene expression was astrocytic. The observed propensity of rh43-CAG-EGFP to transduce astrocytes, along with the observation that AAV8-CAG-EGFP transduced a small number of astrocytes, led to the investigation of whether the use of cell-specific promoters in these two serotypes would enhance transgene expression in glial cell populations. The results show that it is possible to transduce specific glial cell populations by changing the promoter. Use of the GFAP promoter resulted in widespread astrocytic transduction with both serotypes. Use of the MBP promoter resulted in wide-spread oligodendroglial transduction, although this was more readily observed following intrastriatal infusion of AAV 8 where 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 x 10⁹ vg) showed less widespread glial transduction (Figure 6 C, D). Importantly, use of these promoters resulted in only low-level neuronal transgene expression accompanying the observed glial transduction.

Importantly, use of these promoters resulted in minimal neuronal expression - transgene expression was almost exclusively glial. However, use of the promoters is not limited to GFAP or MBP. Other promoters such as adenosine kinase, aspartoacylase promoters, JC virus early promoter, SlOOB, vimentin, CAR2, CD44, GLUL, PDGFRA, RLBPl, SLCl A3 or parts thereof, for any gene which is highly or relatively specifically expressed within glial or subglial populations can be used.

Whilst the ability to transduce glial cells with AAV in vitro has been shown previously [36, 37], this has not translated into reliable, wide-spread transduction of these cell types in vivo. It has also been noted that vector stocks prepared by CsCl density gradients may contain significant levels of contaminating proteins resulting in altered tropism profiles when compared to iodixanol density gradient-purified stocks. The study undertaken by Klein et al [26] showed that astroglial transduction was obtained following infusion of AAV8-CMV-GFP purified by CsCl density gradient. However when the same expression cassette was packaged but purified using an iodixanol density gradient as used in the present invention, resulting in higher purity vector stock, transgene expression reverted back to neuronal, suggesting that the tropism was influenced by the presence of contaminants rather than serotype. This clearly indicates that due to the presence of contaminants in earlier studies, the observed astroglial transduction was unlikely to be reproducible. Vector stocks in the current study were all purified concurrently by iodixanol density gradient and were free from visible protein contamination (SDS-PAGE/ Coomassie blue - data not shown) suggesting that the observed variations in tropism resulted from differences in promoter. Thus, if all the vector stocks are purified to a high standard (no contaminating proteins, as it is possible to get with iodixanol purification), and the standard constitutive CAG promoter is used, this allows selection of serotypes (eg. Rh43 and AAV8) that have a natural propensity to transduce astrocytes. Since all iodixanol-purified vector stocks are equally pure, then packaging the same serotype (eg. Rh43) with different promoters allows the contribution of promoter to tropism/transduction to be determined.

Astrocytes have traditionally been considered as merely neuronal support cells, however it is becoming evident that astrocytes contribute to the pathogenesis of neurodegenerative disorders [14, 15, 16] and may be an ideal cellular target for gene therapy, especially given that neuronal loss and astroglial proliferation are common characteristics of neurodegenerative diseases. The ability to genetically manipulate astrocytes in situ means that alternative gene therapy strategies for treatment of neurodegenerative diseases may be explored e.g transgenic neurotrophins may be more effective if secreted from astrocytes. The ability to efficiently transduce oligodendrocytes with AAV8 and rh43 will be useful in developing treatments for leukodystrophies such as Canavan disease - this has been limited by the fact that AAV preferentially targets neurons e.g. AAV-mediated neuronal overexpression of ASPA did not improve pathology or behavioural deficits in a rat model of Canavan disease [43].

Through the studies described herein it has been clearly demonstrated for the first time that infusion of bb2, cy5, rh20 and rh39 into rodent brain results in the transduction of a larger volume of brain tissue than AAV8. These findings further confirm the potential of these serotypes as both therapeutic agents and for disease modelling and functional genomics studies, especially as wide-spread transduction was obtained with a relatively low vector dose. Without wishing to be bound by theory or any particular mechanism of action, the ability to alter the tropism of both AAV8 and rh43 by varying the cellular promoter means that reliable wide-spread transduction of glial cell populations, in the absence of significant neuronal transduction, is possible and expands the potential utility of AAV to treatment of diseases with glial cell pathology. However, it will be understood that neuronal transduction need not be absent, as long as there is significant and/or enhanced astroglial transgene expression. Further, in some specific instances it may be necessary to restrict expression to astrocytes (e.g. astrocyte-specific gene knock down to see what contribution astrocytic gene expression makes to a particular disease). Such a selection is now possible based on the present disclosure.

A preferred embodiment of the invention will now be described in more detail by reference to the following non-limiting examples.

EXAMPLES EXAMPLE 1 : Vector production EGFP (Clontech) was cloned into an AAV expression plasmid (developed by department of Molecular Medicine and Pathology, The University of Auckland, New Zealand) under the control of the CAG (hybrid CMV-chicken β-actin) promoter and containing WPRE (woodchuck hepatitis virus post-transcriptional-regulatory element -J Donello, J Virol (1998) 72:5085-5092), and bovine growth hormone polyadenylation signal flanked by AAV2 inverted terminal repeats (ITRs). The final vectors ends up with a capsid specific to the AAV serotype but has AAV2 ITRs.

