WO2019104311A1 - Compositions and methods for suppressing neurological disease - Google Patents

Compositions and methods for suppressing neurological disease Download PDF

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Publication number
WO2019104311A1
WO2019104311A1 PCT/US2018/062580 US2018062580W WO2019104311A1 WO 2019104311 A1 WO2019104311 A1 WO 2019104311A1 US 2018062580 W US2018062580 W US 2018062580W WO 2019104311 A1 WO2019104311 A1 WO 2019104311A1
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Prior art keywords
polypeptide
cyp4gl
mutation
als
polynucleotide
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PCT/US2018/062580
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French (fr)
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Robert REENAN
Asli Sahin
Yiannis SAVVA
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Brown University
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Publication of WO2019104311A1 publication Critical patent/WO2019104311A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • ALS Amyotrophic lateral sclerosis
  • fALS familial ALS
  • sALS sporadic ALS
  • ALS a common feature of ALS is hypermetabolism, dysregulation of lipids, and wasting. Many mouse models have been developed to help unravel the mechanisms of ALS motor neuron death, but to date, have resulted in candidate gene approaches (educated guesses) that do not extend lifespan of affected mice.
  • the present invention features compositions and methods useful for reducing or ameliorating an effect of a mutation causing familial amyotrophic lateral sclerosis (fALS) as well as treatment for sporadic ALS (sALS) cases that share many common features of fALS.
  • fALS familial amyotrophic lateral sclerosis
  • sALS sporadic ALS
  • the invention provides an isolated polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
  • the invention provides an isolated Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
  • the invention provides an isolated human Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
  • the invention provides an isolated polynucleotide encoding a polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
  • the invention provides an isolated polynucleotide encoding a Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
  • the invention provides an isolated polynucleotide encoding a human Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
  • the Cyp4gl polypeptide is a Cyp4gl polypeptide of an invertebrate or vertebrate.
  • the vertebrate or invertebrate is Drosophila melanogaster, Homo sapiens, Tenebrio molitor, Aedes aegypti, Diaphorina citri, Blatella germanica, Nasonia vitripennis, Spodoptera frugiperda, Oryctolagus cuniculus, Canis lupus, Gallus gallus, Xenopus tropicalis, or Danio rerio.
  • the polypeptide has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide or a human Cyp4gl polypeptide.
  • the mutation is any one or more of mutations G92E, D112N, S143N, and M333I. In some embodiments, the mutation is S138N.
  • polypeptide contains any one of the following sequences:
  • the mutation is a gain-of-function or loss-of-function mutation.
  • the mutation suppresses a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the mutation associated with ALS is SOD1-G85R.
  • the invention provides an expression vector containing the isolated polynucleotide of any one of the aspects delineated herein.
  • the invention provides a therapeutic composition containing the polypeptide, polynucleotide or vector of any one of the aspects delineated herein.
  • the composition further contains a pharmaceutically acceptable excipient.
  • the composition further contains a vehicle for intracellular delivery of the polypeptide, polynucleotide, or vector.
  • the invention provides a host cell or host organism containing the isolated polynucleotide or expression vector of any one of the aspects delineated herein.
  • the host cell or host organism is an amyotrophic lateral sclerosis (ALS) model or contains a mutation and/or misexpression associated with ALS.
  • the host cell or host organism is mammalian.
  • the invention provides a Drosophila melanogaster mutant containing a Cyp4gl polynucleotide and/or polypeptide having a mutation in any one or more of amino acid positions 92, 112, 143, and 333.
  • the mutation is a missense mutation.
  • the mutation is any one or more of mutations G92E, D112N, S143N, and M333I.
  • the Drosophila melanogaster mutant further contains a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the mutation and/or misexpression associated with ALS has a lethal effect.
  • the mutation associated with ALS is SOD1-G85R.
  • the Cyp4gl mutation suppresses the lethal effect of the mutation and/or misexpression associated with ALS. In some other embodiments, the suppression is partial, nearly complete, or complete.
