WO2019186567A1 - Méthodes de traitement de maladies neurodégénératives - Google Patents

Méthodes de traitement de maladies neurodégénératives Download PDF

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WO2019186567A1
WO2019186567A1 PCT/IL2019/050365 IL2019050365W WO2019186567A1 WO 2019186567 A1 WO2019186567 A1 WO 2019186567A1 IL 2019050365 W IL2019050365 W IL 2019050365W WO 2019186567 A1 WO2019186567 A1 WO 2019186567A1
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Prior art keywords
promoter
cell
sequence
activated
nucleic acid
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PCT/IL2019/050365
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Inventor
Daniel DEITCH
Mor PASI
Liat TSORAN
Sagi ANGEL
Einan FARHI
Mor SELA
Avital BAILEN
Re'em SADEH
Nitzan KEIDAR
Ori TULCHINSKY
Ramon Y. BIRNBAUM
Lital Alfonta
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B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
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Publication of WO2019186567A1 publication Critical patent/WO2019186567A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/1101—IkappaB kinase (2.7.11.10)
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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

  • the present invention is in the field of molecular and cellular biology, and is directed to methods of use such as for treating neurodegenerative diseases.
  • astrocytes In both sporadic amyotrophic lateral sclerosis (SALS) and familial ALS (FALS), non-neuronal cells, such as astrocytes and microglia, directly contribute to motor neuronal damage and cell death. Under disease or injury conditions, astrocytes change their normal morphology and function, gaining a new neurotoxic function and rapidly killing motor neurons, thus termed“reactive astrocytes”. Reactive astrocytes have an altered gene expression profile compared to quiescent astrocytes.
  • activated microglia cells are required to induce astrocytes to become reactive, by secreting cytokines, such as interleukin 1 alpha (IL-la) and tumor necrosis factor alpha (TNF-a), which is mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB).
  • cytokines such as interleukin 1 alpha (IL-la) and tumor necrosis factor alpha (TNF-a)
  • NF-KB nuclear factor kappa-light-chain-enhancer of activated B cells
  • the present invention is directed to methods of inhibition of reactive gliosis in a subject.
  • methods of the present invention are directed to targeted inhibition of an activated microglia cell and not of a resting microglia cell.
  • methods of the present invention are directed to targeted apoptosis of an activated astrocyte cell and not of a resting astrocyte cell.
  • methods of the present invention are directed to targeted inhibition of an activated microglia cell and not of a resting microglia cell, and to apoptosis of an activated astrocyte cell and not of a resting astrocyte cell.
  • methods of the present invention utilize promoter polynucleotide sequences predominantly active in an activated microglia cell, an activated astrocyte cell, or both, and not in a resting microglia cell, a resting astrocyte cell, or both. In some embodiments, methods of the present invention are directed to treating or ameliorating a neurodegenerative disease in a subject in need thereof.
  • a method for inhibiting reactive gliosis in a subject comprising administering to the subject a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • a method for inhibiting reactive gliosis in a subject comprising administering to the subject a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is operably coupled to a promoter sequence comprising the EMR1 promoter, and wherein the antisense sequence is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is operably coupled to a promoter sequence comprising the EMR1 promoter, and wherein the antisense sequence is transcribed, or expressed in
  • the antisense sequence is transcribed from the first polynucleotide under the regulation of a first promoter sequence which is predominantly activated in the activated microglia cell and not in a resting microglia cell.
  • the first promoter comprises the EGF-like module- containing mucin-like hormone receptor-like 1 (EMR1) promoter sequence.
  • EMR1 mucin-like hormone receptor-like 1
  • the antisense sequence being transcribed from the first polynucleotide inhibits the expression of the IKKB in an activated microglia cell and not in a resting microglia cell.
  • the composition further comprises a second polynucleotide encoding an apoptosis-inducing polypeptide, wherein the apoptosis- inducing polypeptide is expressed in an activated astrocyte cell and not in a resting astrocyte cell.
  • the composition comprises a clustered regularly interspaced short palindromic repeat associated protein 9 system (CRISPR/Cas9).
  • the apoptosis-inducing polypeptide is a reversed-Caspase
  • a second promoter sequence is used for trans-activation of the expression of the apoptosis-inducing protein predominantly in the activated astrocyte cell and not in a resting astrocyte cell.
  • the second promoter comprises the six-transmembrane epithelial antigen of prostate family member 4 (Steap4) promoter or the tissue inhibitor of metalloproteinase 1 (Timpl) promoter.
  • the second polynucleotide comprises a cis promoter inducing the expression of the polypeptide predominantly in the activated astrocyte and not in the resting astrocyte cell.
  • the cis promoter is an exogenous promoter.
  • the cis promoter sequence comprises the adenovirus major late promoter (MLP).
  • MLP adenovirus major late promoter
  • the first promoter sequence, the second promoter sequence, or both are mammalian promoters.
  • the mammalian is a human.
  • the subject is afflicted with a neurodegenerative disease.
  • the neurodegenerative disease is selected from the group consisting of: Huntington's disease, Alzheimer's disease, Parkinson’s disease, and a brain injury.
  • administering is by intrathecal injection.
  • the administering further comprises utilizing an adeno- associated viral delivery system.
  • composition comprising one or more vectors comprising: a first nucleic acid sequence encoding at least one CAS polypeptide, wherein the first nucleic acid sequence is operably coupled to a first promoter sequence comprising the EMR1 promoter; a second nucleic acid sequence encoding a sgRNA complementary to a target nucleotide comprising the IKKB sequence, wherein the second nucleic acid sequence is operably coupled to the first promoter, wherein the second nucleic acid sequence comprises one ribozyme sequence upstream of the sgRNA and one ribozyme sequence downstream of the sgRNA; a third nucleic acid sequence encoding at least one dCAS polypeptide, wherein the third nucleic acid sequence is operably coupled to a second promoter sequence comprising any one of the Steap4 promoter or the Timpl promoter; a fourth nucleic acid sequence encoding a sgRNA complementary to a target nucleot
  • the dCAS polypeptide is a dCAS9 fused to a transcription regulator.
  • the third promoter is an exogenous promoter.
  • the exogenous promoter sequence comprises the MLP.
  • the transcription regulator is a transcription activator.
  • Fig. 1 is a vertical graph showing the effect of inhibitor of nuclear factor kappa- B kinase subunit beta (IKKb) suppression on tumor necrosis factor alpha (TNFa) production.
  • IKKb nuclear factor kappa- B kinase subunit beta
  • TNFa tumor necrosis factor alpha
  • Fig. 2 is a vertical bar graph showing the results of a dual Luciferase assay in C8-D30 astrocyte cells to detect the activation of the six-transmembrane epithelial antigen of prostate family member 4 promoter (pSTEAP4), or the tissue inhibitor of metalloproteinase 1 promoter (pTIMPl).
  • the dual luciferase reporter vectors pGL3 included the promoter TIMP1 or STEAP4 downstream of the Firefly luciferase gene.
