WO2024236193A1 - Vecteur - Google Patents

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Publication number
WO2024236193A1
WO2024236193A1 PCT/EP2024/063802 EP2024063802W WO2024236193A1 WO 2024236193 A1 WO2024236193 A1 WO 2024236193A1 EP 2024063802 W EP2024063802 W EP 2024063802W WO 2024236193 A1 WO2024236193 A1 WO 2024236193A1
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Prior art keywords
vector
sequence
seq
mir
expression
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Inventor
Vania Broccoli
Simone BIDO
Diana GAMBARÈ
Melania NANNONI
Alice CALDERONI
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Consiglio Nazionale delle Richerche CNR
Ospedale San Raffaele SRL
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Consiglio Nazionale delle Richerche CNR
Ospedale San Raffaele SRL
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Publication of WO2024236193A1 publication Critical patent/WO2024236193A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • lentiviral vector comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10 or 11.
  • the vector e.g. lentiviral vector
  • the vector comprises or consists of SEQ ID NO: 10 or 11.
  • the vector is a AAV vector.
  • the vector e.g. AAV vector
  • the vector comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 35.
  • the vector comprises or consists of SEQ ID NO: 35.
  • the vector is in the form of a viral vector particle.
  • the viral vector particle is a VSV-g pseudotyped viral vector particle.
  • the vector is replication-incompetent.
  • the vector is an AAV vector particle, wherein the AAV vector particle comprises a capsid selected from the group consisting of an AAV9; AAV9 PHP.B; AAV9 PHP.eB; and AAVrh10 capsid.
  • the vector is an AAV vector particle for astrocyte-specific expression.
  • the AAV vector particle may, for example, be of a serotype for astrocyte-specific expression.
  • the AAV vector particle displays tropism for astrocytes.
  • the invention provides use of the vector, cell or pharmaceutical composition of the invention for the manufacture of a medicament for treating or preventing Parkinson’s
  • the invention provides a method of treating or preventing Parkinson’s disease comprising administering the vector, cell or pharmaceutical composition of the invention to a subject in need thereof.
  • the vector, cell or pharmaceutical composition is administered to a subject systemically or locally.
  • the vector, cell or pharmaceutical composition is administered to a subject intracranially or intraparenchymally.
  • the vector, cell or pharmaceutical composition is administered to a subject intracerebrally.
  • the vector, cell or pharmaceutical composition is used in combination with another treatment for Parkinson’s disease, for example administration of levodopa, administration of a dopamine agonist, administration of a catechol-O-methyltransferase (COMT) inhibitor, administration of a monoamine oxidase-B (MAO-B) inhibitor, and/or deep brain stimulation (DBS).
  • the vector, cell or pharmaceutical composition is used in combination with dopamine replacement therapy (DRT).
  • C SNpc infected tissue with LV-miRTag-GFP and stained for the measurement of GFP colocalization with astrocytes (GFAP), microglia (IBA1), neurons (NeuN) and oligodendrocytes (Olig2). The degree of colocalization indicates the selective expression of GFP in microglia.
  • FIGURE 2 Immunohistochemical analysis of the total number of the dopaminergic neurons stained with TH and counted with unbiased stereology. ***p ⁇ 0.001.
  • FIGURE 3 (A) Schematic of a platform using four copies of GAS before a Mx1 minimum promoter (B) Immunofluorescence images demonstrating enhanced GFP expression in microglia primary cultures infected with LV-4GAS-mMX1-GFP in response to IFN gamma treatment. (C) RT- PCR quantification of IL-10 transcription in microglia primary cultures infected with LV-4GAS- mMX1-IL10 and expressed as fold change vs IFN gamma untreated cells.
  • FIGURE 4 (A-C) Examination of microglial activation and phagocytic function in the SNpc carried out by immunodecoration for CD68, IBA1 and a marker for ⁇ SYN aggregates pS129 ⁇ SYN. The quantification of CD68 particles within individual cells was performed in terms of the number per unit area (B) and their average size (C). The magnified inset in (A) highlights the co- localization of ⁇ SYN aggregates with CD68-positive particles. (D, E) Expression of human ⁇ SYN and presence of ⁇ SYN aggregates within the nigral tissue in the different conditions, quantified as the percentage of the stained area.
  • FIGURE 5 In vitro phagocytosis assay using primary microglia infected with LV:Nluc, LV: ⁇ gIL10, LV:SNCA and LV:SNCA/ ⁇ gIL10 and seeded with fluorescent ⁇ SYN pre-formed fibrils (Fluor488-PFFs). Phagocytosis is measured by the area occupied by Fluor488-PFFs signal within microglia (IBA1).
  • B, D, E In vitro phagocytosis assay employing primary microglia infected with LV:Nluc, LV: ⁇ gIL10, LV:SNCA and LV:SNCA/ ⁇ gIL10 and seeded with pHrodo- labelled synaptosomes.
  • the invention provides a vector for central nervous system (CNS)-specific expression, wherein the vector comprises a transgene encoding interleukin 10 (IL-10).
  • the “central nervous system” as used herein means the nervous system consisting of the brain and spinal cord.
  • the “peripheral nervous system” as used herein means the components of the nervous system outside of the central nervous system.
  • the peripheral nervous system consists of the nerves and ganglia outside of the brain and spinal cord.
  • blood brain barrier means the highly selective semi- permeable membrane barrier which separates the circulating blood from the brain and extracellular fluid in the central nervous system. It is formed by the selectivity of tight junctions between endothelial cells.
  • the blood-brain barrier occurs along all capillaries of the brain and consists of tight junctions.
  • brain endothelial cells may refer to the cerebral endothelial cells that present the interface between the blood and the central nervous system, i.e. blood-brain barrier endothelial cells.
  • the brain endothelial cells may form the blood-brain barrier.
  • the vector is for microglia-specific expression.
  • Microglia are a type of glial cell located in the central nervous system, and account for about 10-15% of cells in the brain.
  • Microglia are the resident macrophage cells that act as the main form of immune defence in the central nervous system.
  • Macrophages are innate immune cells that clear tissue from pathogens or other biological material. In adult mammals, macrophages are found in all tissues where they display great anatomical and functional diversity. In tissues, they are organized in defined patterns with each cell occupying its own territory.
  • Microglia contribute to brain maintenance, for example by scavenging for plaques, damaged or unnecessary neurons and synapses, and infectious agents.
  • the skilled person is readily able to identify cells such as macrophages and microglia, for example using markers that may be expressed on a cell surface.
  • ionized calcium-binding adaptor molecule 1 Iba1
  • Iba1 ionized calcium-binding adaptor molecule 1
  • CD11b and CD45 markers may be used to distinguish microglia from macrophages. Resting microglia are CD11b hi CD45 low , whereas macrophages are CD11b hi CD45 hi .
  • the vector may be a central nervous system (CNS)-specific expression vector, preferably a microglia-specific expression vector.
  • CNS central nervous system
  • CNS-specific expression and microglia- specific expression may refer to the preferential or predominant expression of a transgene in the CNS or microglia, respectively, as compared to other cells (e.g. blood, lung and bone marrow cells).
  • at least 50% of transgene expression occurs in the microglia.
  • at least 60%, 70%, 80%, 90% or 95% of transgene expression occurs in the microglia.
  • the transgene is substantially exclusively expressed in the microglia.