  • the invention provides a method of reducing or ameliorating or preventing an effect of a mutation associated with a neurode generative disease characterized by an energy deficit in a subject.
  • the method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby suppressing an effect of a mutation associated with a neurode generative disease characterized by an energy deficit in the subject.
  • the invention provides a method of modulating lipid metabolism in a subject.
  • the method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby modulating lipid metabolism in the subject.
  • the invention provides a method of treating a neurodegenerative disease characterized by an energy deficit in a subject.
  • the method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby treating a neurodegenerative disease characterized by an energy deficit in the subject.
  • the composition is the therapeutic composition of any one of the aspects delineated herein.
  • the polypeptide or polynucleotide is the polypeptide, polynucleotide, or vector of any one of the aspects delineated herein.
  • the subject has a mutation associated and/or misexpression with amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the mutation associated with a neurological disease or the mutation associated with ALS is SOD1-G85R or C9orf72 repeat expansion.
  • the subject is human.
  • the methods of any one of the aspects delineated herein further contain the step of administering to the subject an effective amount of an agent that decreases a level or activity of Cyp4gl polypeptide.
  • the agent is an inhibitory nucleic acid that inhibits expression of Cyp4gl polypeptide.
  • the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the method contains the steps of (a) contacting a polypeptide with a candidate agent, where the polypeptide is a Cyp4gl polypeptide, and (b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity, where an alteration in activity indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the method contains the steps of (a) contacting a polypeptide with a substrate, where the polypeptide is a Cyp4gl polypeptide having enzymatic activity, and (b) detecting a reaction product of the polypeptide contacted with the substrate, where detection of a reaction product indicates the substrate and/or reaction product is a modulator a neurodegenerative disease characterized by an energy deficit.
  • the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the method contains the steps of (a) contacting a cell or organism with a candidate agent, and (b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity, where an alteration in the level or activity indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the method contains the steps of (a) contacting a cell or organism with a candidate agent, and (b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism containing a Cyp4gl polynucleotide or polypeptide having a mutation, where a similarity in the phenotypes indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
  • the activity is binding of the polypeptide to the candidate agent or enzymatic activity of the polypeptide.
  • the activity or enzymatic activity is omega-hydroxylation of a fatty acid.
  • the substrate is a fatty acid.
  • the phenotype is lipid metabolism.
  • the cell or organism contacted with the candidate agent is an amyotrophic lateral sclerosis (ALS) model or contains a mutation and/or misexpression associated with ALS.
  • the candidate agent inhibits an activity of Cyp4gl polypeptide.
  • the candidate agent suppresses an ALS phenotype.
  • the cell or organism is the host cell or host organism of any one of the aspects delineated herein.
  • ALS amyotrophic lateral sclerosis
  • dementia dementia
  • Parkinson’s disease Huntington’s disease
  • cytochrome P450 (CYP) polypeptide By“activity of a cytochrome P450 (CYP) polypeptide” is meant lipid metabolism modulating activity. In some embodiments, the activity is omega-hydroxylation of fatty acids.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule including RNA-based molecules that can act as inhibitory molecules including siRNA or antisense RNAs, or polypeptide, or fragments thereof.
  • an agent that decreases a level or activity of Cyp4gl polypeptide is administered to a subject.
  • the agent that decreases a level or activity of Cyp4gl polypeptide is a siRNA.
  • the agent that decreases a level or activity of Cyp4gl polypeptide is a small molecule compound (e.g., HET0016).
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • an analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a corresponding naturally -occurring polypeptide, while having certain biochemical modifications that enhance or change the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
  • An analog may include an unnatural amino acid.
  • biological sample any liquid, cell, or tissue obtained from a subject.
  • NP 525031 (Drosophila melanogaster) or NCBI Accession No. NP 997235 (human) and having an activity of a cytochrome P450 (CYP) polypeptide.