  • the activities of Firefly luciferase and Renilla luciferase were measured sequentially, and the Firefly luciferase/Renilla luciferase ratio was calculated and normalized relative to cells co-transfected with the empty pGL3 and Renilla vectors. Data represent the mean ⁇ S.E. of three biological replicates. The results show that cells which were co transfected with the pGL3-pTIMPl vector, or pGL3-pSTEAP4 had 27.5-fold higher or 8.l4-fold higher luciferase activity, respectively, compared to cells that were co transfected with the empty vector.
  • Figs. 3A-3B are micrographs showing that C8-D30 astrocyte cells co-transfected with lentivirus construct comprising the pTimpl-VP64, and pSynt expressing mCherry downstream to the pro-apoptotic protein reverse-Caspase 3. Images were taken 24 hours post co-transfection. (3A) is a bright field image, and (3B) is a fluorescent micrograph of the C8-D30 astrocyte cells post dead Cas9 activation, showing mCherrry expression, therefore also indicating expression of the pro-apoptotic protein revesrse-Caspase3. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to a method of treating reactive gliosis in a subject by inhibiting a microglia cell, an astrocyte cell, or both, in the subject.
  • the method comprises inhibition of an active microglia cell and not of a resting or quiescent microglia cell.
  • the method further comprises induction of apoptosis of an activated astrocyte cell and not of a resting or quiescent astrocyte cell.
  • the method comprises inhibition of an activate microglia cell and induction of apoptosis of an activated astrocyte cell.
  • the method of the present invention utilizes a first promoter polynucleotide sequence predominantly active in a first type of an activated cell for driving the expression of a specific polynucleotide predominantly in the first type of an activated cell and not in a resting cell of the same cell type.
  • the method further utilizes a second or a third promoter polynucleotide sequence predominantly active in a second type of an activated cell for driving the expression of a specific polynucleotide predominantly in a second type of an activated cell and not in a resting cell of the same cell type.
  • methods of the present invention are directed to treating or ameliorating a neurodegenerative disease in a subject in need thereof.
  • the present invention is directed to a method for inhibiting reactive gliosis in a subject, comprising administering to the subject a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • An antisense sequence as described herein comprises any one of: antisense oligonucleotide, ribozyme, external guide sequence (EGS) oligonucleotide, siRNA compound, single- or double-stranded RNA interference (RNAi) compound such as siRNA compound, modified bases/locked nucleic acid (LNA), antagomir, peptide nucleic acid (PNAs), or any other oligomeric compound or oligonucleotide mimetic capable of hybridizing to at least a portion of the target nucleic acid, such as the IKKB gene or a transcript thereof, and modulate its function.
  • RNAi RNA interference
  • LNA locked nucleic acid
  • PNAs peptide nucleic acid
  • the antisense sequence comprises an antisense RNA, antisense DNA, chimeric antisense oligonucleotide, antisense oligonucleotide comprising modified linkages, micro interfering RNA (miRNA), and a short hairpin RNA (shRNA).
  • miRNA micro interfering RNA
  • shRNA short hairpin RNA
  • Interfering RNA refers to any double stranded or single stranded RNA sequence, capable— either directly or indirectly (i.e., upon conversion)— of inhibiting or down regulating gene expression by mediating RNA interference.
  • Interfering RNA includes but is not limited to siRNA and shRNA.
  • RNAi refers to the selective degradation of a sequence-compatible messenger RNA transcript.
  • the term“shRNA” refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem.
  • the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • siRNA refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner.
  • the small RNA can be, for example, about 18 to 21 nucleotides long.
  • the invention is directed to a method for inhibiting reactive gliosis in a subject, comprising administering to the subject a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is operably linked to a promoter sequence comprising the EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1) promoter, and wherein the antisense sequence is transcribed, or expressed in an activated microglia cell and not in a resting microglia cell, thereby inhibiting reactive gliosis in the subject.
  • a pharmaceutical composition comprising a first polynucleotide encoding an antisense sequence complementary to the inhibitor kappa B kinase-b (IKKB) gene or a transcript thereof, wherein the first polynucleotide is operably linked to a promoter sequence compris
  • the sequence of the IKKB gene or protein sequence is known in the art.
  • the IKKB is a mammalian IKKB.
  • the IKKB is a murine IKKB.
  • the IKKB is a primate IKKB.
  • the IKKB is a human IKKB.
  • Non-limiting examples of IKKB are given by accession numbers: AF088910.1, NM_00l556.3, NM_001190720.2, NC_005l00.4, NM_l74353.2 [041]
  • "reactive gliosis” refers to a nonspecific change of glial cells resulting in the formation of a glial scar.
  • gliosis effects include, but not limited to, inhibition of axon regeneration, induction of neurotoxins' secretion (such as TNF-a, nitric oxide, etc.) and of excitotoxic glutamate, restricted recovery and even further declining of clinical symptoms.
  • the term "glial cell” encompasses a microglia, an astrocyte, an oligodendrocyte, and an ependymal cell.
  • non specific changes to glial cell include, but are not limited to, cell proliferation.
  • cell proliferation comprises cell hypertrophy.
  • non-specific change of a glial cell is attributed to damages to the CNS.
  • damage to the CNS comprises a physical trauma.
  • damage to the CNS comprises a disease or disorder.
  • CNS disease or disorder includes: multiple sclerosis (MS), autoimmune inflammatory disorders, retinal gliosis, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and others.
  • methods of the present invention are directed to treating or preventing a neurodegenerative disease or disorder in a subject.
  • the term "neurodegenerative disease or disorder” refers to a progressive loss of structure or function of neurons.
  • a neurodegenerative disease or disorder comprises neuronal cell death.
  • a neurodegenerative disease or disorder comprises destruction of neuro-muscular junctions.
  • a neurodegenerative disease or disorder comprises destruction of motor-end-plates.
  • a neurodegenerative disease or disorder comprises loss of neural regulation on muscular activity.
  • a neurodegenerative disease or disorder comprises loss of voluntary muscular neurostimulation.
  • a neurodegenerative disorder is selected from: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), age-related macular degeneration (AMD), and other brain injuries.
  • brain injury refers to any injury to the brain tissue of a living organism comprising traumatic and non-traumatic brain injury.
  • non-traumatic brain injury include, but are not limited to, anoxic and hypoxic brain.
  • traumatic brain injuries include, but are not limited to, concussion, coup-countercoup, diffuse axonal and penetration.
  • brain injury includes, but is not limited to, primary or secondary brain injury.
  • inhibiting reactive gliosis is by at least 5%, 10%, 25%, 35%, 50%, 60%, 75%, 90%, 95% or 99%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In one embodiment, inhibiting reactive gliosis is by 2-5%, 4-10%, 8-25%, 17-35%, 25-50%, 40-60%, 45- 75%, 70-90%, 85-95%, or 90-99%. Each possibility represents a separate embodiment of the invention.
  • promoters that are differentially activated in a specific cell type or tissue. Differential activation of promoters can cause their cognate gene products to be aberrantly expressed, e.g., up-regulated or down- regulated, in a particular cell type or tissue type, or under a particular condition.