  • expression of the transgene in microglia transduced by the vector may be greater than expression of the transgene in other cells transduced by the vector; (ii) the transgene may be substantially not expressed in cells other than the microglia, when transduced by the vector; (iii) the transgene may be substantially not expressed in neurons, oligodendrocytes and/or astrocytes, when transduced by the vector; (iv) expression of the transgene in microglia may be at least five times greater than expression in neurons, oligodendrocytes and/or astrocytes, when transduced by the vector; and/or (v) the transgene may be substantially not expressed in neurons, oligodendrocytes and/or astrocytes when transduced by the vector.
  • Expression of the transgene may be determined by any suitable method known to the skilled person, for example any of the methods as disclosed herein.
  • expression of the transgene in microglia transduced by the vector may be greater than expression of the transgene in other cells transduced by the vector.
  • expression of the transgene in microglia transduced by the vector may be at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than in other cells transduced by the vector.
  • the transgene is substantially not expressed in cells other than the microglia, when transduced by the vector.
  • the percentage of the cells other than the microglia which express the transgene may be 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, or 0%.
  • expression of the transgene in cells other than the microglia may be undetectable.
  • the transgene is substantially not expressed in neurons, oligodendrocytes and/or astrocytes, when transduced by the vector.
  • the percentage of neurons, oligodendrocytes and/or astrocytes which express the transgene may be 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, or 0%.
  • expression of the transgene in neurons, oligodendrocytes and/or astrocytes may be undetectable.
  • the transgene is substantially only expressed in microglia.
  • the percentage of the cells types other than microglia which express the transgene may, for example, be 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, or 0%.
  • expression of the transgene in cell types other than microglia may be undetectable.
  • expression of the transgene in microglia may be at least five times greater than expression in neurons, oligodendrocytes and/or astrocytes, when transduced by the vector.
  • expression of the transgene in microglia may be at least ten times greater, at least twenty times greater, or at least fifty times greater than expression in neurons, oligodendrocytes and/or astrocytes.
  • the transgene may be substantially not expressed in neurons, oligodendrocytes and/or astrocytes when transduced by the vector.
  • the percentage of neurons, oligodendrocytes and/or astrocytes which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%.
  • expression of the transgene in neurons, oligodendrocytes and/or astrocytes may be undetectable.
  • the vector of the invention may comprise one or more expression control sequence.
  • the transgene is operably linked to one or more expression control sequence.
  • an “expression control sequence” is a nucleotide sequence that controls expression of a transgene, e.g. to facilitate and/or increase expression in some cell types d/ d i i th ll t
  • the expression control sequence and the transgene may be in any suitable arrangement in the vector, for example providing that the expression control sequence is operably linked to the transgene.
  • operably linked means that the parts (e.g.
  • the expression control sequence may be a central nervous system (CNS)-specific expression control sequence, particularly a microglia-specific expression control sequence (e.g. such that the vector specifically expresses a transgene in the CNS, particularly microglia).
  • CNS central nervous system
  • Expression control sequences include promoters, enhancers, and 5’ and 3’ untranslated regions (e.g. miRNA target sequences).
  • the one or more expression control sequence may comprise one or more miRNA target sequence.
  • the one or more expression control sequence comprises a promoter and one or more miRNA target sequence.
  • the one or more expression control sequence comprises a promoter, one or more gamma activated sequence (GAS) and one or more miRNA target sequence.
  • the vector may, for example, comprise from 5’ to 3’: a promoter; the transgene; and one or more miRNA target sequence.
  • the vector may, for example, comprise from 5’ to 3’: one or more GAS; a promoter; the transgene; and one or more miRNA target sequence.
  • MicroRNA target sequences In some embodiments, the vector comprises one or more miRNA target sequence.
  • the transgene encoding IL-10 is operably linked to the one or more miRNA target sequence.
  • MicroRNA (miRNA) genes are scattered across all human chromosomes, except for the Y chromosome. They can be either located in non-coding regions of the genome or within introns of protein-coding genes. Around 50% of miRNAs appear in clusters which are transcribed as polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are usually transcribed from polymerase-II promoters, generating a so-called primary miRNA transcript (pri-miRNA). This pri-miRNA is then processed through a series of endonucleolytic cleavage steps, performed by two enzymes belonging to the RNAse Type III family, Drosha and Dicer.
  • pri-miRNA primary miRNA transcript
  • miRNA precursor a stem loop of about 60 nucleotides in length, called miRNA precursor (pre-miRNA)
  • pre-miRNA a stem loop of about 60 nucleotides in length
  • DGCR8 DiGeorge syndrome critical region gene
  • Dicer performs a double strand cut at the end of the stem loop not defined by the Drosha cut, generating a 19-24 bp duplex, which is composed of the mature miRNA and the opposite strand of the duplex, called miRNA*.
  • RISC RNA-induced silencing complex
  • This strand is usually the one whose 5’ end is less tightly paired to its complement, as was demonstrated by single-nucleotide mismatches introduced into the 5’ end of each strand of siRNA duplexes.
  • miRNAs that support accumulation of both duplex strands to similar extent.
  • MicroRNAs trigger RNAi, very much like small interfering RNAs (siRNA) which are extensively used for experimental gene knockdown. The main difference between miRNA and siRNA is their biogenesis.
  • the guide strand of the small RNA molecule interacts with mRNA target sequences preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. It has been shown that nucleotides 2-8 counted from the 5' end of the miRNA, the so-called seed sequence, are essential for triggering RNAi. If the whole guide strand sequence is perfectly complementary to the mRNA target, as is usually the case for siRNAs and plant miRNAs, the mRNA is endonucleolytically cleaved by involvement of the Argonaute (Ago) protein, also called “slicer” of the small RNA duplex into the RNA-induced silencing complex (RISC).
  • Ago Argonaute
  • DGRC DiaGeorge syndrome critical region gene 8
  • TRBP TAR (HIV) RNA binding protein 2
  • RISC effector complex RISC
  • RNAi acts through multiple mechanisms leading to translational repression.
  • Eukaryotic mRNA degradation mainly occurs through the shortening of the polyA tail at the 3’ end of the mRNA, and de-capping at the 5’ end, followed by 5’-3’ exonuclease digestion and accumulation of the miRNA in discrete cytoplasmic areas, the so called P-bodies, enriched in components of the mRNA decay pathway.
  • Expression of the nucleic acid sequence encoding the transgene may be regulated by one or more endogenous miRNA using one or more corresponding miRNA target sequence.
  • the target sequence may be fully or partially complementary to the miRNA.
  • the term “fully complementary”, as used herein, may mean that the target sequence has a nucleic acid sequence which is 100% complementary to the sequence of the miRNA which recognises it.
  • the term “partially complementary”, as used herein, may mean that the target sequence is only in part complementary to the sequence of the miRNA which recognises it, whereby the partially complementary sequence is still recognised by the miRNA.
  • a partially complementary target sequence in the context of the present invention is effective in recognising the corresponding miRNA and effecting prevention or reduction of transgene expression in cells expressing that miRNA.
  • a partially complementary miRNA target sequence may be fully complementary to the miRNA seed sequence.
  • the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-124 target sequence.
  • the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-31 target sequence.
  • the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-338-3p target sequence.