  • CYP cytochrome P450
  • Exemplary Cyp4gl polypeptide sequences at NCBI Accession No. NP 525031 and NP 997235 are provided below:
  • NP 525031 (Drosophila melanogaster)
  • Cyp4gl nucleic acid, gene or gene-containing fragment” or“Cyp4gl polynucleotide” is meant a nucleic acid molecule encoding a Cyp4gl polypeptide.
  • Exemplary Cyp4gl polynucleotide sequences are provided at NCBI Accession No. NM 080292 (Drosophila melanogaster) and No. NM 207352 (human), which are reproduced below:
  • GGGGCC GGGGCC repeat in the C9orf72 gene, which encodes the C9orf72 polypeptide.
  • a healthy subject or a subject that does not have ALS or a neurological disease, there are usually a few repeats of the hexanucleotide.
  • subject having fALS or sALS there may be up to hundreds or thousands of repeats of the hexanucleotide.
  • An exemplary sequence of the C9orf72 gene is provided at NCBI Accession No. NG 031977 (Homo sapiens).
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • diseases include neurological diseases, including neurodegenerative diseases associated with or characterized by an energy deficit such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD), Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease.
  • ALS amyotrophic lateral sclerosis
  • FTD fronto-temporal dementia
  • Parkinson’s disease Huntington’s disease
  • Alzheimer’s disease Alzheimer's disease
  • an effective amount is meant the amount that is required to ameliorate the symptoms and/or progression of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, stage of disease, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • the invention provides a number of targets that are useful for the development of highly specific drugs to beat or a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • inhibitory nucleic acid is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene.
  • a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.
  • an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Puri y denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally -occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally -occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • misexpression is meant an alteration in expression level of a polypeptide or polynucleotide in a cell relative to expression level in a control (e.g., cells from a healthy subject or cells from a subject that does not have amyotrophic lateral sclerosis (ALS) or a neurological disease).
  • Misexpression may be an overexpression (i.e., positive alteration in expression level) or underexpression (i.e., negative alteration in expression level) of a polynucleotide or polypeptide.
  • overexpression of TDP-43 is a misexpression associated with ALS.
  • mutation is meant a change in a polypeptide or polynucleotide sequence relative to a wild-type reference sequence.
  • Exemplary mutations include point mutations, missense mutations, amino acid substitutions, and frameshift mutations.
  • a missense mutation in a Cyp4gl polynucleotide causes an amino acid substitution in a Cyp4gl polypeptide (e.g., a human Cyp4gl polypeptide) .
  • a mutation in Cyp4gl polypeptide or polynucleotide suppresses an effect of a mutation causing or associated with amyotrophic lateral sclerosis (ALS) (in particular, an effect of a SOD1 G85R allele).
  • ALS amyotrophic lateral sclerosis
  • A“loss-of- fimction mutation” is a mutation that decreases or abolishes an activity or function of a polypeptide.
  • A“gain-of-function mutation” is a mutation that enhances or increases an activity or function of a polypeptide.
  • a gain-of-function mutation in a Cyp4gl polypeptide increases enzymatic activity of the Cyp4gl polypeptide (e.g., increases its ability to perform omega-hydroxy lation of fatty acids) or causes an alternative product lipid to be synthesized as opposed to the normal product.
  • mutation and/or misexpression associated with amyotrophic lateral sclerosis or“mutation associated with a neurological disease” is meant any mutation causing or correlated with ALS or a neurological disease in a subject.
  • exemplary mutations associated with ALS or a neurological disease include, without limitation, mutations in SOD1 (e.g., SOD1 G85R, SOD1 H71Y) or expansion of C9orf72 repeat.
  • neurodegenerative disease characterized by an energy deficit is meant a disease characterized by or associated with progressive loss of function or structure of neurons, including death of neurons.
  • the neuron is a motor neuron.
  • Examples of neurode generative disease characterized by an energy deficit include, without limitation, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
  • Exemplary neurodegenerative diseases amenable to treatment with a composition delineated herein include, but are not limited to, ALS, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.