  • the cell and tissue types are eukaryotic cells, including animals, plants, fungi, and other eukaryotic cells.
  • the cell or tissue type can be from a mammal, yeast, insect, bovine, porcine, murine, equine, canine, feline, avian, piscine, ovine, insect, simian, and/or human.
  • the tissue or cell type is a tissue or cell from the cerebrum, cerebellum, or any other part of the central nerve system (CNS).
  • CNS central nerve system
  • an activated cell in a cell undergoing activation refers to a cell's transition from a resting state to a pro- inflammatory activation state.
  • a resting cell is a quiescent cell.
  • a quiescent cell is a cell under homeostatic conditions.
  • an activated cell is afflicted by homeostatic disturbances.
  • an activated cell is a result of homeostatic disturbance.
  • an activated cell originates from a quiescent cell following homeostatic disturbances affecting the quiescent cell.
  • activated state and quiescent state are different stages throughout the life of a given cell.
  • a cell in its activated state may differ from the same cell during its quiescent state by several aspects, non-limiting examples of which include alterations in: gene expression, proteome, epigenome, polarity, migration, chemoattraction, cytotoxicity, and other cell characteristics.
  • an activated cell can be deactivated and return to its quiescent state.
  • activated cell deactivation is achieved by homeostasis restoration.
  • an activated cell disclosed in the present invention encompasses a microglia cell, an astrocyte cell, and an oligodendrocyte cell.
  • the present invention is directed to methods of targeting a microglia cell.
  • the present invention is directed to methods of targeting an activated microglia cell.
  • Microglial activation refers to cases when infection, injury or disease occur in the brain and affect nerve cells, microglia in the central nervous system become "active," causing inflammation in the brain, similar to the manner in which white blood cells act in the rest of the body.
  • microglia act like the monocyte phagocytic system.
  • activated microglia can generate large quantities of superoxide anions, with hydroxyl radicals, singlet oxygen species and hydrogen peroxide being a downstream product, any of which can be assayed in the preparations utilized in such methods of the invention.
  • Reactive microglia may be characterized by at least one of the following characteristics: (1) their cell bodies becoming larger, their processes becoming shorter and thicker, (2) an increase in the staining for several molecular activation markers, including Iba-l, (3) proliferation and clustering, (4) production and secretion of inflammatory mediators, including pro -inflammatory (e.g., interleukin (IL)-l, IL-6, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and tumor necrosis factor-a) and anti-inflammatory (e.g., IL-10, IL-lra) cytokines, as well as additional inflammatory mediators (e.g., prostaglandins), (5) production and secretion of various neuroprotective factors, including brain-derived neurotrophic factor (BDNF) and insulin growth factor- 1 (IGF-1), 6) production and secretion of chemo-attractive factors (chemokines), which recruit microglia from within the brain to specific brain
  • microglial activation is determined in at least one brain region or area, such as in the hippocampal dentate gyrus (DG), in the prelimbic cortex or in any depression-related area.
  • an activated microglia cell and not a resting microglia cell promotes inflammation by expressing inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB).
  • an activated microglia cell comprises an activated EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1) promoter.
  • EMR1 epidermatitise subunit beta
  • an activated microglia cell comprises an activated Chemokine CX3C motif receptor 1 (CX3CR1) promoter.
  • an activated microglia cell comprises an activated integrin alpha M (ITGAM) promoter. In one embodiment, an activated microglia cell comprises an activated toll-like receptor 4 (TLR4) promoter. In one embodiment, an activated microglia cell comprises an activated cluster determinant 14 (CD 14) promoter.
  • IGAM activated integrin alpha M
  • TLR4 activated toll-like receptor 4
  • CD 14 activated cluster determinant 14
  • any promoter sequence which is active predominantly in an activated microglia cell and not in a resting microglia cell can be used to drive the expression of a vector, such as disclosed herein (e.g., comprising a CAS protein) in an activated microglia cell and not in a resting microglia, and therefore used, for example, for treating a neurodegenerative disease in a subject in need thereof.
  • a vector such as disclosed herein (e.g., comprising a CAS protein) in an activated microglia cell and not in a resting microglia, and therefore used, for example, for treating a neurodegenerative disease in a subject in need thereof.
  • methods of the present invention are directed to inhibiting pro-inflammatory activity predominantly in an activated microglia cell and not a resting microglia cell.
  • the methods are directed to inhibiting IKKB pathway predominantly in an activated microglia cell and not a resting microglia.
  • inhibiting IKKB pathway predominantly in a microglia cell and not in a resting microglia is achieved by utilizing a promoter sequence predominantly active in an activated microglia cell and not in a resting microglia.
  • a promoter sequence predominantly active in an activated microglia cell and not in a resting microglia is utilized for driving the expression of an IKKB inhibitory-compound.
  • an inhibitory-compound is a polynucleotide.
  • an IKKB inhibitory-polynucleotide is inhibiting IKKB transcription.
  • inhibiting transcription comprises gene knock-down, gene knock-out, or both.
  • gene knock-down or gene knock out results in the reduction of mRNA transcript molecules, which can be determined using any available method known to anyone of ordinary skill in the art.
  • an IKKB inhibitory- polynucleotide is inhibiting IKKB translation.
  • an IKKB inhibitory-compound is inhibiting IKKB folding.
  • an IKKB inhibitory-polynucleotide is mutating the IKKB gene.
  • an IKKB inhibitory-polynucleotide is mutating the IKKB promoter. In some embodiments, an IKKB inhibitory-polynucleotide is knocking-out the IKKB gene. In some embodiments, an IKKB inhibitory-polynucleotide is knocking-out the IKKB promoter.
  • a promoter sequence predominantly active in an activated microglia cell and not in a resting microglia cell is the EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1) promoter sequence. In one embodiment, human EMR1 promoter is homologous to the murine F4/80.
  • EMR1 is encoded by the ADGRE1 gene.
  • EMR1 and “ADGRE1” are interchangeable.
  • EMR1 promoter and “ADGRE1 promoter” are synonymous, so as their polynucleotide sequence. Any promoter sequence known in the art as being predominantly active in an activated microglia can be used to drive expression of an inhibitory-polynucleotide in an activated microglia cell according to the methods of the present invention.
  • an IKKB inhibitor can be any molecule selected from: a polypeptide, a small organic molecule and an inorganic molecule.
  • the present invention is directed to methods of targeting an activated astrocyte cell.
  • astrocyte cells activation is in response to a CNS disease or disorder, including but not limited to stroke, trauma, tumorigenesis, or neurodegenerative disease.
  • an activated astrocyte cell contributes to the brain lesion.
  • an activated astrocyte cell provides an ischemic environment.
  • an activated astrocyte cell is involved in disrupting the blood-brain barrier.
  • an activated astrocyte cell promotes an inflammatory response.
  • an activated astrocyte cell generates metabolic, excitotoxic, and oxidative stress, thereby contributing to a condition of reactivated gliosis.