  • the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-124 target sequence and at least one miR-31 target sequence. In one aspect, the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-124 target sequence and at least one miR-338-3p target sequence. In one aspect, the invention provides a vector comprising a transgene encoding interleukin 10 (IL-10) and at least one miR-124 target sequence, at least one miR-31 target sequence and at least one miR-338-3p target sequence. Including more than one copy of a miRNA target sequence may increase the effectiveness of the system.
  • the protein- coding sequence may be operably linked to more than one miRNA target sequence, which may or may not be different.
  • the miRNA target sequences may be in tandem, but other arrangements are envisaged.
  • the polynucleotide may, for example, comprise 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequences. Copies of miRNA target sequences may be separated by a spacer sequence.
  • the spacer sequence may comprise, for example, at least one, at least two, at least three, at least four or at least five nucleotide bases.
  • the number of copies of each of the miRNA target sequences is independently selected from the group consisting of: one, two, three, and four.
  • the vector comprises one miR-124 target sequence. In some embodiments, the vector comprises two miR-124 target sequences. In some embodiments, the vector comprises three miR-124 target sequences. In preferred embodiments, the vector comprises four miR-124 target sequences. In some embodiments, the vector comprises one miR-31 target sequence. In some embodiments, the vector comprises two miR-31 target sequences. In some embodiments, the vector comprises three miR-31 target sequences. In preferred embodiments, the vector comprises four miR-31 target sequences. In some embodiments, the vector comprises one miR-338-3p target sequence. In some embodiments, the vector comprises two miR-338-3p target sequences. In some embodiments, the vector comprises three miR-338-3p target sequences.
  • the vector comprises four miR-338-3p target sequences. In preferred embodiments, the vector comprises four miR-124 target sequences, four miR-31 target sequences and four miR-338-3p target sequences.
  • An exemplary miR-124 target sequence is: TATTGCCTTATTTC (SEQ ID NO: 1)
  • the miR-124 target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1.
  • the miR-124 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 1.
  • the miR-124 target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 1.
  • An exemplary miR-338-3p target sequence is: CAACAAAATCACTGATGCTGGA (SEQ ID NO: 2)
  • the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 2.
  • the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 2.
  • the miR-338-3p target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 2.
  • An exemplary miR-31 target sequence is: CAGCTATGCCAGCATCTTGCC (SEQ ID NO: 3)
  • the miR-31 target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 3.
  • the miR-31 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 3.
  • the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-338-3p, miR-124 and miR-31. In some embodiments, the miRNA target sequences or clusters of copies of sequences are, In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-31, miR-124 and miR-338-3p. In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-31, miR-338-3p and miR-124.
  • the vector comprises a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity
  • the polynucleotide comprises a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in neurons. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in astrocytes. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in oligodendrocytes.
  • suppress expression may refer to a reduction of expression in the relevant cell type(s) of a transgene to which the one or more miRNA target sequence is operably linked as compared to transgene expression in the absence of the one or more miRNA target sequence, but under otherwise substantially identical conditions.
  • transgene expression is suppressed by at least 60%, 70%, 80%, 90% or 95%.
  • transgene expression is undetectable.
  • transgene expression is substantially prevented.
  • Both the individual target sequences and the clusters of sequences may be contiguous with one another, separated by spacer sequences, or any combination thereof.
  • the miRNA target sequences are separated by spacer sequences.
  • a “spacer” may be a sequence (e.g. a nucleotide or amino acid sequence) that may be used to separate other sequence elements within a larger polymer. Individual miRNA target sequences or groups of miRNA target sequences may be separated by one or more spacer sequence. In some embodiments, the miRNA target sequences are separated by one or more spacer sequence.
  • the spacer sequence may comprise, for example, at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least thirty nucleotide bases.
  • promoter operably linked to the transgene. In some embodiments, the promoter is a constitutive promoter or an inducible promoter.
  • an “inducible promoter” is a promoter which is only active under specific conditions. For example, expression of the transgene may be induced by a small molecule or drug (e.g. which binds to a promoter, regulatory sequence or to a transcriptional repressor or activator molecule) or by using an environmental trigger.
  • types of inducible promoter include chemically-inducible promoters (e.g. a Tet-on system); temperature-inducible promoters (e.g. Hsp70 or Hsp90-derived promoters); and light-inducible promoters.
  • the promoter is chemically-inducible.
  • a “constitutive promoter” is a promoter which is always active.
  • the promoter is a Ef1 ⁇ promoter.
  • An exemplary Ef1 ⁇ promoter sequence is: CGATAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTA GGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC GAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTT CCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGT ACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGAGTTCGAGGCCTTGCGCTTGCGCTTGCGCTTGCG
  • the promoter comprises or consists of SEQ ID NO: 6.
  • the promoter is a GFAP promoter.
  • a GFAP promoter may target expression to astrocytes.
  • An exemplary GFAP promoter sequence is: AACATATCCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTG AGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCC CCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCA CAGTGCCTGCTTCCCGCCGCACCCCAGCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGG CTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTG CCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGT
  • the promoter comprises or consists of SEQ ID NO: 34.
  • Gamma activated sequence GAS
  • the vector further comprises one or more gamma activated sequence (GAS) operably linked to the transgene.
  • GAS may be triggered by interferon gamma signalling and may be harnessed to render transgene expression inducible, for example by interferon gamma.
  • An exemplary GAS is: TTCCCGGAA (SEQ ID NO: 7)
  • the GAS comprises or consists of a nucleotide sequence that has at In some embodiments, the GAS comprises or consists of SEQ ID NO: 7.
  • the vector further comprises one, two, three or four GASs operably linked to the transgene. In some embodiments, the vector further comprises four GASs operably linked to the transgene. TTCCCGGAATTCCCGGAATTCCCGGAATTCCCGGAA (SEQ ID NO: 12) In some embodiments, the vector further comprises a nucleotide sequence that has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the vector further comprises SEQ ID NO: 12. Other expression control sequences The vector of the present invention may further comprise one or more regulatory elements which may act pre- or post-transcriptionally.
  • the transgene is operably linked to one or more regulatory elements which may act pre- or post-transcriptionally.
  • the one or more regulatory elements may facilitate expression of the transgene in microglia.
  • a “regulatory element” is any nucleotide sequence which facilitates expression of a polypeptide, e.g. acts to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory elements include for example promoters, enhancer elements, post- transcriptional regulatory elements and polyadenylation sites.
  • Post-transcriptional regulatory elements The vector of the invention may comprise one or more post-transcriptional regulatory element.
  • the transgene is operably linked to one or more post-transcriptional regulatory element.
  • the post-transcriptional regulatory element may improve gene expression.
  • the vector of the invention may comprise a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element
  • the transgene is operably linked to a WPRE.
  • Suitable WPRE sequences will be well known to those of skill in the art (see, for example, Zufferey et al. (1999) Journal of Virology 73: 2886-2892; Zanta-Boussif et al. (2009) Gene Therapy 16: 605-619).
  • the WPRE is a wild-type WPRE or is a mutant WPRE.
  • the WPRE may be mutated to abrogate translation of the woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.
  • WHX woodchuck hepatitis virus X protein
  • the WPRE comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 13 or a fragment thereof.
  • the WPRE comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 13 or a fragment thereof.
  • the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 13 or a fragment thereof.
  • the transgene is operably linked to a polyadenylation sequence.
  • a polyadenylation sequence may be inserted after the transgene to improve transgene expression.
  • a polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognised by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tail is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.