  • “obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • phenotype associated with amyotrophic lateral sclerosis or“ALS phenotype” is meant any observable or measurable characteristic exhibited by an organism having ALS.
  • ALS phenotypes include, without limitation, weakened muscles, motor neuron death, metabolic wasting and shortened lifespan.
  • a "reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • Lor polypeptides the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • siRNA is meant a double stranded RNA.
  • an siRNA is 18, 19, 20, 21,
  • dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream.
  • siRNAs are used to downregulate mRNA levels or promoter activity.
  • SOD1 polypeptide is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to GenBank Accession No. CAG46542 (Homo sapiens), NCBI Accession No. NP 001261700, or NCBI Accession No. NP 476735 (various isoforms in Drosophila melanogaster), and having an activity of superoxide dismutase (e.g., antioxidant activitiy or catalysis of dismutation of a superoxide radical).
  • superoxide dismutase e.g., antioxidant activitiy or catalysis of dismutation of a superoxide radical.
  • CAG46542 is provided below: 1 matkavcvlk gdgpvqgiin feqkesngpv kvwgsikglt eglhgfhvhe fgdntagcts
  • S0D1 polynucleotide is meant a nucleic acid molecule encoding a SOD1 polypeptide.
  • An exemplary SOD1 polynucleotide sequence is provided at GenBank Accession No. CR541742, which is reproduced below:
  • telomere binding By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • SDS sodium dodecyl sulf
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT,
  • GAP GAP, or PILEUP/PRETTYBOX programs.
  • Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
  • TDP-43 polypeptide is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to NCBI Accession No. NP 031401.1 and DNA-binding activity.
  • the exemplary TDP-43 polypeptide sequence at NCBI Accession No. NP 031401.1 is provided below:
  • TDP-43 polynucleotide is meant a nucleic acid molecule encoding a TDP-43 polypeptide.
  • An exemplary TDP-43 polynucleotide sequence is provided at NCBI Accession No. NM 007375.3, which is reproduced below:
  • subject is meant a multicellular organism, including, but not limited to, a vertebrate (e.g. a human or a non-human mammal such as bovine, equine, canine, ovine, feline) or invertebrate organism (e.g., a fly or worm).
  • a vertebrate e.g. a human or a non-human mammal such as bovine, equine, canine, ovine, feline
  • invertebrate organism e.g., a fly or worm
  • the subject is a vertebrate or invertebrate.
  • the subject is human.
  • the subject is Drosophila melanogaster.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIG. 1 is a schematic representation of an unbiased forward genetic screen described herein.
  • Forward genetics is the process of identifying a phenotype with a genotype.
  • a fly (Drosophila melanogaster) model of amyotrophic lateral sclerosis (ALS) was generated by introduction of SOD1 ALS-causing mutations into the fly genome.
  • Such human rapid- progressing mutations are lethal in the fly, in particular, the G85R SOD1 allele.
  • Flies die late in development or early in adult life with a profound nerve degeneration.
  • a forward genetic screen using random chemical mutagenesis was performed. The goal was to identify second-site mutations in genes that would restore viability of G85R when mutated.
  • FIGS. 2A-2C are schematic representations of a forward genetic screen designed to identify suppressors for SODf lethality. Shown in FIG. 2A is a screen designed such that all progeny of the mutagenesis cross have a genotype that is ultimately lethal, unless the progeny carries a mutation in another gene that suppresses the lethal effect of the SOD1 G85R allele.
  • FIG. 2B males heterozygous for G85R (line A) are starved, fed 25mM EMS, and mated to another line of heterozygous G85R (line B) unmutagenized females.
  • FIG. 2C shows representative progeny as an outcome of this cross. G85R homozygotes will only survive this treatment if they carry a potential dominant suppressor mutation (asterisk). The balancer homozygous flies will die due to the recessive lethal gene on the TM3,hs-hid balancer chromosome, and G85R homozygous flies will die due to the lethality of the G85R allele.