HEK293 cells (Microbix) were co-transfected with three plasmids - AAV plasmid, appropriate helper plasmid encoding rep and cap genes, and adenoviral helper pFΔ6 - using standard CaPO₄ transfection (Source of helper plasmids - University of Pennsylvania, James Wilson). Cells were harvested 60 hours following transfection, and cell pellets lysed with 0.5% sodium deoxycholate (Sigma) and 50U/ml Benzonase (Sigma). Cell lysates were clarified by centrifugation at 5000g for 30min at 4⁰C (discard pellet, retain supernatant). AAV vectors were purified from the clarified cell lysate by ultracentrifugation through an iodixanol (Sigma) density gradient as follows: 9ml of cell lysate was loaded into a 34ml tube. This was underlaid with 8.5ml of 15% iodixanol containing IM NaCl in PBS-MK, 6ml of 25% iodixanol in PBS-MK, 5ml of 40% iodixanol in PBS-MK and 5ml of 54% iodixanol in PBS-MK. The gradient was subjected to ultracentrifugation at 243,00Og for 90min at 18⁰C. A needle and syringe was stuck through the side of the tube and 4ml of iodixanol containing AAV removed and diluted with 12ml of PBS-MK. This was concentrated down to lOOul using a 4ml 100,000MWCO concentrator (Millipore). The concentrated AAV vector was washed with PBS-MK, removed from the concentrator and sterilised using a 0.2um 13mm syringe filter. [8]. Vectors were titered using real-time PCR (ABI Prism 7700) and purity of vector stocks was confirmed by running a lOμl sample on SDS-PAGE and staining with Coomassie blue.

For the initial study comparing transduction volume obtained with each serotype, all vector stocks were diluted to 1.5 x 10¹² viral genomes (vg)/ml) and 3μl injected into each site (total of 4.5 x 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 560 bp CMV (human cytomegalovirus immediate-early promoter), 2,200 bp GFAP (human glial fibrilliary acidic protein, generously provided by M. Brenner; GenBank accession number M67446) or 1,350 bp MBP (mouse myelin basic protein; GenBank accession number M24410) promoter. Vectors were titer-matched to 1 x 10¹³ and 1.5 x 10¹² vg/ml and 3ul injected into the striatum or hippocampus (giving 3 x 10¹⁰ and 4.5 x 10⁹ vg per injection respectively). EXAMPLE 2: Infusion of vectors Animal studies were approved by The University of Auckland Animal Ethics

Committee. 250-30Og male Sprague-Dawley rats were used for all studies. Animals were anaesthetized with sodium pentobarbitone (75mg/kg, i.p.) and placed in a Kopf stereotaxic frame. AAV vectors were infused unilaterally into the brain using the following stereotaxic co-ordinates: Hippocampus: flat skull - anterior-posterior (AP) - 4.0mm, medial-lateral (ML) 2.1mm, and dorsal- ventral (DV) 4.3mm from skull surface, bregma = zero); striatum AP +1.4mm, ML 2.5mm, DV -5.5mm; substantia nigra AP - 5.2mm, ML 2.4mm, DV 8.1mm. 3μl vector was infused at 70nl/min. Following infusion the needle was left in place for 5min prior to being slowly retracted from the brain. Animals were killed three weeks after AAV infusion. Three-five animals were injected per vector per injection site.

EXAMPLE 3: Immunohistochemistry

Rats were euthanised with pentobarbitone and perfused transcardially with 60ml saline followed by 60ml 10% neutral buffered formalin (Sigma) (4% paraformaldehyde in 0.1 mo 1/1 phosphate buffer may also be used). Brain tissue was post-fixed for 24h in 10% neutral buffered formalin (or 4% paraformaldehyde), cryoprotected in increasing concentrations (10, 20, 30%) of sucrose (BDH) in PBS and cut into 40μm free-floating sections using a cryostat. Alternate sections were selected for immunohistochemistry as described below, or mounted for examination of native GFP fluorescence. Immunostaining for EGFP was done according to the following protocol (described in Lawlor et al, 2007, MoI Neurodegener 2:11 ). Sections were washed in IxPBS containing 0.2% Triton (PBS-T), and incubated in I⁰AH₂O₂ in 50% methanol for 30min to bind endogenous peroxidase present in the tissue. Sections were washed extensively in IxPBS-T. 200μl of primary antibody (anti-GFP, Abeam, ab290) diluted 1:20,000 in immunobuffer (IxPBS-T containing 1% normal goat serum, 0.4mg/ml methiolate or thimerosol) was applied overnight at room temperature on a rocking table. The following day sections were washed in IxPBS-T and incubated in 200μl of biotinylated anti-rabbit (Sigma; diluted 1 :250 in immunobuffer) for 3hr at room temperature. Following further washes in IxPBS-T, sections were incubated in 200μl ExtrAvidin Peroxidase (Sigma; diluted 1 :250 in immunobuffer) for 2 hr at room temperature. Sections were washed in PBS-T and antibody binding was visualised using 3', 3-diaminobenzidine (DAB; Sigma, St. Louis, MO) at 0.5mg/ml DAB in 0.1 M phosphate buffer with 0.01% H₂O₂ or vector VIP substrate kit (as per manufacturer's instructions; Vector Labs, Burlingame, CA, USA).