  • Suitable polyadenylation sequences will be well known to those of skill in the art (see, for example, Schambach et al. (2007) Molecular Therapy 15: 1167-1173; Choi et al. (2014) Molecular Brain 7: 1-10).
  • Exemplary polyadenylation sequences include the bGH poly(A) signal sequence and SV40pA signal sequence.
  • Kozak sequence The vector of the invention may comprise a Kozak sequence.
  • the transgene is operably linked to a Kozak sequence.
  • a Kozak sequence may be inserted before the start codon to improve the initiation of translation.
  • Suitable Kozak sequences will be well known to the skilled person (see, for example, Kozak (1987) Nucleic Acids Research 15: 8125-8148).
  • the Kozak sequence comprises or consists of a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 14 or a fragment thereof. In some embodiments, the Kozak sequence comprises or consists of the nucleotide sequence SEQ ID NO: 14 or a fragment thereof. GCCACC (SEQ ID NO: 14) Other cis-acting elements
  • the vector (e.g. lentiviral vector) of the invention may comprise any other suitable cis-acting elements, such as one or more of a rev response element (RRE); a retroviral psi packaging element; a primer binding site (PBS); a TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts.
  • RRE rev response element
  • PBS primer binding site
  • TAR TAT activation region
  • splice donor and acceptor sites and central and terminal polypurine tracts.
  • LTRs Long terminal repeats
  • the vector (e.g. lentiviral vector) of the invention may comprise one or more long terminal repeat (LTR). LTRs are responsible for proviral integration and transcription. Typically, a naturally occurring LTR comprises U3, R, and U5 regions.
  • the vector (e.g. lentiviral vector) may comprise a 5’ LTR and/or a 3’ LTR.
  • the vector (e.g. lentiviral vector) may comprise a 5’ LTR and a 3’ LTR.
  • a 5’ LTR comprises R and U5 regions, and optionally comprises a U3 region.
  • a 3’ LTR comprises U3, R and U5 regions.
  • a LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • a LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • a LTR comprises or consists of the nucleotide sequence SEQ ID NO: 15 or a fragment thereof.
  • SEQ ID NO: 15 TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTT GCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGAC CCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (SEQ ID NO: 15)
  • the vector e.g.
  • lentiviral vector of the invention may comprise one or more self-inactivating long terminal repeat (SIN-LTR).
  • SIN-LTR may comprise a deletion that abolishes transcription of the full-length virus after it has incorporated into a host cell.
  • a 3’ SIN-LTR may comprise a deletion in the U3 region removing the promoter/enhancer elements (see, for example, Zufferey et al. (1998) Journal of Virology 72: 9873-9880). This deletion is copied into the 5’ LTR after reverse transcription, thereby making the gene expression in target cells dependent on an internal promoter of choice. Suitable SIN-LTR sequences will be well known to the skilled person (see, for example, Zufferey et al.
  • the 5’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 16 or a fragment thereof.
  • the 5’ LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 16 or a fragment thereof.
  • the 5’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 16 or a fragment thereof.
  • the 5’ LTR and/or the 3’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • the 5’ LTR and/or the 3’ LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • the 5’ LTR and/or the 3’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 15 or a fragment thereof.
  • the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15 or a fragment thereof.
  • the 5’ LTR and the 3’ LTR comprise or consist of the nucleotide sequence SEQ ID NO: 15 or a fragment thereof.
  • the vector (e.g. lentiviral vector) of the invention may comprise a primer binding site (PBS).
  • a PBS is a cis-acting element where a primer may bind to initiate reverse transcription of the RNA genome (see, for example, Lanchy et al. (1998) Journal of Biological Chemistry 273: 24425-24432). Suitable retroviral PBSs will be well known to the skilled person.
  • a PBS comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 17 or a fragment thereof.
  • a PBS comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17 or a fragment thereof.
  • a PBS comprises or consists of the nucleotide sequence SEQ ID NO: 17 or a fragment thereof.
  • the vector (e.g. lentiviral vector) of the invention may comprise a retroviral psi packaging element.
  • a retroviral psi packaging element is a cis-acting element which is involved in regulating the process of packaging the retroviral RNA genome into the viral capsid during replication (see, for example, McBride et al. (1997) Journal of Virology 71: 4544-4554).
  • a retroviral psi packaging element may form part of the 5’ region of the gag gene. Suitable retroviral psi packaging elements will be well known to the skilled person.
  • a retroviral psi packaging element comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 18 or a fragment thereof.
  • a retroviral psi packaging element comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 18 or a fragment thereof.
  • a retroviral psi packaging element comprises or consists of the nucleotide sequence SEQ ID NO: 18 or a fragment thereof.
  • the vector e.g.
  • lentiviral vector of the invention may comprise a rev response element (RRE).
  • RRE is a cis-acting element that enables the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell (see, for example, Pollard et al. (1998) Annual Review of Microbiology 52: 491-532). Suitable RRE sequences will be well known to the skilled person.
  • a RRE comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 19 or a fragment thereof.
  • a RRE comprises or consists of the nucleotide sequence SEQ ID NO: 19 or a fragment thereof.
  • the vector (e.g. lentiviral vector) of the invention may comprise a central polypurine tract (cPPT).
  • a cPPT may allow initiation of plus-strand synthesis (see, for example, Follenzi et al. (2000) Nature Genetics 25: 217-222). Suitable cPPT sequences will be well known to the skilled person.
  • a cPPT comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 20 or a fragment thereof.
  • a cPPT comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 20 or a fragment thereof.
  • a cPPT comprises or consists of the nucleotide sequence SEQ ID NO: 20 or a fragment thereof.
  • the vector of the invention comprises a transgene encoding interleukin 10 (IL-10).
  • Interleukin 10 (IL-10) is a cytokine produced by a variety of immune cells, including T cells, B cells, and macrophages, as well as non-immune cells, such as astrocytes and neurons.
  • IL-10 serves as a potent antiinflammatory mediator by suppressing the expression of pro inflammatory cytokines, such as IL-1, IL-6 and TNF ⁇ , and promoting the expression of anti- inflammatory cytokines, such as IL-1 receptor antagonist (IL-1Ra) and IL-10 itself. Additionally, IL-10 has profound impact on microglia behaviour toward a phagocytic phenotype. In some embodiments, the IL-10 is human IL-10.
  • an exemplary Interleukin-10 amino acid sequence is: MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKE SLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKS KAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 8)
  • the IL-10 comprises or consists of an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 8 or a fragment thereof.
  • the IL-10 comprises or consists of an amino acid sequence that has at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 8 or a fragment thereof.
  • the IL-10 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or a fragment thereof.
  • a fragment and/or variant of IL-10 may retain IL-10 activity (e.g. the activity of SEQ ID NO: 8).
  • a fragment and/or variant of IL-10 may act as an anti-inflammatory mediator by suppressing the expression of pro-inflammatory cytokines, such as IL-1, IL-6 and TNF ⁇ , and/or promoting the expression of anti-inflammatory cytokines, such as IL-1 receptor antagonist (IL-1Ra) and IL-10.
  • IL-1Ra IL-1 receptor antagonist
  • a fragment and/or variant of IL-10 may have the same or similar activity to IL-10, for example may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the activity of IL-10 (e.g. the IL-10 of SEQ ID NO: 8).
  • the transgene encoding IL-10 comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the transgene encoding IL-10 comprises or consists of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8.