  • a resting astrocyte cell does not generate metabolic, excitotoxic, and oxidative stress, thereby does not contribute to a condition of reactivated gliosis.
  • an activated astrocyte cell generates at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more metabolic, excitotoxic, oxidative stress, or both, compared to a resting astrocyte cell, or any value and range therebetween.
  • an activated astrocyte cell generates 20-40%, 30-50%, 45-60%, 55-70%, 55-80%, 65-90%, or 88-100% more metabolic, excitotoxic, oxidative stress, or both, compared to a resting astrocyte cell.
  • methods of the present invention are directed to inhibiting the activity of an activated astrocyte cell and not a resting or quiescent astrocyte cell.
  • the methods are directed to inducing specific cell death predominantly of an activated astrocyte cell and not of a resting astrocyte cell.
  • specific cell death is cell apoptosis.
  • specific cell apoptosis predominantly of an active astrocyte cell and not of a resting astrocyte cell is achieved by utilizing a promoter sequence predominantly active in an activated astrocyte cell and not of a resting astrocyte cell for driving the expression of an apoptotic polypeptide predominantly in the activated astrocyte cell and not in the resting astrocyte cell.
  • Non-limiting examples of promoters predominantly active in an activated astrocyte cell and not in a resting astrocyte cell include, but are not limited to the six- transmembrane epithelial antigen of prostate family member 4 (Steap4) promoter sequence, tissue inhibitor of metalloproteinase 1 (Timpl) promoter sequence, ipocalin- 2 (LCN2), Alpha- l-antichymotrypsin (Serpina3) and Plasma protease Cl inhibitor (Serpingl).
  • Any promoter sequence known in the art as being predominantly active in an activated astrocyte cell and not in a resting astrocyte cell can be used to drive expression of an apoptotic polypeptide in the activated astrocyte cell and not in the resting astrocyte cell according to the methods of the present invention.
  • methods of the present invention are directed to an apoptosis-inducing compound.
  • an apoptosis-inducing compound is a polypeptide.
  • the apoptotic polypeptide is encoded by a polynucleotide contiguous to a polynucleotide comprising a promoter sequence.
  • the polynucleotide comprising a promoter sequence is driving the expression of the polynucleotide encoding an apoptotic polypeptide predominantly in a distinct cell type.
  • a distinct cell type is an activated cell type.
  • an activated cell type is selected from: a microglia cell, an astrocyte cell, and an oligodendrocyte cell.
  • an apoptotic polypeptide comprises endogenous or exogenous polypeptides capable of promoting regulated cell death (i.e., pro-apoptotic).
  • pro-apoptotic polypeptides include but are not limited to, Bcl-2-associated X protein (BAX), Bcl-2-associated death promoter (BAD), Bcl-2-associated death promoter Bcl-2 homologous antagonist killer (BAK), BH3 interacting domain death agonist (BID), Cysteine-aspartic proteases or cysteine aspartases or cysteine-dependent aspartate-directed proteases (i.e., Caspase), apoptosis inducing factor (AIF), cytochrome C, and others.
  • a Caspase is Caspase 3.
  • Caspase 3 is a reversed-Caspase 3. Any polynucleotide sequence known in the art as encoding a pro-apoptotic polypeptide can be used to promote apoptosis predominantly in a reactivated astrocyte according to the methods of the present invention.
  • targeting refers to any action preventing the undesired activity of a specific molecule, cell or tissue.
  • a targeted molecule include a polynucleotide, a polypeptide, a protein, a lipid, a small organic molecule, a hydrophobic molecule, a hydrophilic molecule, an inorganic molecule, and others.
  • targeting is achieved by any one of hybridizing, binding, competing, blocking, digesting, degrading, or any combination thereof.
  • the terms “preventing”, “blocking”, “reducing”, inhibiting” and “eliminating” are interchangeable.
  • a vector of the invention comprises a clustered regularly interspaced short palindromic repeat associated protein 9 system (CRISPR/Cas9).
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeat associated protein 9 system
  • DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art.
  • Type II Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III.
  • a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II.
  • Cas9 the single effector enzyme
  • the Type II effector system consists of a long pre- crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracer RNA important for gRNA processing.
  • the tracer RNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNA that remain associated with the tracer RNA and Cas9.
  • the enzyme of the present invention such as Cas9, unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a "protospacer" sequence in the target DNA and the remaining spacer sequence in the crRNA.
  • Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3' end.
  • PAM protospacer-adjacent motif
  • different protospacer-adjacent motif can be utilized.
  • the S. pyogenes system requires an NGG sequence, where N can be any nucleotide.
  • S. thermophilus Type II systems require NGGNG (Horvath and Barrangou, 2010) and NNAGAAW (Deveau, Barrangou et al. 2008).
  • Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that may serve to identify additional useful PAMs and expand the set of CRISPR-targetable.
  • sgRNA single guide RNA
  • crRNA CRISPR RNA
  • Exemplary DNA binding proteins may have nuclease activity function to nick or cut double stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains with each domain responsible for cutting or nicking a particular strand of the double stranded DNA.
  • Exemplary polypeptide sequences having nuclease activity known to those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
  • exemplary DNA binding proteins are those that in nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
  • Cas9 generates a blunt-ended double- stranded break 3bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand.
  • PAM protospacer-adjacent motif
  • Cas9 proteins are known to exist in many Type II CRISPR systems including, but not limited to the following: Methanococcus maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465; Thermomono spora
  • Cas9 any Cas9 known in the art may be utilized in the systems and methods described herein.
  • the Cas9 e.g., Sacas9 as described below
  • Sacas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains.
  • the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
  • Target Finder e.g., E-CRISP
  • RGEN Tools Cas-OFFinder
  • CasFinder Flexible algorithm for identifying specific Cas9 targets in genomes
  • CRISPR Optimal Target Finder e.g., CRISPR Optimal Target Finder
  • Non-limiting examples of cas9 include SpCas9 and SaCas9.
  • An non-limiting exemplary S. pyogenes Cas9 protein sequence is set forth in SEQ ID NO: 1:
  • SpCas9 comprises the nucleotide sequence
  • SaCas9 comprises the nucleotide sequence
  • the method of the invention utilizes a dead- Cas9 (dCas9).
  • dCas9 refers to a Cas9 nuclease-null variant that is altered or otherwise modified to inactivate the nuclease activity.
  • Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain.
  • modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e.
  • nuclease-null DNA binding protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the nuclease-null DNA binding protein retains the ability to bind to DNA even though the nuclease activity has been inactivated.
  • the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity.
  • the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may have one or more or all of the nuclease sequences exhibiting nuclease activity inactivated.
  • a DNA binding protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains.
  • a DNA binding protein nickase Such a modified or altered DNA binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding protein cuts or nicks only one strand of double stranded DNA.
  • the DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase.
  • An exemplary DNA binding protein is an RNA guided DNA binding protein nuclease of a Type II CRISPR System, such as a Cas9 protein or modified Cas9 or homolog of Cas9.
  • An exemplary DNA binding protein is a Cas9 protein nickase.
