WO2010071454A1 - Vecteurs viraux adéno-associés et leurs utilisations - Google Patents

Vecteurs viraux adéno-associés et leurs utilisations Download PDF

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WO2010071454A1
WO2010071454A1 PCT/NZ2009/000290 NZ2009000290W WO2010071454A1 WO 2010071454 A1 WO2010071454 A1 WO 2010071454A1 NZ 2009000290 W NZ2009000290 W NZ 2009000290W WO 2010071454 A1 WO2010071454 A1 WO 2010071454A1
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transduction
aav vector
cells
aav8
disease
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Patricia Alice Lawlor
Deborah Young
Matthew John During
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Auckland Uniservices Limited
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • 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).
  • CNS central nervous system
  • 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.
  • 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,
  • 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.
  • rAd recombinant Adenovirus
  • rAAV recombinant adeno associated virus
  • 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].
  • AAV2 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].
  • 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].
  • 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.
  • 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.
  • AAV adeno- associated viral
  • the present invention provides a purified AAV vector comprising a promoter that enhances transduction of non-neuronal brain tissue.
  • the present invention provides use of a vector according to the first or second aspect for gene therapy.
  • 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.
  • 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;
  • step (b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.
  • the present invention provides a AAV vector stock prepared by the method of the fourth or fifth aspects.
  • 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.
  • 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.
  • transduction of brain tissue occurs in vivo.
  • the adeno-associated viral vector is preferably able to evade pre-existing immunity.
  • the adeno-associated viral vector is recombinant.
  • the adeno-associated viral vector is non-human.
  • the adeno-associated viral vector is of primate origin.
  • the brain tissue is human brain tissue.
  • 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.
  • RNA interference short hairpin RNA, micro RNA
  • antisense or ribozyme sequences are provided.
  • 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.
  • NPY neuropeptide Y
  • EAAT2 excitatory amino acid transporter 2
  • 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.
  • 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.
  • 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,
  • the brain cells are glial cells such as astrocytes and/or oligodendrocytes.
  • the brain cells are human brain cells.
  • 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.
  • the neurological disorder is epilepsy and/or depression however it will be understood that the present invention is not limited to these disorders.
  • 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.
  • the present invention provides a method for selecting an AAV vector that preferentially transduces non-neuronal brain cells, comprising the steps of:
  • step (b) purifying the AAV vector in step (a) to obtain a AAV vector stock that preferentially transduces non-neuronal brain cells.
  • the present invention provides a method for selecting an AAV vector that transduces s desired brain cell type, comprising the steps of:
  • step (b) purifying the AAV vector in step (a) to obtain a AAV vector stock that transduces the desired brain cell type.
  • 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.
  • 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.
  • FIG. 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 ⁇ 10 9 viral genomes of each serotype.
  • FIG. 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 10 9 vg of rh43 into the striatum (F) resulted in low-level astrocytic transduction. Infusion of 3 x 10 10 vg of rh43 into the striatum (G) and hippocampus (H) resulted in increased numbers of transduced astrocytes, along with transduction of neurons.
  • Figure 4 Altering the promoter changed the tropism of AAV 8 and rh43 in the striatum and hippocampus. Examples shown are AAV8 (3 x 10 10 vg).
  • AAV8 (3 x 10 10 vg).
  • A-F CAG promoter
  • 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).
  • FIG. 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 10 10 vg) resulted in wide-spread astrocytic transduction in both the hippocampus (A) and striatum (B), and extending into corpus callosum (CC) (B).
  • 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 10 10 vg.
  • A A
  • rh43 B
  • the extent of transgene expression with the MBP and GFAP promoters was reduced, (C).
  • 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 10 9 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.
  • 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.
  • 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.
  • AAV having cell specific, rather than constitutively active promoters specifically target non-neuronal brain cells i.e. glial cells.
  • 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.
  • 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.
  • 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.
  • CsCl and sucrose gradients
  • CsCl and sucrose gradients
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GFAP glial flbrilliary acidic protein
  • MBP myelin basic protein
  • 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.
  • 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.
  • 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.
  • obtaining widespread glial cell transduction required the infusion of a high dose of vector (3 * 10 10 vg).