  • An exemplary nucleotide sequence encoding Interleukin-10 is: ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGG CACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAG ATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAG TCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCT GGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGG AGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGC AAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAG TG
  • the transgene encoding IL-10 comprises or consists of SEQ ID NO: 9.
  • VECTORS A vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • the vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid and/or facilitating the expression of the protein encoded by a segment of nucleic acid.
  • Vectors may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.
  • Vectors comprising polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.
  • Transfection may refer to a general process of incorporating a nucleic acid into a cell and includes a process using a non-viral vector to deliver a polynucleotide to a cell.
  • Transduction may refer to a process of incorporating a nucleic acid into a cell using a viral vector.
  • the vector of the invention is a viral vector.
  • the vector of the invention may be a lentiviral vector, although it is contemplated that other viral vectors may be used.
  • suitable viral vectors include those described in Lundstrom, et al. (2016) Diseases 6: 42.
  • suitable viral vectors include a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an alphaviral vector, a flaviviral vector, a rhabdoviral vector, a measles viral vector, a Newcastle disease viral vector, a poxviral vector and a picornaviral vector.
  • the vector of the invention may be in the form of a viral vector particle.
  • the viral vector of the invention is in the form of a lentiviral vector particle.
  • the vector may be an integrating viral vector or a non-integrating viral vector.
  • Retroviral and lentiviral vectors The vector of the invention may, for example, be a retroviral vector or a lentiviral vector.
  • the vector of the invention may, for example, be a retroviral vector particle or a lentiviral vector particle.
  • a retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified.
  • MMV murine leukaemia virus
  • HTLV human T-cell leukaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • FuSV Fujinami sarcoma virus
  • Mo-MLV Moloney murine leukaemia virus
  • FBR MSV FBR murine osteosarcoma virus
  • Mo-MSV Moloney murine sarcoma virus
  • Abelson murine leukaemia virus A-MLV
  • MC29 avian myelocytomatosis virus-29
  • AEV avian erythroblastosis virus
  • Retroviruses may be broadly divided into two categories, “simple” and “complex”.
  • Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.
  • the basic structure of retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components – these are polypeptides required for the assembly of viral particles.
  • Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • these genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are responsible for proviral integration and transcription.
  • LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.
  • the LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5.
  • U3 is derived from the sequence unique to the 3’ end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA.
  • U5 is derived from the sequence unique to the 5’ end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • a defective retroviral vector genome gag, pol and env may be absent or not functional.
  • at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective.
  • Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome.
  • Lentivirus vectors are part of the larger group of retroviral vectors.
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV).
  • non-primate lentiviruses examples include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • the lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • lentiviral vector is a vector which comprises at least one component part derivable from a lentivirus. Suitably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the lentiviral vector may be a “primate” vector.
  • the lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans).
  • non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.
  • lentivirus-based vectors HIV-1- and HIV-2-based vectors are described below.
  • the HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE.
  • Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm.
  • full-length HIV-1 RNAs accumulate in the nucleus.
  • a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE.
  • Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.
  • Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.
  • the viral vector used in the invention has a minimal viral genome.
  • minimal viral genome it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell.
  • the vector lacks a functional gag-pol and/or env gene
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell.
  • These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).
  • the vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted.
  • SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors.
  • the transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.
  • the vector may be integration-defective. Integration defective lentiviral vectors can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.
  • the vector is an integration-defective lentiviral vector. In some embodiments, the vector is an integration-proficient lentiviral vector. Suitably, the vector is VSV-G-pseudotyped. In some embodiments, the vector is a VSV-G- pseudotyped lentiviral vector particle.
  • Adeno-associated virus In one aspect, the vector is an adeno-associated viral (AAV) vector. In one aspect, the vector is an adeno-associated viral (AAV) vector particle.
  • AAV adeno-associated virus
  • AAV adeno-associated viral vector
  • AAV adeno-associated viral vector particle.
  • the use of AAV as a vector may be an alternative to the use of a lentiviral vector comprising the one or more miRNA target sequence.
  • the AAV vector may lack miRNA target sequences.
  • the AAV vector may be of a serotype for astrocyte-specific expression and/or may comprise an astrocyte-specific promoter.
  • Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.
  • the AAV particle may comprise an AAV genome or a fragment or derivative thereof.
  • An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle.
  • Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle.
  • the AAV genome of the AAV vector of the invention is typically replication-deficient.
  • the AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form.
  • the use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.
  • the AAV genome may be from any naturally derived serotype, isolate or clade of AAV.
  • the AAV genome may be the full genome of a naturally occurring AAV.
  • AAVs occurring in nature may be classified according to various biological systems. Commonly, AAVs are referred to in terms of their serotype.
  • a serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies.
  • a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype.
  • AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, and also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain.
  • the AAV is AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43.
  • the AAV comprises a capsid protein derived from an AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 capsid protein. Reviews of AAV serotypes may be found in Choi et al. (2005) Curr. Gene Ther.5: 299-310 and Wu et al. (2006) Molecular Therapy 14: 316-27.
  • sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC 005889 AY388617 AAV capsid protein
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises a peptide comprising or consisting of the amino acid sequence PGV
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises a peptide comprising or consisting of the amino acid sequence AGPGVPGRF (SEQ ID NO: 22), or a variant thereof having up to two amino acid substitutions, additions or deletions.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises a peptide comprising or consisting of the amino acid sequence NGVRSVG (SEQ ID NO: 23), or a variant thereof having up to two amino acid substitutions, additions or deletions.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises a peptide comprising or consisting of the amino acid sequence AGNGVRSVG (SEQ ID NO: 24), or a variant thereof having up to two amino acid substitutions, additions or deletions.
  • the AAV capsid protein is an AAV VP1 capsid protein.
  • the AAV is AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43.
  • the AAV is AAV9.
  • AAV9 capsid protein amino acid sequence is: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA KTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMA SGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDN AYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTS TVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTG NNFQFSY
  • corresponding to”, “reference to” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence may refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as that of an AAV capsid protein, can be aligned to a reference sequence by introducing gaps to optimise residue matches between the two sequences.
  • Example AAV capsid sequences that comprise the peptide of the disclosure are: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA KTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMA SGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDN AYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGV
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, In some embodiments, the capsid protein comprises or consists of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the capsid protein comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 28.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29.
  • the capsid protein comprises or consists of the amino acid sequence of SEQ ID NO: 28.
  • the capsid protein comprises or consists of the amino acid sequence of SEQ ID NO: 29.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutation A587G, wherein the amino acids are numbered with reference to SEQ ID NO: 25.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutation Q592A, wherein the amino acids are numbered with reference to SEQ ID NO: 25. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutations A587G and Q592A, wherein the amino acids are numbered with reference to SEQ ID NO: 25. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutation W503A, wherein the amino acids are numbered with reference to SEQ ID NO: 25.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutations A587G and W503A, wherein the amino acids are numbered with reference to SEQ ID NO: 25. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutations Q592A and W503A, wherein the amino acids are numbered with reference to SEQ ID NO: 25. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutations A587G, Q592A and W503A, wherein the amino acids are numbered with reference to SEQ ID NO: 25.
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutation K449R, wherein the amino acids are numbered with reference to SEQ ID NO: 25. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein further comprises the mutations A587G, Q592A, W503A and K449R, wherein the amino acids are numbered with reference to SEQ ID NO: 25.