  • An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System which lacks nuclease activity.
  • An exemplary DNA binding protein is a nuclease-null Cas9 protein.
  • Cas9 is altered to reduce, substantially reduce or eliminate nuclease activity.
  • Cas9 nuclease activity is reduced, substantially reduced or eliminated by altering the RuvC nuclease domain or the HNH nuclease domain.
  • the RuvC nuclease domain is inactivated.
  • the HNH nuclease domain is inactivated.
  • the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • Cas9 proteins are provided where the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • nuclease-null Cas9 proteins are provided insofar as the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • a Cas9 nickase is provided where either the RuvC nuclease domain or the HNH nuclease domain is inactivated, thereby leaving the remaining nuclease domain active for nuclease activity. In this manner, only one strand of the double stranded DNA is cut or nicked.
  • nuclease-null Cas9 proteins are provided where one or more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins.
  • the altered amino acids include D10 and H840 of SEQ ID NO: 1.
  • the altered amino acids include D839 and N863 of SEQ ID NO: 1.
  • one or more or all of D10, H840, D839 and H863 of SEQ ID NO: 1 are substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity.
  • one or more or all of D10, H840, D839 and H863 OF SEQ ID NO: 1 are substituted with alanine.
  • a Cas9 protein having one or more or all of D10, H840, D839 and H863 of SEQ ID NO: 1 substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity, such as alanine is referred to as a nuclease-null Cas9 or dCas9 and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection.
  • nuclease activity for a dCas9 may be undetectable using known assays, i.e. below the level of detection of known assays.
  • the Cas9 protein, Cas9 protein nickase or nuclease null Cas9 includes homologs and orthologs thereof, which retain the ability of the protein to bind to the DNA and be guided by the RNA.
  • the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. pyogenes and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology thereto, or any value and range therebetween, and being a DNA binding protein, such as an RNA guided DNA binding protein. Each possibility represents a separate embodiment of the invention.
  • homology is 15-35%, 20- 45%, 35-55%, 40-65%, 55-75%, 70-95% or 80-99% homology.
  • an engineered Cas9-sgRNA system enables RNA-guided genome regulation in cells by tethering transcriptional activation domains to either a nuclease-null Cas9 or to guide RNAs.
  • complementarity of a polynucleotide, such as an antisense polynucleotide as disclosed herein, sgRNA, to a target nucleotide, such as the IKKB gene or transcript thereof, is at least 75%, 85%, 90%, 95% 97%, 99% or 100% complementary, or any range an value therebetween.
  • a polynucleotide such as an antisense polynucleotide as disclosed herein, sgRNA
  • target nucleotide such as the IKKB gene or transcript thereof
  • complementarity of a polynucleotide, such as an antisense polynucleotide as disclosed herein, sgRNA, to a target nucleotide, such as the IKKB gene or transcript thereof, is 70-85%, 80-90% 92- 97%, 95-99%, or 97-100%.
  • promoter refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • active promoter refers to a condition within a cell that allows for the transcription of a nucleotide gene product, which is situated in the correct location and orientation in relation to the promoter.
  • the promoter is a cis-promoter.
  • cis-promoter refers to cases wherein the promoter sequence and the target sequence for expression reside within the same polynucleotide sequence, such as a plasmid DNA.
  • an activated cis-promoter directly activates the expression of its expression target.
  • an activated cis-promoter directly regulates the expression of its expression target.
  • the promoter is a trans-promoter.
  • trans-promoter refers to cases wherein the promoter sequence and a target sequence for expression reside within distinct polynucleotide sequences, such as different plasmids.
  • an activated trans-promoter indirectly activates the expression of an expression target.
  • an activated trans-promoter indirectly regulates the expression of its expression target.
  • an activate trans-promoter drives the expression of an expression target by producing active elements required for expression of the expression target.
  • an activated trans-promoter promotes or induces the expression of a transcription factor, an activation element, a ligand, or any equivalent thereof, which is required for the expression of the expression target.
  • the activation of a trans-promoter provides the required elements, such as a transcription factor, a ligand, etc., required for the activation of a cis-promoter.
  • the activation of a cis-promoter by means of a trans-promoter activation promotes or induces the expression of an expression target.
  • any promoter utilized according to the present invention is a cis-promoter, a trans-promoter, or both.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 1-30 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the terms “promoter”, “active promoter” and “additional promoter elements” are collectively referred to as “promoter”.
  • the promoter refers to a genomic polynucleotide sequence of a mammal.
  • a mammal include, but not limited to, a human or a non- human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
  • Cas9-sgRNA complex targets an enhancer.
  • enhancers refers to genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • the sgRNA is flanked by nucleic acids sequences encoding a ribozyme.
  • two ribozyme sequences flank the sgRNA, i.e., one ribozyme sequence upstream of the sgRNA and one ribozyme sequence downstream of the sgRNA.
  • the primary transcript undergoes a self- catalyzed cleavage to precisely release the designed sgRNA.
  • ribozyme refers to a RNA molecule motif that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule.
  • the minimal ribozyme sequence that is required for the self-cleavage reaction includes approximately 13 conserved or invariant "core" nucleotides, most of which are not involved in forming canonical Watson-Crick base-pairs.
  • the core region is flanked by Stems I, II and III, which are in general made of canonical Watson-Crick base-pairs but are otherwise not constrained with respect to sequence.
  • nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II).
  • RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
  • one or more transcriptional regulatory proteins or domains are joined or otherwise connected to a nuclease- deficient Cas9 or one or more single guide RNAs (sgRNA).
  • the transcriptional regulatory domains correspond to targeted loci. Accordingly, aspects of the present disclosure include methods and materials for localizing transcriptional regulatory domains to targeted loci by fusing, connecting or joining such domains to either dCas9 or to the sgRNA.
  • an endogenous gene can be any desired gene, referred to herein as a target gene.
  • a dCas9-fusion protein capable of transcriptional activation is provided.
  • a VP64 activation domain is joined, fused, connected or otherwise tethered to the C terminus of dCas9.
  • the term "VP64 activation domain" (VP64) as used herein refers to a transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16, amino acids 437-447, DALDDFDLDML; SEQ ID NO: 4) connected with glycine-serine linkers. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator.
  • activation (or activator) domains include any known transcriptional activation domain, including, without limitation, p65, MyoDl, HSF1, RTA or SET7/9.
  • the transcriptional regulatory domain is provided to the site of target genomic DNA by the dCas9 protein.
  • a dCas9 fused to a transcriptional regulatory domain is provided within a cell along with one or more guide RNAs.
  • the dCas9 with the transcriptional regulatory domain fused thereto bind at or near target genomic DNA.
  • the one or more guide RNAs bind at or near target genomic DNA.
  • the transcriptional regulatory domain regulates expression of the target gene.
  • a dCas9-VP64 fusion activated transcription of reporter constructs when combined with sgRNAs targeting sequences near the promoter, thereby displaying RNA-guided transcriptional activation.