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 immuno labelling: once the area of maximal EGFP transgene expression had been identified, sections were selected for immunostaining with antibodies to the following phenotypic markers (using manufacturers recommended protocols): anti-NeuN (to detect neurons; Chemicon, 1:2000), anti-GFAP (astrocytes; Sigma, 1 :2000), CAII (oligodendrocytes; S Ghandour), 1 :1000). Sections were hydrogen peroxide-treated and primary antibodies applied overnight as detailed above. Sections were then washed extensively in PBS-T and the appropriate fluorescent Cy3 -conjugated secondary antibody (Jackson Labs) applied at 1 :250 in immunobuffer for 3hr at room temperature. Sections were again extensively washed in PBS-T prior to mounting onto slides. EXAMPLE 4: Stereoloev

The volume of brain tissue transduced was quantified stereo logically using the Cavalieri estimator in Stereo Investigator (MicroBrightfield). The area within the target structure containing EGFP-positive immunoreactivity was outlined and markers placed at a grid size of 1 OOμm to estimate the area of transduction within each section. The area in every 12^th 40μm section was measured (4-11 sections per brain measured, depending on brain structure and vector), then averaged and multiplied by the rostro-caudal 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 stereo logical 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 ^χ40 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. EXAMPLE 5: Statistics

Results were analysed by ANOVA with Fisher's LSD used for post-hoc comparisons (SPSS), with significance set at p<0.05. General laboratory procedures not specifically described in this specification can be found in the general molecular biology texts including, for example, Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY. EXAMPLE 6: EGFP transgene expression following intra-striatal, intra-hippocampal and intra-nigral infusion of vectors (Figure 1).

Rats injected with AAV vectors were killed 3 weeks following infusion and brains processed immunohistochemically for detection of EGFP. (A) Transduction volume (mm³) following vector infusion. Every 12^th section was used to measure the transduction volume according to the Cavalieri estimator. Overall, transduction volumes observed following infusion of cy5, rh20 and rh39 with were greater than that achieved with AAV8 in all structures (striatum: ANOVA PO.001; LSD post-hoc cy5>AAV8, p=0.002; rh20>AAV8, pO.OOl, 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). Use of bb2 resulted in a larger striatal transduction volume than that observed with AAV8 but this transduction was restricted to one neuronal sub-type. Transduction with bb2 in the hippocampus and SN was more restricted than that seen with AAV8. Use of rh43 resulted in restricted astrocytic expression in the striatum and no measurable transduction in the hippocampus or SN at this titer. Bars represent mean + SEM, n=3-5 per treatment. (B) The number of sections containing EGFP immunoreactive staining was used to estimate the rostro-caudal spread (mm) of each vector. This was significantly different between the serotypes only in the striatum (ANOVA P=O.003; cy5>AAV8 p= 0.009; rh20>AAV8 p=0.001 ; rh39>AAV8 p=0.009). 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 x 10⁹ vg of each serotype. Transduction obtained with AAV8 was wide-spread throughout the striatum. More wide-spread transduction was observed following infusion of cy5, rh20 or rh39, but transduction was not confined to the target area, extending into the cortex. Minimal transduction of striatum was obtained with bb2 or rh43. (D) The extent of transduction obtained by infusing 4.5 x 10⁹ vg of each serotype into the hippocampus. Following infusion into the hippocampus, transduction with AAV8 was wide-spread throughout the principal cell layers of the ipsilateral hippocampus, with some fibre staining evident in the contralateral hippocampus. More wide-spread ipsilateral transduction was obtained with cy5, rh20 and rh39, with intense fibre staining and some cell bodies in the contralateral hemisphere. (E) Transduction of the SNpc following infusion of 4.5 x 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. Wide-spread transduction of neurons of the SNpc was obtained following infusion of AAV8. Greater transduction of SNpc neurons was observed with cy5, rh20 and rh39 although transduction was not confined to target area, extending upwards to the thalamus. Minimal transduction was seen with bb2 and rh43. EXAMPLE 7: Density of transduction following intrastriatal infusion of new adeno- associated viral (AAV) serotypes (Figure 2). (A) Higher magnification views of the striatum show the density of transduction obtained following infusion of 4.5 x 10⁹ viral genomes of each serotype. (B) The total number of transduced cells within the striatum was counted using unbiased stereo logical 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.

EXAMPLE 7: Infusion of new serotypes cy5, rh20 and rh39 resulted in transduction of neurons within the striatum (A), hippocampus (C, D) and SN (B) (Figure 3). Transduction following bb2 infusion was detectable only by use of immunohistochemistry using anti-GFP (E). Infusion of 4.5 x 10⁹ vg of rh43 into the striatum (F) resulted in low- level astrocytic transduction. Infusion of 3 x 10¹⁰ vg of rh43 into the striatum (G) and hippocampus (H) resulted in increased numbers of transduced astrocytes, along with transduction of neurons. (I) Infusion of 4.5 x 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. EXAMPLE 8: Altering the promoter chenged the tropism of AAV 8 and rh43 in the striatum and hippocampus (Figure 4).

Altering the promoter changed the tropism of AAV 8 and rh43 in the striatum and hippocampus. Examples shown are AAV8 (3 x 10¹⁰ vg). Use of the CAG promoter (A- F) resulted in wide-spread neuronal EGFP expression with transduced cells morphologically consistent with a neuronal phenotype - cells had a rounded soma with multi-polar axonal and dendritic projections (A, D). This was confirmed by co- localisation of EGFP transgene and the neuronal marker NeuN within cells (B, E; co- labelled cells appear yellow), and lack of co-localisation between EGFP and the astrocytic marker GFAP, (C, F). Use of AAV 8 with the CMV promoter resulted in predominantly neuronal transduction (G, H, J, K) with minimal astrocytic transduction (I, L). Use of the GFAP promoter resulted in wide-spread astrocytic transduction (M, P), - these cells had large soma surrounded by multiple, highly-branched processes, however these processes were shorter than those observed on neurons. These cells were confirmed to be astrocytes by the lack of EGFP/NeuN co-localisation in (N, Q) and the large number of GFP/GFAP co-labelled cells, appearing yellow (O, R). Use of the MBP promoter resulted in oligodendroglial transduction within the striatum and hippocampus (S, V). Transduced cells had smaller cell bodies than either neurons or astrocytes with no cellular processes, and EGFP expression co-localised with the oligodendroglial marker CAII (U, X), rather than with NeuN (T, W). Scale bar on v = 20μm (applies to A, D, G, J, M, P, S). Scale bar on x = 50μm (applies to B, C, E, F, H, I, K, L, N, O, Q, R, T, U, W).