  • Example AAV capsid proteins of the invention are: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA KTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMA SGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDN AYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTS TVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTG NNFQFSYEFENVP
  • the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein is encoded by a nucleotide sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the AAV vector particle comprises an AAV capsid protein, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 33.
  • the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR).
  • ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell.
  • the AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle.
  • the rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof.
  • the cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle.
  • a promoter may be operably linked to each of the packaging genes.
  • promoters include the p5, p19 and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571).
  • the p5 and p19 promoters are generally used to express the rep gene
  • the p40 promoter is generally used to express the cap gene.
  • the AAV genome used in the AAV vector of the invention may therefore be the full genome used to prepare an AAV vector or vector particle in vitro. However, while such a vector may in principle be administered to patients, this will rarely be done in practice.
  • the AAV genome will be derivatised for the purpose of administration to patients.
  • Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art.
  • Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (2007) Virology Journal 4: 99, and in Choi et al. and Wu et al., referenced above.
  • Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function.
  • a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more.
  • ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR.
  • a preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome.
  • the AAV comprises at least one, such as two, AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 ITRs.
  • the AAV comprises at least one, such as two, AAV serotype 2 ITRs.
  • the one or more ITRs will preferably flank the nucleotide sequence of interest (which may also be referred to as a transgene) at either end.
  • the inclusion of one or more ITRs is preferred to aid concatamer formation in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases.
  • ITR elements will be the only sequences retained from the native AAV genome in the derivative.
  • a derivative will preferably not include the rep and/or cap for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome.
  • reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.
  • the following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes.
  • derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome.
  • Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.
  • the invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome.
  • the invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.
  • the AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype.
  • the AAV particles of the invention also include mosaic forms wherein a mixture of capsid proteins from two or more different serotypes makes up the viral capsid.
  • the AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface.
  • ligands may include antibodies for targeting a particular cell surface receptor.
  • the vector is a non-viral particle.
  • the vector is a non-viral particle, wherein the non-viral particle comprises a peptide, wherein the peptide: (a) comprises or consists of the amino acid sequence PGVPGRF (SEQ ID NO: 21), or a variant thereof having up to two amino acid substitutions, additions or deletions; or (b) comprises or consists of the amino acid sequence NGVRSVG (SEQ ID NO: 23), or i t th f h i t t i b tit ti dditi d l ti
  • the vector is a non-viral particle, wherein the non-viral particle comprises a peptide, wherein the peptide: (a) comprises or consists of the amino acid sequence AGPGVPGRF (SEQ ID NO: 22), or a variant thereof having up to two amino acid substitutions, additions or deletions; or (b) comprises or consists of the amino acid sequence AGNGVRSVG (SEQ ID NO:
  • the non-viral particle is a nanoparticle. In some embodiments, the non- viral particle is a lipid nanoparticle. In some embodiments, the non-viral particle is a liposome.
  • EXEMPLARY VECTORS the vector comprises from 5’ to 3’: the promoter; the transgene; and the one or more miRNA target sequence. In some embodiments, the vector comprises from 5’ to 3’: the Ef1 ⁇ promoter; the transgene encoding IL-10; and the one or more miRNA target sequence. In some embodiments, the vector comprises from 5’ to 3’: the Mx1 minimal promoter; the transgene encoding IL-10; and the one or more miRNA target sequence.
  • the vector comprises from 5’ to 3’: the one or more GAS; the Mx1 minimal promoter; the transgene encoding IL-10; and the one or more miRNA target sequence.
  • Exemplary vectors include: CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAAT ATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGTTTTCCAATGATGAGCACTTT TAAA
  • the vector comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 10 or a fragment thereof.
  • the vector comprises or consists of the nucleotide sequence SEQ ID NO: 10 or a fragment thereof.
  • the vector comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 11 or a fragment thereof.
  • the vector comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 11 or a fragment thereof.
  • the vector comprises or consists of the nucleotide sequence SEQ ID NO: 11 or a fragment thereof.
  • a further exemplary vector is: TTGGCCACTCCCTCTCTCTGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACTAGTAACATAT CCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCAGGG CCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCC TGCTTCCCGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCAT TGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGC TGTGGCGCCACCGGCGGTGGAGAACAAG
  • the vector comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 35 or a fragment thereof.
  • the vector comprises or consists of the nucleotide sequence SEQ ID NO: 35 or a fragment thereof. VARIANTS, DERIVATIVES, ANALOGUES, HOMOLOGUES AND FRAGMENTS
  • the invention also encompasses variants, derivatives, analogues, homologues and fragments thereof.
  • a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions.
  • a variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.
  • derivative as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.
  • analogue as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.
  • amino acid substitutions may be made, for example from 1, 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues. Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • Conservative substitutions may be made, for example according to the table below.
  • Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • the term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence.
  • the term “homology” can be equated with “identity”.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • the homologues will can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences. Percent homology may be calculated over contiguous sequences, i.e.
  • one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time.
  • This is called an “ungapped” alignment.
  • ungapped alignments are performed only over a relatively short number of residues.
  • BLAST and FASTA are available for offline and online searching. However, for some applications, it is preferred to use the GCG Bestfit program.
  • Another tool, BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).
  • the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • BLOSUM62 the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • percent homology preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay.
  • “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.
  • Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion either side of the insertion site may be made.
  • the flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • Codon optimisation The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.
  • the invention provides a method of treatment comprising administering the vector, cell or pharmaceutical composition of the invention to a subject in need thereof.
  • the invention provides a method for treating dopaminergic neurons by administering the vector of the invention. These neurons are vital to diverse brain functions, including: voluntary movement, reward, addiction and stress. Progressive loss of dopaminergic neurons is responsible for neurodegenerative diseases, such as Parkinson’s Disease.
  • the invention provides a method of treating or preventing Parkinson’s disease comprising administering the vector, cell or pharmaceutical composition of the invention to a subject in need thereof.
  • the treatment of mammals, particularly humans, is preferred.
  • the method of treatment provides the transgene to the central nervous system (CNS) of the subject.
  • the method of treatment provides the transgene to the microglia of the subject.
  • the method of treatment provides an improvement in learning and/or cognitive function in the subject.
  • Methods for measuring learning and/or cognitive function are known to the skilled person. For example, in humans the General Practitioner Assessment of Cognition (GPCOG) test may be used.
  • Alternative cognitive tests include but are not limited to the Mini Mental State Examination (MMSE), The Six-item Cognitive Impairment Test (6CIT), Abbreviated Mental Test (AMT) and Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE).
  • PHARMACEUTICAL COMPOSITIONS AND INJECTED SOLUTIONS Although the agents for use in the invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • a “pharmaceutical composition” may refer to a preparation which is stable and in a form which is acceptable to the patient.
  • the medicaments, for example vectors and vector particles, of the invention may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the medicament, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabiliser or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, e.g. intravenous or intra-arterial.
  • the pharmaceutical composition is typically in liquid form.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, serum albumin may be used in the composition.
  • PF68 pluronic acid
  • serum albumin may be used in the composition.
  • the active ingredient may be in the form of an aqueous solution which is pyrogen- free, and has suitable pH, isotonicity and stability.
  • aqueous solution which is pyrogen- free, and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers
  • the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.