  • a sgRNA-fusion protein capable of transcriptional activation is provided.
  • a VP64 activation domain is joined, fused, connected or otherwise tethered to the sgRNA.
  • the transcriptional regulatory domain is provided to the site of target DNA (e.g., genomic or exogenic DNA) by the sgRNA.
  • a sgRNA fused to a transcriptional regulatory domain is provided within a cell along with a dCas9 protein. The dCas9 binds at or near target genomic DNA.
  • the one or more guide RNAs with the transcriptional regulatory protein or domain fused thereto bind at or near target genomic DNA.
  • the transcriptional regulatory domain regulates expression of the target gene.
  • a dCas9 protein and a sgRNA fused with a transcriptional regulatory domain activates transcription of reporter constructs, thereby displaying RNA-guided transcriptional activation.
  • the transcriptional regulator protein or domain is a transcriptional activator. According to some embodiments, the transcriptional regulator protein or domain upregulates transcription of the target nucleic acid. According to some embodiments, the transcriptional regulator protein or domain is a transcriptional repressor. According to some embodiments, the transcriptional regulator protein or domain downregulates expression of the target nucleic acid. Transcriptional activators and transcriptional repressors can be readily identified by one of skill in the art based on the present disclosure. Gene products
  • target sequence refers to any nucleic acid sequence that is recognizable by a CAS/RNA i.e., Cas9-sgRNA complex.
  • the target sequence of the present invention is a gene.
  • the target sequence is a regulatory element such as, but not limited to a promoter or an enhancer.
  • the target sequence encodes a protein. In some embodiments the target sequence encodes a functional RNA molecule such as, but not limited to siRNA, miRNA, tRNA, ribozyme etc.
  • the target sequence is a genomic sequence.
  • genomic sequence refers to any nucleotide sequence that is found in the genome of an organism.
  • the term“genome” used herein refers collectively to the nuclear genome and the mitochondrial genome.
  • genomic sequence refers to a human genomic sequence.
  • genomic sequence refers to a mammalian genomic sequence.
  • genomic sequence refers to a eukaryotic genomic sequence. In the context of this invention the genome includes both the genes and the non-coding sequences of the DNA/RNA.
  • the target sequence is an exogenous nucleic acid sequence.
  • exogenous sequence or “exogenous promoter” refers to any sequence that does not appear in the genome of the subject and/or the cells and/or the tissue to be employed by the system and methods on the present invention.
  • exogenous sequence is a synthetic nucleic acid sequence.
  • the exogenous sequence may be derived from a biological sample including, but not limited to a tissue sample, a blood sample, a modified organism, etc.
  • an exogenous sequence is an exogenous promoter polynucleotide sequence.
  • an exogenous promoter is used for driving expression of a gene in a mammalian cell.
  • Exogenous promoters for use in expression in mammalian cells are well known within the art, and include, but not limited to, cytomegalovirus (CMV) promoter, adenovirus major late promoter (MLP), SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, 35S RNA and 19S RNA promoters of CaMV, coat protein promoter to TMV.
  • the exogenous promoter is the adenovirus major late promoter (MLP).
  • methods of the present invention comprise utilizing a promoter polynucleotide for driving the expression of a gene predominantly in a reactivated cell.
  • the term "expression of a gene predominantly in an activated cell” refers to any gene product (e.g., RNA, mRNA, or a peptide) that is found or produced primarily in the activated cell compared to a resting or quiescent cell of the same cell type, or any other cell type.
  • a predominantly expressed gene product is primarily secreted from the activated cell compared to a resting or quiescent cell of the same type, or any other cell type.
  • a predominantly expressed gene product is found primarily or primarily secreted from the activated cell of one subtype compared to a resting or quiescent cell of the same subtype, or any other activated cell of a different subtype.
  • predominantly expressed refers to increased mRNA transcribed level of the gene as indicated herein in an activated cell as described herein compared to a resting cell as described herein.
  • a resting cell is a non-activated cell.
  • increased mRNA level is a result of increased activation of an activated cell specific gene promoter sequence.
  • increased mRNA level is increased number of mRNA transcript molecules.
  • increased mRNA level is increased mRNA stability.
  • increased mRNA level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater in an activated cell compared to a resting cell of the same cell type, or any other cell type, or any value and range therebetween.
  • increased mRNA level is 5-10%, 8- 20%, 15-30%, 20-40%, 35-50%, 40-60%, 55-70%, 65-80%, 75-90% or 85-100% greater in an activated cell compared to a resting cell of the same cell type, or any other cell type.
  • Methods of detecting gene overexpression are well known to any one of ordinary skill in the art, and include non-limiting examples, such as quantitative real time RT-PCR, qualitative real-time RT-PCR, and others.
  • predominantly expressed refers to increased levels of a translated peptide in an activated cell compared to a resting cell of the same cell type.
  • increased levels of a translated peptide refer to increased levels of a peptide in the producing activated cell compared to a resting cell of the same cell type.
  • increased levels of a translated peptide refer to increased levels of the peptide in the cytosol of the producing reactivated cell compared to the cytosol of a resting cell of the same cell type.
  • increased levels of a translated peptide refer to increased levels of the peptide in the secretory pathway of the producing activated cell compared to the resting cell of the same cell type.
  • increased levels of a translated peptide refer to increased levels of sorting the peptide to secretory vesicles. In some embodiments, increased levels of a translated peptide refer to increased secretion of the peptide from the producing activated cell compared to the resting cell of the same cell type. In some embodiments, increased levels of a translated peptide refer to a peptide primarily secreted from an activated cell compared to a resting cell of the same cell type, or any other cell type.
  • increased peptide level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater in an activated cell compared to the a resting cell of the same cell type, or any other cell type, or any value and range therebetween.
  • increased protein level is 5-10%, 8- 20%, 15-30%, 20-40%, 35-50%, 40-60%, 55-70%, 65-80%, 75-90% or 85-100% greater in a reactivated cell compared to a resting cell of the same cell type, or any other cell type.
  • increased protein level is 5-10%, 8- 20%, 15-30%, 20-40%, 35-50%, 40-60%, 55-70%, 65-80%, 75-90% or 85-100% greater in a reactivated cell compared to a resting cell of the same cell type, or any other cell type.
  • Each possibility represents a separate embodiment of the invention.
  • Methods of detecting increased peptide production are well known to the skilled artisan, and include non-limiting examples, such as immunohistochemistry, immunocytochemistry, Enzyme-linked immunosorbent assay (ELISA, e.g., direct, indirect, 'sandwich', etc.), westem-blot, dot-blot, and others.
  • ELISA Enzyme-linked immunosorbent assay
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double- stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • a“plasmid” refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vectors Another type of vector, wherein virally-derived DNA or RNA sequences are present in the virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno- associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfecting into host cells.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as“expression vectors”.
  • expression vectors Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • viral vectors are used as delivery system of polynucleotides.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • a recombinant expression vector“operably linked” or“operably coupled” is intended to mean that the indicated nucleotide sequence or gene is linked to the regulatory element(s) in a manner that induces or allows the expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • additional elements such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • the term "recombinant" as a modifier of viral vector means that the compositions (e.g., AAV or sequences) have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature.