EXAMPLE 10: Immunohistochemical detection of EGFP transgene in the hippocampus and striatum following infusion of AAV 8 and rti43 driven by CAG, CMV, GFAP and MBP promoters (Figure 5).

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 x 10¹⁰ vg) resulted in wide-spread astrocytic transduction in both the hippocampus (A) and striatum (B), and extending into corpus callosum (CC) (B). Within the striatum, infusion of rh43 -MBP-EGFP resulted in oligodendroglial transduction, although some astrocytes were also transduced (C). When infused into the hippocampus, a population of hippocampal neurons adjacent to the granule cell (GC) layer were transduced. Examination of transgene expression in the corpus callosum (an area rich in oligodendrocytes) further showed that use of the MBP promoter resulted in oligodendroglial transgene expression with both rh43 (E) and AAV8 (F). Scale bars: a, c-f: 50μm; b lOOμm.

(G) Low magnification views of both ipsi- and contra- lateral EGFP expression in hippocampus of AAV8- and rh43-injected brains. Upper panel - use of AAV8-CAG resulted in extensive ipsilateral neuronal transduction, confirmed by the detection of significant contralateral fibre staining. In contrast EGFP transgene expression in contralateral fibres 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). Scale bar = 200μm. AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; str, striatum.

EXAMPLE 11 : The effect of promoter on the amount of EGFP transgene expression with AAV8 and rh43 (Figure 6). 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 AAV8 (A) and rh43 (B) driven by the various promoters at a dose of 3 x 10¹⁰ vg. When injected at a lower dose (4.5 x 10⁹ vg), the extent of transgene expression with the MBP and GFAP promoters was reduced, (C). Transduction volume (mm³) in the striatum following infusion of 3 x 10¹⁰ vg AAV8 and rh43 vectors driven the CAG, CMV, GFAP, and MBP promoters (D). 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. EXAMPLE 12: Density of transduction following intrastriatal infusion of AAV 8 and rh43 (Figure 7). (A) Higher magnification views of the striatum show the density of transduction obtained following infusion of 3 * 10⁹ viral genomes of AAV 8 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. EXAMPLE 13: Immunohistochemical detection of EGFP transgene in the striatum and hippocampus following infusion of AAV9 vectors driven by CAG and GFAP and promoters (Figure 8). Infusion of AAV9-CAG-GFP resulted in widespread (low mag) neuronal transduction (high mag) in the striatum, whilst infusion of AAV9-GF AP-GFP resulted in widespread astrocytic transgene expression. 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 (A). Low magnification views of both ipsi- and contra- lateral EGFP expression in hippocampus of AAV9-injected brains (B). Upper panel - use of AAV9-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 AAV9-GFAP-injected tissue, despite extensive ipsilateral transgene expression, confirming further that the majority of transduced cells are astrocytes. EXAMPLE 14: Widespread transgene expression following infusion of new serotypes cy5, rh20 and rh39

Vector stocks encoding the EGFP reporter under control of the chicken β- actin/CMV hybrid (CAG) promoter were titer matched to 1.5 x 10¹² genomes/ml and 3μl (total of 4.5 x 10⁹ viral genomes, vg) injected unilaterally into the striatum, hippocampus or SN. Rats were killed three weeks post-infusion and brain tissue examined immunohistochemically for EGFP expression. 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. As seen in Fig. IA-C, use of AAV8 resulted in widespread transduction (~10mm³) within the striatum. Two to three fold higher transduction was observed following infusion of cy5, rh20 or rh39 (ANOVA PO.001; cy5>AAV8, p=0.002; rh20>AAV8, p<0.001, rh39>AAV8, p=0.026; Fig. IA), however transgene expression with these serotypes was not confined to the striatum, extending into the cortex, medial forebrain bundle and septum of the injected hemisphere (Fig. 1C). AAV8 immunoreactivity extended for 2.2mm around the injection site, with cy5, rh20 and rh39 immunoreactivity extending significantly further than this (3.2-3.5mm; ANOVA P=0.003; cy5>AAV8 p= 0.009; rh20>AAV8 p=0.001 ; rh39>AAV8 p=0.009), Fig. IB.

Whilst infusion of bb2 resulted in no visible EGFP fluorescence, however immunohistochemistry for EGFP showed that bb2 -mediated transduction extended over a greater portion of the striatum (~15.5mm³), than observed with AAV8. Although this transduction was wide-spread, it was confined to one neuronal subtype called medium spiny neuron (Fig. 2A, E). As these cells were sparsely spread they were able to be quantified, with an average of 46+/-6 bb2-transduced cells observed per striatal section. Intrastriatal infusion of rh43 resulted in the least transgene expression of all serotypes (Fig. IA-C).