  • kits comprising the vector, cells and/or pharmaceutical composition of the present invention.
  • said kits are for use in the methods and used as described herein, e.g., the therapeutic methods as described herein.
  • kits comprise instructions for use of the kit components.
  • ADMINISTRATION in some embodiments, the vector, cell or pharmaceutical composition is administered to a subject systemically or locally.
  • systemic delivery or “systemic administration” as used herein, means that the agent of the invention is administered into the circulatory system, e.g. to achieve broad distribution of the agent.
  • topical or local administration restricts the delivery of the agent to a localised area, e.g. intracerebral administration entails direct injection into the brain.
  • the vector, cell or pharmaceutical composition is administered intravenously. In some embodiments, the vector, cell or pharmaceutical composition is administered intra- arterially. In some embodiments, the vector, cell or pharmaceutical composition is administered to a subject intracranially or intraparenchymally. In some embodiments, the vector, cell or pharmaceutical composition is administered to a subject intracerebrally.
  • DOSAGE The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound the age body weight general health sex diet mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.
  • subject refers to either a human or non-human animal. Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g.
  • mice, rats or guinea pigs), pigs and cats The non-human animal may be a companion animal.
  • the subject is a human.
  • DISEASES Neurodegenerative diseases are diseases which primarily affect neurons. These diseases result in the progressive loss of structure or function of nerve cells (neurons) including neuronal cell death. This causes problems with movement (called ataxias), or mental functioning (called dementias).
  • the vector, cell or pharmaceutical composition of the invention may be administered to a subject in a method for treating and/or delaying progression of a neurodegenerative disease.
  • the subject may have a neurodegenerative disease, i.e. has been diagnosed as having the neurodegenerative disease or is suspected as having the neurodegenerative disease, or may be asymptomatic.
  • the subject may have a genetic predisposition to the neurodegenerative disease.
  • the subject may have one or more family members with a neurodegenerative disease.
  • the neurodegenerative disease is Parkinson’s disease.
  • the neurodegenerative disease is a Parkinson’s disease-related disorder.
  • the Parkinson’s disease-related disorder is Parkinson’s dementia.
  • the Parkinson’s disease-related disorder is dementia with Lewy bodies.
  • the neurodegenerative disease is lower body Parkinson’s syndrome.
  • the neurodegenerative disease is Alzheimer’s disease, frontotemporal dementia, Pick disease, Multiple systemic atrophy (MSA) or multiple sclerosis.
  • the neurodegenerative disorder is associated with Lewy bodies.
  • Lewy bodies are abnormal aggregates of protein that develop inside nerve cells and may be found within the substantia nigra or within the cortex. Lewy bodies comprise alpha-synuclein associated with other proteins which may include ubiquitin, neurofilament protein and alpha B crystallin. Lewy neurites may be associated with Lewy bodies. In some embodiments, the neurodegenerative disease is dementia with Lewy bodies. In some embodiments, alpha-synuclein aggregates are decreased, for example in the substantia nigra pars compacta (SNpc). The decrease may be, for example, by at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%.
  • Amounts of alpha-synuclein aggregates may be determined using methods known in the art, for example as described herein.
  • microglial phagocytic capacity is increased. The increase may be, for example, by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5- fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold or at least about 10-fold.
  • Microglial phagocytic capacity may be determined using methods known in the art, for example as described herein.
  • the subject may have Parkinson’s disease and dementia.
  • the subject may have Parkinson’s disease and Parkinson’s dementia.
  • the present invention provides a cell comprising the vector of the invention.
  • the cell may be an isolated cell.
  • the cell may be a human cell, suitably an isolated human cell.
  • the cell may be any cell type known in the art.
  • Method of making a cell The vector of the present invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.
  • the vector of the present invention is introduced into the cell by transfection or transduction.
  • the present invention provides a method of making the cell of the invention. The method may comprise introducing the vector of the invention into the cell, for example by transfection or transduction.
  • the cell may be from a sample isolated from a subject.
  • the cell may be further
  • the cell of the present invention may be generated by a method comprising the following steps: (i) isolation of a cell-containing sample from a subject or provision of a cell- containing sample; and (ii) transduction or transfection of the cell-containing sample with the vector of the invention, to provide a population of engineered cells.
  • the cells may be cultured prior to, or after, introducing the vector of the invention.
  • the steps may be performed in a closed and sterile cell culture system.
  • EXAMPLE 1 RESULTS IL-10 is efficiently and specifically expresser in microglia cells
  • this method enabled high cell-specific expression of the therapeutic gene.
  • RNA levels in primary cultures infected with LV-miRtag-GFP to test the efficient and selective production of the green fluorescent protein (GFP) induced by the miRNA detargeting platform.
  • GFP green fluorescent protein
  • IL-10 human interleukin- 10
  • LV-IL10 LV- miRtag-IL10
  • IL-10 is an effective neuroprotective agent in experimental model of Parkinson's disease (PD)
  • PD Parkinson's disease
  • SNc substantia nigra pars compacta
  • aS alpha-synuclein
  • mice were divided into two groups: the IL-10 group received bilateral SNc injections of LV-IL10, and the SNCA + IL10 group received an initial injection of LV-IL10 followed by an injection of LV-SNCA three weeks later.
  • Ten weeks following LV-SNCA injection there was no discernible decline in the number of tyrosine hydroxylase (TH) positive neurons in the SNc of SNCA + IL10 mice, demonstrating that LV-IL10 overexpression produced a prolonged neuroprotective effect.
  • TH tyrosine hydroxylase
  • elevated IL-10 expression may offer neuroprotection against PD.
  • Fig 2 The work shows that IL-10 overexpression in microglia can have a neuroprotective impact in an animal model of PD.
  • LV-inducible platform responds to extracellular IFN-gamma by inducing IL-10 transcription
  • GAS gamma-activated sequences
  • DISCUSSION In response to IFN gamma signaling, the data described here show the establishment of an inducible platform for the release of IL-10 a strong anti-inflammatory cytokine The success of IL-10 expression and release from microglia was increased through the use of miRNA detargeting, which forms the foundation of this platform.
  • the promoter region of gamma activated sequences (GAS) allowed for the activation of IL-10 production in response to IFN gamma. Given that IFN gamma is known to be released during inflammation and to be able to contribute to persistent neurodegeneration, this trait is very important. By designing a platform that responds to IFN gamma, we can potentially limit this harmful effect and instead promote neuroprotection.
  • METHODS Viral vector and lentivirus production
  • the mirTAG and IL-10 sequences have been inserted in a third-generation lentiviral backbone, downstream of the GAS and Mx1 minimal promoter.
  • MirTAG is made up of three sequences that are repeated four times and are each complementary to miRNAs 124, 338, and 31.
  • Green fluorescent protein (GFP) was initially cloned along with the mirTAG sequence as a reporter to evaluate the expression specificity.
  • the human IL-10 sequence was then inserted after the GFP fragment had been broken up by enzymatic digestion.
  • Lentiviral particles that were VSVg-coated and replication-incompetent were packaged in 293T cells.
  • the day before the infection 7.5 million 293T cells were plated in a petri dish with a 150mm diameter.
  • the traditional CaCl2 transfection method was used.
  • the medium was collected after 30 hours, filtered (0.44 m cellulose acetate), and centrifuged for 2 hours at 50,000 g.