  • a particular example of a recombinant vector, such as an AAV vector would be where a polynucleotide that is not normally present in the wild-type viral (e.g., AAV) genome is inserted within the viral genome.
  • a recombinant polynucleotide would be where a heterologous polynucleotide (e.g., gene) encoding a protein is cloned into a vector, with or without 5', 3' and/or intron regions that the gene is normally associated within the viral (e.g., AAV) genome.
  • a heterologous polynucleotide e.g., gene
  • AAV a heterologous polynucleotide
  • sequences such as polynucleotides and polypeptides
  • sequences including polynucleotides and polypeptides are expressly included in spite of any such omission.
  • Recombinant vector e.g., AAV
  • a recombinant vector e.g., AAV
  • a recombinant vector can be based upon any AAV genome, such as AAV-l, -2, -3, -4, - 5, -6, -7, -8, -9, -10, -11, -rh74, -rhlO or AAV-2i8, for example.
  • Such vectors can be based on the same of strain or serotype (or subgroup or variant), or different from each other.
  • a recombinant vector e.g., AAV
  • plasmid based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector.
  • the vector may be a DNA plasmid delivered via non-viral methods or via viral methods.
  • the viral vector may be a retroviral vector, a herpes viral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector.
  • the heterologous nucleotide sequence may encode a therapeutic gene product.
  • the heterologous nucleotide sequence may encode a non-therapeutic detectable and/or selectable gene product.
  • the promoters may be active in mammalian cells. At least one of said promoters may be a viral promoter. In some embodiments, one promoter may direct expression of both said heterologous gene sequence and said Cas9 coding region.
  • the heterologous gene sequence may further comprise a coding sequence for a protein degradation tag.
  • the present invention is directed to a composition
  • a composition comprising one or more vectors comprising: a first nucleic acid sequence encoding at least one CAS polypeptide, wherein the first nucleic acid sequence is flanked by a first promoter sequence comprising the EMR1 promoter; a second nucleic acid sequence encoding a sgRNA complementary to a target nucleotide comprising the IKKB sequence, wherein the second nucleic acid sequence is flanked by the first promoter, wherein the second nucleic acid sequence comprises one ribozyme sequence upstream of the sgRNA and one ribozyme sequences downstream of the sgRNA; a third nucleic acid sequence encoding at least one dCAS polypeptide, wherein the third nucleic acid sequence is flanked by a second promoter sequence comprising any one of the Steap4 promoter or the Timpl promoter; a fourth nucleic acid sequence encoding a sgRNA complementary to a
  • the composition is a pharmaceutical composition.
  • a pharmaceutical composition comprising a therapeutically effective amount of at least one of the aforementioned polynucleotides or vectors comprising thereof, is for use in treating reactive gliosis condition or disorder in a subject.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier or diluent.
  • the term“pharmaceutical composition”, as used herein, refers to: an inhibitory- polynucleotide targeting a gene, or an mRNA transcribed therefrom, predominantly transcribed, expressed, or produced by an activated microglia cell, a pro-apoptotic encoding-polynucleotide predominantly expressed by an activated astrocyte cell, or any combination thereof, with chemical components such as diluents or carriers that do not cause unacceptable adverse side effects and that do not inhibit reactive gliosis.
  • a“therapeutically effective amount” or“an amount effective” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutic result may be, e.g., lessening of symptoms, prolonged survival, improved mobility, improved social and vocational functioning, and the like.
  • a therapeutic result need not be a“cure.”
  • a therapeutic result may also be prophylactic.
  • the prophylactically effective amount will be less than the therapeutically effective amount.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.
  • pharmaceutically acceptable means suitable for administration to a subject, e.g., a human.
  • pharmaceutically acceptable can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • compositions can take the form of solutions, suspensions, emulsions, colloidal dispersions, emulsions (oil-in-water or water- in-oil), sustained-release formulations and the like.
  • a polynucleotide encoding an antisense sequence complementary to a molecule predominantly produced by an activated microglia cell (e.g., IKKB) and a pro-apoptotic encoding-polynucleotide expressed predominantly in an activated astrocyte cell, expression vectors comprising thereof, and compositions comprising them, can be delivered systemically or intrathecally via a carrier means.
  • Carrier means for delivering microglial modulators and compositions to microglia cells are known in the art and include, for example, encapsulating the composition in a liposome moiety.
  • Another means for delivery comprises attaching the microglial modulator to a protein or nucleic acid that is targeted for delivery to the target cell.
  • U.S. Pat. No. 6,960,648 and Published U.S. Patent Application Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes.
  • the route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intrathecal, intradermal, intravenous, intramuscular, intralesional, subcutaneous, and any other mode of injection as known in the art.
  • the pharmaceutical composition is administered intrathecally to a subject in need thereof.
  • the route of administration is improved by encapsulating the pharmaceutical agent in nanoparticles, such as to protect the encapsulated drug from biological and/or chemical degradation, and/or to facilitate transport to the CNS thereby targeting reactivated microglia and astrocytes.
  • compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the compounds of the present invention and optionally other compounds.
  • the compositions comprise from about 0.01% to about 10.0% w/v of a subject compound, more preferably from about 0.1% to about 2.0.
  • the pharmaceutical compositions are administered by intrathecal, intravenous, or subcutaneous injection of a liquid preparation.
  • liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical composition is administered intrathecally, and is thus formulated in a form suitable for intrathecal administration.
  • the pharmaceutical composition is administered intravenously, and is thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical composition is administered subcutaneously, and is thus formulated in a form suitable for subcutaneous administration.
  • compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention is formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically.
  • formulation is dependent upon the route of administration chosen.
  • injectables of the invention are formulated in aqueous solutions.
  • injectables, of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers with optionally, an added preservative.
  • compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcy stine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed.
  • the compositions also comprise, in some embodiments, local anesthetics or other actives.
  • the compositions can be used as sprays, mists, drops, and the like.
  • compositions for intrathecal administration include aqueous solutions of the active preparation in water-soluble form.
  • suspensions of the active ingredients are prepared as appropriate oily or water-based injection suspensions.
  • Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension also contains suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
  • a liposome see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
  • the pharmaceutical composition delivered in a controlled release system is formulated for intravenous infusion, liposomes, or other modes of administration.
  • a controlled release system can be placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (1990).
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.
  • a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • preparation of effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosages vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975)].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved. In some embodiments, the course of treatment is everlasting. In another embodiment, said dosing can depend on severity and responsiveness of the condition to be treated. In one embodiment, administering is at least once.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the term "therapeutically effective amount” refers to a concentration of a polynucleotide encoding an antisense sequence complementary to a molecule predominantly produced by an activated microglia cell (e.g., IKKB) and a pro- apoptotic encoding-polynucleotide expressed predominantly in an activated astrocyte cell, expression vectors comprising thereof, and compositions comprising them, effective to treat a disease or disorder in a mammal.