EGFP-positive fibres were observed in striatal projection areas (globus pallidus and SNpr) following AAV8, cy5, rh20 and rh39 infusion. Retrograde transport of vector to the SN was also observed following intra-striatal infusion of AAV8, cy5, rh20 and rh39 with EGFP-immunoreactive cell bodies observed in SNpc. Intra-striatal infusion of bb2 resulted in few positive fibres within the globus pallidus, and no observed transduction of SNpc, consistent with the sparse transduction of neurons observed within the striatum. Use 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 (DG), hilus, CAl, CA2, CA4 - with EGFP- immunoreactive fibres and cell bodies also observed in the contralateral hippocampus. Overall, cy5 and rh39 transduced a significantly larger portion of the hippocampus than AAV8 (ANOVA P=0.006; cy5>AAV8, p=0.028; rh39>AAV8, p=0.017, Fig. IA), with transduced neurons present in all principal layers of the hippocampus (Fig 1 D). Intense staining of fibres 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 - EGFP-positive immunoreactivity extended 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 a small sub-population of neurons, with no fibre staining in the contralateral hippocampus. Infusion of rh43 resulted in minimal transduction (<5 cells transduced throughout the hippocampus) and no volume measurement was possible. There was no difference in rostro-caudal spread between serotypes (P=0.571, Fig. IB), with AAV8, cy5, rh20 and rh39 all spreading a distance of ~4mm around the hippocampal injection site and more limited spread observed with bb2 (3.2mm).

Projection areas for the hippocampus include the nucleus accumbens and septum. EGFP immunoreactivity was not detected in the nucleus accumbens with any serotype. EGFP immunoreactive fibres were observed in both ipsi-and contralateral septum following AAV8 infusion into the hippocampus. This fibre staining was observed to a greater degree with cy5, rh20, and rh39. EGFP-positive immunoreactivity was not observed in these projection areas in bb2 and rh43 -injected brains, in agreement with the sparse hippocampal transduction observed.

As seen in Fig. IE, infusion of AAV 8 resulted in extensive transgene expression in the SN, extending into the thalamus around the needle tract. Infusion of cy5, rh20 and rh39 resulted in greater transgene expression than obtained with AAV8 (Fig. IE) with EGFP-positive immunoreactivity evident throughout the SN and extending into the entire thalamus of the injected hemisphere (ANOVA P=0.021 ; rh20>AAV8, p=0.03; rh39>AAV8, p=0.05, Fig. IA). There was no difference in rostro-caudal spread (ANOVA P=0.977), with infusion of these serotypes resulting in transgene expression extending ~2mm around injection site, Fig. IB. Use of bb2 resulted in transduction of a small number of neurons within the SN, detectable only by GFP immunostaining. Given the minimal nature of the 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. With the exception of rh43, infusion of these serotypes into the striatum, hippocampus and SN resulted in overwhelmingly neuronal transduction. Wide-spread trunsduction, regardless of whether it is neuronal or glial, may also be useful for treatment of some brain disorders. The present invention enables selection of appropriate vectors based on their propensity towards transduction of certain cell types and selection of appropriate promoters to target specific cell types. Without pure vector stocks as described herein such a selection could not be made reproducibly.

Examination of both EGFP fluorescence and EGFP immunostaining showed that transduced cells were morphologically consistent with a neuronal phenotype (examples of cy5, rh20 and rh39 in Fig. 3A-D). The presence of fibre staining in the contralateral hippocampus following hippocampal infusion (Fig. ID) and striatal EGFP expression in SN-injected brains further confirmed the neuronal phenotype of transduced cells (data not shown). Notably bb2 transduced only a sub-type of neuron within the striatum - these cells were determined to be a sub-population of medium spiny neuron (as determined by the observation of dendritic spines on EGFP-immunoreactive cells). 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 the titer used (Fig. 3F) - these cells were morphologically distinct from neurons with large cell bodies and multiple, highly-ramified processes. The observation that intra-striatal infusion of rh43 resulted in exclusive, but limited, astrocytic transduction led to the investigation of whether injecting an increased number of vector genomes would result in more widespread astrocytic transduction. Additional animals were injected with 3 x 10¹⁰ vg of rh43 -CAG-EGFP. However, whilst more widespread astrocytic transduction was observed at this titer, this was accompanied by significant neuronal transduction in both the striatum (Fig. 3G) and hippocampus (Fig. 3H). As discussed above, as long one achieves significant astroglial transgene expression, then some accompanying neuronal transduction will not matter, and may in fact be beneficial. Altered tropism of AAV 8 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 EGFP epifluorescence, only by immunohistochemistry Fig 31) led to the investigation of the effect of varying the promoter on transgene expression in these two serotypes. In order to optimize targeted glial cell transduction, further AAV8 and rh43 vectors were generated, driven by the cell-specific promoters GFAP (glial fϊbrilliary acidic protein) and MBP (myelin basic protein) and compared this to transduction obtained using the constitutive viral promoters, CAG and CMV (cytomegalovirus).

Each vector (3 x 10¹⁰Vg) was infused into the striatum and hippocampus and brain tissue examined immunohistochemically for transgene expression three weeks post-infusion.

The phenotype of transduced cells varied markedly depending on the promoter used. Overall, transduction with AAV8 resulted in wide-spread 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 - co-labelling results presented in Fig. 4 are from AAV8-injected brains. It has been noted previously that immunohistochemical detection of GFP is more sensitive than quantification of visible EGFP fluorescence so whilst the images of visible EGFP fluorescence in Fig. 4 depict the predominant cell type transduced with each promoter, results presented in Fig. 5 show additional transduction of other cell populations detectable only after immunohistochemistry with anti-GFP.

Striatal or hippocampal infusion of 3 x 10¹⁰vg AAV8-CAG-EGFP (Fig 4A-F) resulted in wide-spread neuronal EGFP expression (Fig 4A, D), confirmed by co- localisation of EGFP with the neuronal marker NeuN (Fig. 4B, E), and lack of EGFP/GFAP co-localisation (Fig. 4C, F). Within the hippocampus neuronal transduction was additionally confirmed by the presence of wide-spread contralateral fibre staining (Fig. 5G). As previously mentioned (Fig. 3G, H), at this titer intra-striatal infusion of rh43-CAG-EGFP resulted in wide-spread transduction of both neurons and astrocytes.