  • Brains injected with lower titers (4.5 x 10 9 vg) showed less widespread glial transduction ( Figure 6 C, D).
  • use of these promoters resulted in only low-level neuronal transgene expression accompanying the observed glial transduction.
  • promoters resulted in minimal neuronal expression - transgene expression was almost exclusively glial.
  • 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.
  • 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.
  • 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].
  • 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.
  • neuronal transduction need not be absent, as long as there is significant and/or enhanced astroglial transgene expression.
  • 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.
  • 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 ⁇ -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.
  • AAV expression plasmid developed by department of Molecular Medicine and Pathology, The University of Auckland, New Zealand
  • CAG hybrid CMV-chicken ⁇ -actin promoter
  • WPRE woodchuck hepatitis virus post-transcriptional-regulatory element -J Donello, J Virol (1998)
  • HEK293 cells (Microbix) were co-transfected with three plasmids - AAV plasmid, appropriate helper plasmid encoding rep and cap genes, and adenoviral helper pF ⁇ 6 - using standard CaPO 4 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 4 0 C (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 18 0 C.
  • 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 lO ⁇ l sample on SDS-PAGE and staining with Coomassie blue.
  • 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 40 ⁇ m 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 I 0 AH 2 O 2 in 50% methanol for 30min to bind endogenous peroxidase present in the tissue. Sections were washed extensively in IxPBS-T. 200 ⁇ l 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.
  • primary antibody anti-GFP, Abeam, ab290
  • immunobuffer immunobuffer
  • 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 OO ⁇ m to estimate the area of transduction within each section.
  • the area in every 12 th 40 ⁇ m 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 ⁇ m section (4-9 sections per brain) using a ⁇ 40 objective and 100 ⁇ m 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 mm 3 of striatal tissue.
  • Rats injected with AAV vectors were killed 3 weeks following infusion and brains processed immunohistochemically for detection of EGFP.
  • 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 10 9 vg of rh43 into the striatum (F) resulted in low- level astrocytic transduction. Infusion of 3 x 10 10 vg of rh43 into the striatum (G) and hippocampus (H) resulted in increased numbers of transduced astrocytes, along with transduction of neurons.
  • A- F CAG promoter
  • 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).
  • 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 20 ⁇ m (applies to A, D, G, J, M, P, S).
  • Scale bar on x 50 ⁇ m (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).
  • 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 10 10 vg.
  • A A
  • rh43 B
  • the extent of transgene expression with the MBP and GFAP promoters was reduced, (C).
  • Transduction volume (mm 3 ) in the striatum following infusion of 3 x 10 10 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 * 10 9 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.
  • AAV9-CAG-GFP 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.
  • 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 ⁇ - actin/CMV hybrid (CAG) promoter were titer matched to 1.5 x 10 12 genomes/ml and 3 ⁇ l (total of 4.5 x 10 9 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.
  • 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.
  • 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.
  • DG hippocampus - dentate gyrus
  • CA2 CA4 - with EGFP- immunoreactive fibres and cell bodies also observed in the contralateral hippocampus.
  • 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.
  • 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.
  • 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).
  • 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).
  • AAV8 and rh43 vectors were generated, driven by the cell-specific promoters GFAP (glial f ⁇ brilliary acidic protein) and MBP (myelin basic protein) and compared this to transduction obtained using the constitutive viral promoters, CAG and CMV (cytomegalovirus).
  • GFAP glial f ⁇ brilliary acidic protein
  • MBP myelin basic protein
  • Each vector (3 x 10 10 Vg) was infused into the striatum and hippocampus and brain tissue examined immunohistochemically for transgene expression three weeks post-infusion.
  • transduction with AAV8 resulted in wide-spread visible EGFP fluorescence
  • 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.
  • 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.
  • Fig 4M-R EGFP fluorescence was observed in astrocytes only (Fig.
  • 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.
  • 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.
  • AAV2 Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant
  • 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.
  • Promoters and serotypes targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.
  • Adeno- associated virus type 4 targets ependyma and astrocytes in the subventricular zone and RMS. Gene Ther 12: 1503-1508.
  • Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59-65. 43. Klugmann, M.,chtlein, 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.

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Abstract

La présente invention porte sur des vecteurs viraux et, en particulier, sur des vecteurs viraux adéno-associés ayant des capacités améliorées de transfert de gènes dans les cellules du système nerveux central (CNS). L'invention a été développée principalement pour un transfert de gènes dans les cellules gliales et en outre pour le traitement de maladies associées à une pathologie des cellules gliales.
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