  • the term“a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition.
  • the terms“subject” or“individual” or“animal” or“patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
  • adjectives such as“substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended.
  • the word“or” in the specification and claims is considered to be the inclusive“or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.
  • each of the verbs, “comprise,”“include” and“have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • the terms “comprises”, “comprising”, “containing”, “having” and the like can mean “includes”, “including”, and the like; “consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • the terms “comprises,” “comprising, “having” are/is interchangeable with “consisting”.
  • pX60l-AAV-CMV :NLS-SaCas9-NLS-3xHA- bGHpA
  • U6 ::BsaI-sgRNA
  • the promoter sequence of F4/80 (349 bp) was amplified from C57black mouse genome.
  • gRNA guide RNAs
  • gRNAs - gMLP were synthetized by IDT ⁇ . Sense and antisense oligonucleotides of each gRNA were subcloned into Master template (synthetized by Synthezza Bioscience ⁇ ) containing variety of restriction sites, followed by bovine growth hormone - bGH Poly A sequence (as been used in IGEM BGU 2015 - "Boomerang").
  • Promoters - Steap 4 and Timpl were amplified from C57Black mouse genome. cDNAs of Cas9 were subcloned into the Master Boomerang template.
  • plasmid that contains the Sa-dCas9-VPR coding DNA sequence under steap4 or timpl promoters.
  • the following plasmid is used: pX600-AAV- CMV::NLS-SaCas9-NLS-3xHA-bGHpA (http://www.addgene.org/68495/).
  • plasmid that contains Ribozyme flanked gRNA under steap4 or timpl promoters that targets the sequence complementary to a 20 nucleotides sequence in a synthetic promoter of the Rev-Casapase3.
  • plasmid pX60l-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI- sgRN A (http s ://w w w . addgene . org/61591/).
  • Modified plasmid that contains the coding DNA sequence of a recombinant protein with an auto-proteolytic capability, termed Rev-Caspase3, under a synthetic promoter.
  • the promoter is the same as in option 1 (mentioned above) with minor changes in Protospacer adjacent motif (PAM) sequence (from AGGCT to NNGRR (N for any nucleotide and R for a purine nucleotide).
  • PAM Protospacer adjacent motif
  • mice cell lines C8-D30, astrocytes; and Nsc34, motor neurons
  • DMEM Dulbecco's Modified Eagle's Medium
  • fcs fetal calf serum
  • L- glutamine 1% L- glutamine 1%
  • PenStrp 1% PenStrp 1%
  • IKKb suppression reduces TNFa production in activated microglia
  • BV-2 microglia cells were grown to 80% confluency and infected with shIKKb plasmid. Selection of cells for plasmid DNA expression was performed using Puromycin. Cells were treated with LPS for 2 hours to induce microglia activation and cytokine secretion. Suppression of IKKB expression levels using an IKKB antisense polynucleotide was shown to induce a reduction of TNFa production in activated microglia cells compared to control (Fig. 1). EXAMPLE 2
  • C8-D30 astrocyte cells were tested in a dual Luc assay.
  • the dual luciferase reporter vectors pGL3 included the promoter TIMP1 or STEAP4 downstream of the Firefly luciferase gene.
  • the cells were incubated for 48 hours so as to allow translation of Fuciferase enzyme.
  • C8-D30 astrocyte cells co-transfected with lentivirus construct comprising the pTimpl-VP64, and pSynt expressing mCherry downstream to the pro-apoptotic protein reverse-Caspase 3. Twenty four hours post co-transfection. C8-D30 astrocyte cells were shown to express mCherrry (Fig. 3B) under the regulation of a promoter which is specifically activated in activated astrocyte cells. Further, it is safe to say that as mCherry was transcribed downstream to the reverse Caspase3, the latter was also expressed in these cells red , therefore also indicating expression of the pro-apoptotic protein revesrse-Caspase3.
  • the inventors also observed some background red-fluorescence. As cells undergoing cell death, such as apoptosis, are being degraded, their contents will be released to the media, including the mCHerry polypeptide (lacking a signal peptide for secretion). Thus the non-focused and extra-cellular fluorescence which had been observed by the inventors may indicate that cells expressing the reversed-Caspase 3 have undergone cell death.
  • Dual luciferase reporter assay (Promega) is performed in microglia and astrocyte mouse cells under the regulation of a specific promoter (e.g., F4/80, Steap4 and Timpl).
  • a specific promoter e.g., F4/80, Steap4 and Timpl.
  • Astrocytes are transfected with all three vectors and are activated by exposure to pro-inflammatory cytokines cocktail. Apoptosis rates is compared between the activated astrocytes expressing reversed-Caspase 3 and controls (first control are active astrocytes transfected with a basic plasmid - without any functional gene or promoter; and a second control group receiving no treatment).
  • Microglia are transfected with a plasmid bearing functional gene or promoter (table 1) and activated by exposure to LPS.
  • the differential expression of each of the following genes: TNF- a, IL-l cytokines, NF-kB and IKK-b is compared to controls (first control are reactive microglia transfected with a basic plasmid - without any functional gene or promoter; and a second control group receiving no treatment).
  • ALS multi-cultural system includes a co-culture of motor neurons, active microglia and active astrocytes.
  • the microglia and astrocytes cells are activated by exposure to LPS and pro -inflammatory cytokines cocktail, respectively. Reduction in number and activity of active astrocytes and longer and better survival of motor neurons is compared to the control.

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Abstract

La présente invention concerne une composition et un procédé d'utilisation de celle-ci, par exemple pour traiter une gliose réactive chez un sujet. Le procédé comprend l'administration au sujet d'une composition pharmaceutique comprenant un polynucléotide codant une séquence antisens complémentaire de l'inhibiteur kappa B kinase-bêta (IKKB) ou un transcrit de celui-ci, le polynucléotide étant transcrit, dans une cellule microglie activée et non dans une cellule microglie au repos.
PCT/IL2019/050365 2018-03-29 2019-03-28 Méthodes de traitement de maladies neurodégénératives WO2019186567A1 (fr)

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WO2015069647A1 (fr) * 2013-11-05 2015-05-14 The Research Institute At Nationwide Children's Hospital Compositions et procédés d'inhibition de nf-kb et sod-1 afin de traiter la sclérose latérale amyotrophique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015069647A1 (fr) * 2013-11-05 2015-05-14 The Research Institute At Nationwide Children's Hospital Compositions et procédés d'inhibition de nf-kb et sod-1 afin de traiter la sclérose latérale amyotrophique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU, YANG ET AL.: "IKKbeta dpfirienrv in myeloid cells ameliorates Al7heimer's disease-related symptoms and pathology", JOURNAL OF NEUROSCIENCE, vol. 34, no. 39, 24 September 2014 (2014-09-24), pages 12982 - 12999, XP055640164, Retrieved from the Internet <URL:http://www.jneurosci.org/content/jneuro/34/39/12982.full.pdf> [retrieved on 20190610] *

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