Following infusion of AAV8-CMV-EGFP, visible EGFP fluorescence was observed predominantly in neurons (Fig. 4G-L), although immunohistochemical analysis shows some astrocytes were also transduced. Infusion of rh43-CMV-EGFP into the striatum or hippocampus resulted in EGFP transgene expression in both neurons and astrocytes in the immediate vicinity of the injection site. Astrocytic transgene expression driven by the GFAP promoter Use of the GFAP promoter resulted in wide-spread astrocytic transduction with both serotypes. Following AAV8-GF AP-EGFP infusion (Fig 4M-R), EGFP fluorescence was observed in astrocytes only (Fig. 4M, P), confirmed by co-localisation of EGFP with GFAP (Fig. 40, R), and the lack of EGFP/NeuN co-labelling (Fig. 4N, Q). The lack of contralateral fibre staining following intra-hippocampal infusion (Fig. 5) and the lack of EGFP -positive fibres in striatal projection areas further confirm transgene expression driven by the GFAP promoter as predominantly astrocytic.

However, immunohistochemical detection of EGFP-immunoreactive fibres in AAV8-GF AP-EGFP injected tissue indicates that some neurons were also transduced, detectable only by immunohistochemistry. Following rh43-GFAP-EGFP infusion visible fluorescence was less wide-spread than observed with AAV8, however immunohistochemical results shown in Fig. 5 suggest that transduction was almost exclusively astrocytic, confirmed by the lack of EGFP immunoreactive fibres. The high intensity of EGFP immunostaining in the area of maximal transduction means it was not possible to accurately quantify the ratio of astrocytes to neurons transduced by these serotypes. However observation of EGFP-immunoreactive cells on the margins of the transduced area suggest that 60-70% of transduced cells are astrocytes following AAV8 infusion, 80-90% of transduced cells are astrocytes following infusion of rh43. Use of the MBP promoter results in oligodendro glial transduction

Following infusion of AAV8-MBP-EGFP (Fig 4S-X), wide-spread EGFP fluorescence was observed in oligodendrocytes within both the striatum, along with EGFP-positive fibres within the striosomes (Fig. 4S). The small size and morphology of these transduced cells was consistent with an oligodendroglial phenotype, confirmed by co -localisation of EGFP with the oligodendroglial marker CAII (Fig. 4U) and lack of co- labelling with NeuN (Fig. 4T). Similarly intra-hippocampal infusion of AAV8-MBP- EGFP resulted in transduction of oligodendrocytes within the hilus (Fig. 4V-X). Transgene expression observed following rh43 -MBP-EGFP infusion was less widespread than seen with AAV8 with fluorescence observed in oligodendrocytes only in the immediate vicinity of the injection site. Immunohistochemistry showed that astrocytes within the striatum were also transduced (Fig 5C). Immunohistochemistry for^" EGFP (Fig. 5) showed that microglia within the striatum were also transduced. Following intra-hippocampal infusion of rh43 -MBP-EGFP, native EGFP fluorescence was not visible, however, immunostaining showed that a population of neurons within the DG was transduced (Fig. 5D). Detection of EGFP transgene expression in the corpus callosum, a region rich in glia but devoid of neurons, further demonstrates that AAV8- and rh43- derived expression cassettes under the control of the MBP promoter results in oligodendroglial transduction (Fig. 5E, F). Results presented in Figures 6 and 7 show the extent of transduction obtained with each vector - the volume of EGFP- immunoreactivity and the number of transduced neurons, astrocytes, and oligodendrocytes within the striatum were quantified using stereological methods. Infusion of 3 x 10¹⁰ vg of AAV 8 resulted in wide-spread striatal and hipoocampal transduction regardless of the promoter used (Fig 6A,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 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 x 10⁹ vg, as used in the previous study) driven by the GFAP and MBP promoters resulted in more localized transduction (Fig. 6C, D) with both serotypes.

Although the invention has been described with reference to certain embodiments and examples it will be understood that variants in keeping with the disclosure and the spirit of the invention are also within its scope.

REFERENCES:

I. During, M. J., Young, D., Baer, K., Lawlor, P., and Klugmann, M. (2003). Development and optimization of adeno-associated virus vector transfer into the central nervous system. Methods MoI Med 76: 221-236. 2. Tenenbaum, L., Chtarto, A., Lehtonen, E., VeIu, T., Brotchi, J., and Levivier, M. (2004). Recombinant AAV-mediated gene delivery to the central nervous system. J Gene Med 6 Suppl 1 : S212-222.

3. McCown, T. J. (2005). Adeno-associated virus (AAV) vectors in the CNS. Curr Gene Ther 5: 333-338. 4. During, M. J., et al. (2003). Glucagon-like peptide- 1 receptor is involved in learning and neuroprotection. Nat Med 9: 1173-1179.

5. Szumlinski, K. K., et al. (2005). Homer2 is necessary for EtOH-induced neuroplasticity. J Neurosci 25: 7054-7061.

6. Mouravlev, A., Dunning, J., Young, D., and During, M. J. (2006). Somatic gene transfer of c AMP response element-binding protein attenuates memory impairment in aging rats. Proc Natl Acad Sci USA 103: 4705-4710.

7. Kirik, D., et al. (2002). Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci 22: 2780-2791.

8. Lawlor, P. A., et al. (2007). Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Abeta levels. MoI

Neurodegener 2: 11.