  • Animals Wild type mice were purchased from Jackson Laboratories and housed in the San Raffaele Hospital animal Facility. They were given free access to food and water while being raised in an environment with a 12 hour dark-light cycle, controlled temperature (25°C), and relative humidity (50–60%).
  • lentiviruses were used at a concentration of 1 x 10 9 VP/ml. Mice were sacrificed after 5 or 10 weeks following a specific protocol depending on the type of the analysis. Following the previously mentioned SNc coordinates, the microdialysis probe was inserted following the lentiviral injection. Sutures and a coating of quickly setting cement were used to secure the probe in place. Perfusion fluid CNS (NaCl 147 mmol/L, KCl 2.7 mmol/L, CaCl21,2 mmol/L, MgCl20,85 mmol/L) implemented with 2% bovine serum albumine (BSA) was used to perfuse the probes during the procedure.
  • BSA bovine serum albumine
  • microdialysis The microdialysis probe with a 55 kDA cutoff was implanted 4 weeks after the lentivirus injection. Both surgical techniques were as previously described. The probes were perfused with Perfusion fluid CNS at a flow rate of 1 l/min a day after they were implanted. Samples were taken every 50 minutes after a 15-minute rinse and immediately frozen with dry ice.7-8 hours were spent collecting samples. Animal sacrifices were used to confirm histologically that the probes had been positioned correctly at the conclusion of the experiment. Primary cell culture P2 C57BL mice were dissected, and cortices were collected to create a microglia primary culture.
  • the tissue was first incubated at 37°C for 30 minutes in a solution containing 0.05% Trypsin EDTA (Merck) and 100 g/ml DNAse I (Roche), after which the cells were mechanically separated into a suspension and put through a 40 M strainer. The cells were then resuspended NEAA, and 1x Sodium Pyruvate; all from Merck) and plated in T-75 Matrigel GFR (Growth Factor Reduced, Corning) coated flasks (one flask every two pups). The flasks were incubated for approximately 7 days at 37°C with 5% CO 2 .
  • Flasks were shaken at 400 rpm for four hours at 37°C to separate microglia from the underlying astrocyte layer. Microglia-containing medium was gathered, counted, and seeded in 24-well plates (about 400.000 cells per well). Cells were infected with the desired lentivirus in 1 l/well the following day. Immunostaining Brains from mice were collected and post-fixed the following day after being perfused with freshly prepared 4% PFA in PBS. Brains were successively immersed in a 20% sucrose cryoprotective solution, quickly frozen, and then cut into 50 m slices with a cryostat.
  • Free- floating slices were permeableized for 20 minutes with 2% Triton and 10 minutes with 3% H 2 O 2 and 10% CH3OH PBS solution for the immunofluorescence analysis.
  • Slices were incubated for an hour with a solution of 3% BSA and 0.3% Tween after three washes to saturate the atypical sites.
  • Slices were then incubated with the target primary antibody in a diluted blocking solution (1% BSA) overnight at 4°C. Then, slices were incubated with the appropriate secondary antibody for one hour in the dark the following day, after which they were mounted for imaging.
  • Slices were incubated with a biotinylated secondary antibody for the immunohistochemical analysis after being incubated with the primary antibody overnight.
  • the slices were then successively coupled with avidin/biotinylated enzyme complex and revealed with the DAB solution (Vector Laboratories).
  • Stereological count Five 50 m thick SN slices were collected every 200 m for the entire series of SN slices used for each mouse in order to mark the TH positive cells using an immunohistochemical reaction.
  • the Leica DM400B motorized microscope with Stereo Investigator software (MBF Bioscience) was used to count the cells at a 40x magnification. The total number of TH positive cells was then calculated using the optical fractionator stereological probe.
  • Real-Time PCR TRI reagent (Merck) was used to extract total RNA from cells in accordance with the manufacturer's instructions.
  • the ImProm-II Reverse Transcription System from Promega was then used to reverse-transcribe RNA into cDNA. Titan HotTaq EvaGreen qPCR Mix (BioAtlas), custom-designed oligos, and Real Time PCR were used in the experiment. Results were reported as the 2 ⁇ Ct after expression levels were normalized with respect to 18S expression. Both the dialysate and the nigrostriatal tissue were tested for the presence of human IL10 using the ELISA method. On mice injected with LV mirTAG-IL10 or LV Mock, the microdialysis was carried out as previously described, and the samples obtained were stored at -80°C until needed.
  • mice that had received injections of LV mirTAG-IL10 or LV Mock were then perfused with saline solution to collect tissue samples.
  • the brains were then immediately removed and immediately cut using an acrylic brain matrix (coronal, 1mm) to collect only the SN.
  • the fresh tissue was centrifuged after being mechanically lysed in C+ medium using cOmplete-EDTA free. Before use, the supernatant was collected and kept at -80°C. Using purified and biotinylated antibody pairs, the samples thus obtained were used to perform a standard sandwich ELISA (Becton Dickinson, CA, USA).
  • Blocking Buffer PBS with 2% BSA
  • rhIL-10 R&D Systems, MN, USA
  • the detection threshold was 15 pg/ml.
  • the weight of the tissue that was collected was used to normalize the tissue sample results.
  • EXAMPLE 2 RESULTS IL-10 enhanced phagocytosis efficiently reduces ⁇ SYN aggregate burden in SNpc
  • CD68 a lysosomal associated protein whose levels are associated with phagosome/lysosome activity.
  • CD68+ intracellular puncta were markedly increased in IBA1+ microglia in both IL10 and SNCA WT + IL10 animals (Fig.4A, B).
  • CD68+ cells were also significantly increased in size and co-localized with the pS129 ⁇ SYN+ signal in SNCA WT + IL10 mice (Fig.4A, C), highlighting the recruitment of ⁇ SYN aggregates into the phagosome/lysosome compartment.
  • pS129 ⁇ SYN+ aggregates were quantified in the SNpc tissue of the different animal groups.
  • IL-10 enhanced microglial phagocytic capacity, enabling a significant clearance of ⁇ SYN aggregates in the SNpc tissue.
  • METHODS Phagocytosis assays Primary microglia cultures were submitted to different phagocytosis assays. In one assay, microglia were seeded with fluorescent preformed fibrils of ⁇ SYN (Fluo488-PFFs) at a concentration of 2.3 ⁇ M. In the second assay, microglia were exposed to pHrodo Red SE (Thermo Fischer) labelled-synaptosomes preparation. Labelled-synaptosomes were obtained from isolated wild type adult mouse striata.
  • the tissue was homogenized in Syn-PER reagent (Thermo Fisher) using a glass Dounce homogenizer with 4 ml of Syn-PER reagent.
  • the homogenate was transferred in a 15 ml falcon and centrifuged 1200 x g, 10 min at 4°C. The resulting supernatant was collected and centrifuged at 15,000 x g for 20 min at 4°C.
  • the pellet containing the purified synaptosome fraction was weighted for subsequent treatments.
  • pHrodo was reconstituted in 150 ul DMSO (Merck) and added to synaptosomes resuspended in pH 9 0.1M sodium carbonate buffer (Merck) at a final concentration of 200mg/ml.

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Abstract

L'invention concerne un vecteur d'expression spécifique au système nerveux central (SNC), le vecteur comprenant un transgène codant pour l'interleukine 10 (IL-10), et le transgène étant fonctionnellement lié à une ou plusieurs séquences de régulation d'expression.
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