9. DiFiglia, M., et al. (2007). Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc Natl Acad Sci U S A 104: 17204-17209. 10. Mandel, R. J., and Burger, C. (2004). Clinical trials in neurological disorders using AAV vectors: promises and challenges. Curr Opin MoI Ther 6: 482-490.

I 1. Bankiewicz, K. S., et al. (2006). Long-term clinical improvement in MPTP- lesioned primates after gene therapy with AAV-hAADC. MoI Ther 14: 564-570.

12. Kaplitt, M. G., et al. (2007). Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 369: 2097-2105.

13. Giacobini, E., and Becker, R. E. (2007). One hundred years after the discovery of Alzheimer's disease. A turning point for therapy? J Alzheimers Dis 12: 37-52. 14. Pekny, M., and Nilsson, M. (2005). Astrocyte activation and reactive gliosis. GHa 50: 427-434.

15. Maragakis, N. J., and Rothstein, J. D. (2006). Mechanisms of Disease: astrocytes in neurodegenerative disease. Nat Clin Pract Neurol 2: 679-689. 16. Chen, L. W., Yung, K. L., and Chan, Y. S. (2005). Reactive astrocytes as potential manipulation targets in novel cell replacement therapy of Parkinson's disease. Curr Drug Targets 6: 821-833.

17. Chen, H., McCarty, D. M., Bruce, A. T., and Suzuki, K. (1999). Oligodendrocyte-specific gene expression in mouse brain: use of a myelin- forming cell type-specific promoter in an adeno-associated virus. J Neurosci Res 55: 504-513.

18. Xu, R., et al. (2001). Quantitative comparison of expression with adeno- associated virus (AAV-2) brain-specific gene cassettes. Gene Ther 8: 1323-1332.

19. Mastakov, M. Y., Baer, K., Xu, R., Fitzsimons, H., and During, M. J. (2001). Combined injection of rAAV with mannitol enhances gene expression in the rat brain. MoI Ther 3: 225-232.

20. Mastakov, M. Y., Baer, K., Kotin, R. M., and During, M. J. (2002). Recombinant adeno-associated virus serotypes 2- and 5-mediated gene transfer in the mammalian brain: quantitative analysis of heparin co-infusion. MoI Ther 5: 371-380.

21. Burger, C, Nguyen, F. N., Deng, J., and Mandel, R. J. (2005). Systemic mannitol-induced hyperosmolality amplifies rAAV2-mediated striatal transduction to a greater extent than local co-infusion. MoI Ther 11 : 327-331.

22. Bartlett, J. S., Samulski, R. J., and McCown, T. J. (1998). Selective and rapid uptake of adeno-associated virus type 2 in brain. Hum Gene Ther 9: 1181-1186.

23. Burger, C, et al. (2004). 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. MoI Ther 10: 302-317.

24. Wu, Z., Asokan, A., and Samulski, R. J. (2006). Adeno-associated virus serotypes: vector toolkit for human gene therapy. MoI Ther 14: 316-327.

25. Kwon, L, and Schaffer, D. V. (2007). Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer. Pharm Res. 26. Klein, R. L., Dayton, R. D., Leidenheimer, N. J., Jansen, K., Golde, T. E., and Zweig, R. M. (2006). Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. MoI Ther 13: 517-527.

27. Cearley, C. N., and Wolfe, J. H. (2006). Transduction characteristics of adeno- associated virus vectors expressing cap serotypes 7, 8, 9, and RhIO in the mouse brain.

MoI Ther 13: 528-537.

28. Harding, T. C, et al. (2006). Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8- pseudotyped vectors. Hum Gene Ther 17: 807-820. 29. Taymans, J. M., et al. (2007). Comparative analysis of adeno -associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum Gene Ther 18: 195-206.

30. Gao, G., et al. (2004). Clades of Adeno -associated viruses are widely disseminated in human tissues. J Virol 78: 6381-6388.

31. Gao, G., Vandenberghe, L. H., and Wilson, J. M. (2005). New recombinant serotypes of AAV vectors. Curr Gene Ther 5: 285-297.

32. Klein, R. L., Dayton, R. D., Tatom, J. B., Henderson, K. M., and Henning, P. P. (2007). AAV8, 9, RhIO, Rh43 Vector Gene Transfer in the Rat Brain: Effects of Serotype, Promoter and Purification Method. MoI Ther. 16:89-96.

33. Peden, C. S., Burger, C, Muzyczka, N., and Mandel, R. J. (2004). Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant

AA V2 (rAAV2)-mediated, but not rAAV5 -mediated, gene transfer in the brain. J Virol 78: 6344-6359.

34. Broekman, M. L., Comer, L. A., Hyman, B. T., and Sena-Esteves, M. (2006). Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-I or -2 serotypes for widespread gene delivery to the neonatal mouse brain. Neuroscience 138: 501-510.

35. Reimsnider, S., Manfϊedsson, F. P., Muzyczka, N., and Mandel, R. J. (2007). Time course of transgene expression after intrastriatal pseudotyped rAAV2/l, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat. MoI Ther 15: 1504-1511. 36. Shevtsova, Z., Malik, J. M., Michel, U., Bahr, M., and Kugler, S. (2005).

Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 90: 53-59. 37. Howard, D. B., Powers, K., Wang, Y., and Harvey, B. K. (2007). Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro. Virology.

38. Chen, H., McCarty, D. M., Bruce, A. T., Suzuki, K., and Suzuki, K. (1998). Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus. Gene Ther 5: 50-58.

39. Liu, G., Martins, I. H., Chiorini, J. A., and Davidson, B. L. (2005). Adeno- associated virus type 4 (AA V4) targets ependyma and astrocytes in the subventricular zone and RMS. Gene Ther 12: 1503-1508.

40. Li, S. F., et al. (2006). Intraventricular infusion of rAAVl-EGFP resulted in transduction in multiple regions of adult rat brain: a comparative study with rAAV2 and rAAV5 vectors. Brain Res 1122: 1-9.

41. Cearley, C. N., Vandenberghe, L. H., Parente, M. K., Carnish, E. R., Wilson, J. M., and Wolfe, J. H. (2008). Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain. MoI Ther 16: 1710-1718.

42. Foust, KD, Nurre, E, Montgomery, CL, Hernandez, A, Chan, CM and Kaspar, BK (2009). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59-65. 43. Klugmann, M., Leichtlein, C. B., Symes, C. W., Serikawa, T., Young, D., and During, M. J. (2005). Restoration of aspartoacylase activity in CNS neurons does not ameliorate motor deficits and demyelination in a model of Canavan disease. MoI Ther 11: 745-753.

44. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Møller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sørensen FB, Vesterby A, et al (1988). Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96:379-94.

45. Popovic N, Maingay M, Kirik D, Brundin P. (2005) Lentiviral gene delivery of GDNF into the striatum of R6/2 Huntington mice fails to attenuate behavioral and neuropatho logical changes. Exp Neurol. 193:65-74.

46. Gundersen, H.J., Bagger, P., Bendsten, T. F., Evans, S. M., Korbu, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg,

B., Sorensen, F.B., Vesterby, A., West, M. J., 1988. The new stereological tools: dissector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96, 857 - 881.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

1. A purified adeno-associated viral (AAV) vector stock comprising a vector having a cell specific promoter, wherein the vector preferably transduces non-neuronal brain tissue.

2. A purified AAV vector comprising a promoter that enhances transduction of non-neuronal brain tissue.

3. A purified AAV vector according to claim 1 or claim 2, wherein the non- neuronal brain tissue comprises glial cells

4. A purified AAV vector according to claim 3, wherein the glial cells are selected from astrocytes or oligodendrocytes.

5. A purified AAV vector according to any one of claims 1 to 4, wherein the promoter is selected from the group consisting of GFAP, MBP , adenosine kinase, aspartoacylase promoters, JC virus early promoter, SlOOB, vimentin, CAR2, CD44, GLUL, PDGFRA, RLBPl, SLCl A3 or parts thereof.

6. The purified AAV vector according to any one of claims 1 to 5, further comprising a therapeutic gene or a sequence which reduces expression of a specific target gene.

7. The purified AAV vector according to claim 6, wherein the expression of a specific target gene is reduced by using RNA interference, antisense or ribozyme sequences.

8. The purified AAV vector according to claim 6, wherein the therapeutic gene is selected from the group consisting of neuropeptide Y (NPY), excitatory amino acid transporter 2 (EAAT2) and glutamine synthetase, vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and aspartoacylase (ASPA).

9. The purified AAV vector according to claim 7, wherein target gene whose expression requires reducing is selected from gene encoding adenosine kinase, ion channels, water channels (AQP4), glutamate receptors, inflammatory genes or any gene functioning abnormally in an astrocyte in diseased brain.

10. Use of a vector according to any one of claims 1 to 9 for gene therapy.

11. A method of preparing a purified AAV vector stock for preferential non-neuronal brain tissue transduction comprising purifying a vector having a cell specific promoter, such that transduction is preferably of non-neuronal cells.

12. A method of preparing an AAV vector stock capable of preferential non- neuronal brain cell transduction, comprising the steps:

(a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.

13. AAV vector stock prepared by the method of claim 11 or claim 12.

14. Use of a purified AAV vector according to any one of claims 1 to 9 for the preparation of a medicament for therapeutic or prophylactic treatment of a neurological disorder and/or a neurodegenerative disease by gene therapy.

15. Use according to claim 14, wherein the neurological disorder and/or neurodegenerative disease is associated with glial cell pathology.

16. Use according to claim 14 or claim 15, wherein the neurological disorder and/or a neurodegenerative disease is selected from spinal muscular dystrophy, epilepsy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Canavan disease, amyotrophic lateral sclerosis, spinal cord disease or injury, multiple sclerosis and leukodystrophies.

17. Use according to any one of claims 14 to 16, wherein the medicament preferentially targets glial cells.

18. Use according to any one of claims 15 to 17, wherein the glial cells are astrocytes or oligodendrocytes.

19. Use according to any one of claims 15 to 18, wherein cells are human cells.

20. Method of therapeutic or prophylactic treatment of a neurological disease and/or a neurodegenerative disease by administering to a subject in need thereof an AAV vector according to any one of claims 1 to 9, wherein the neurological disorder and/or a neurodegenerative disease is selected from spinal muscular dystrophy, epilepsy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Canavan disease, amyotrophic lateral sclerosis, spinal cord disease or injury, multiple sclerosis and leukodystrophies.

21. Method for selecting an AAV vector that preferentially transduces non-neuronal brain cells, comprising the steps of:

(a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.

22. Method for selecting an AAV vector that transduces a desired brain cell type, comprising the steps of:

(a) introducing a cell specific promoter into an AAV vector;

(b) purifying the AAV vector in step (a) to obtain a AAV vector stock that transduces the desired brain cell type.

23. The purified vector according to claim 9, wherein the ion channel is a potassium ion channel or a calcium ion channel.

24. The purified vector according to claim 9, wherein the inflammatory genes comprise genes encoding cytokines, interleukins or interferons.

25. The purified vector according to claim 7, wherein RNA interference is by short hairpin RNA or micro RNA.