  • FIG. 3A-3E are schematics and diagrams depicting results of the screen performed and mapping of the mutations as described herein.
  • FIG. 3A is diagram showing a summary of the results of the screen performed. From the screen, five (5) true-breeding suppressed stocks of G85R/G85R were obtained. All five rescuing mutations mapped to the same gene (Cyp4gl).
  • FIGS. 3B-3C show genetic mapping of the lethality of EMS 81 and EMS 102. Genetic crosses were performed to combine the suppressor mutants with a standard mapping chromosome in females. In FIG.
  • 3D-3E show molecular mapping of the lethality of EMS 81 and EMS 102.
  • EMS 81- PR males were crossed to females of five balanced deficiency stocks for the region determined by genetic mapping (BSCdfl-5). Deficiencies that complement EMS 81 are shown in green. Only BSCdf2 (red) failed to complement EMS 81 lethality, mapping it to a ⁇ 260kb region.
  • BSCdf2 red
  • both EMS 81 and EMS 102 balanced females were crossed to males of seven stocks carrying the w + -marked duplications on the third chromsome. Duplications that failed to rescue male lethality are shown in pink.
  • Duplications 2-4 rescued male lethality of both EMS 81 and EMS 102, and limit the molecular interval where they are located.
  • the refined interval contained two genes, A and B (shown at the bottom of FIG. 3E).
  • Gene B had a previously molecularly uncharacterized lethal mutation, l(l)x, which was sequence verified as a 13bp deletion in coding sequence, and EMS 81 failed to complement l(l)x, thus confirming the identity of the gene.
  • Sequence analysis also revealed mutations in both alleles EMS 81 and EMS 102 of the Su(G85R) gene.
  • the Su(G85R) gene is Cyp4gl.
  • FIG. 4 is a sequence diagram showing the mutations in the protein sequence of Cyp4gl. The mutated amino acids are in bold. The amino acid substitutions are listed at the bottom of FIG. 4.
  • FIG. 5 is a sequence diagram showing the mutations in the nucleotide sequence of Cyp4gl.
  • the nucleotide sequence encoding the protein sequence of Cyp4gl is in bold.
  • the mutated codons are underlined.
  • the nucleotide mutations are indicated at the bottom of FIG. 5.
  • FIG. 6 is a diagram depicting the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene.
  • the dCyp4gl (CG3972) gene in Drosophila is an intronless gene.
  • FIG. 7 is an alignment of dCyp4gl and hCYP4V2 (closest human homologue).
  • the box labeled“EMS81” indicates a position of mutation in the mutant line EMS81 obtained in a screen performed herein.
  • the mutation suppressed SOD1 is G85R lethality. The amino acid in this position is conserved between dCyp4gl and hCYP4V2.
  • FIG. 8 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates.
  • the boxes labeled“EMS35/130,”“EMS102,” and“EMS81” indicate positions of suppressor mutations in mutant lines EMS35/130, EMS102, and EMS81 obtained in the screen performed herein, which suppressed SOD1 G85R lethality. Alignment was performed using T-COFFEE, Version_11.00.8cbe486.
  • the Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102. Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow. (1) lBb[19] is almost certainly a null allele of Cyp4gl. The (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote. Thus, without being bound by theory, it is believed that there are loss-of-function and gain-of- function components to the EMS35/130, EMS102, and EMS81 suppressor alleles, which make them novel.
  • FIG. 9 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. The alignment was performed using T-COFFEE,
  • FIG. 10 is an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket. Modeling of the active site of the CYP4A11 protein (related to CYP4V2) showed residues lining the substrate binding pocket and active site involved in omega-hydroxy lation. The EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.
  • FIG. 11 is a schematic showing a structure of the compound HET0016.
  • HET0016 N- hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6) is a potent inhibitor of CYP4V2.
  • FIG. 12 is a table showing the Cyp4 class of enzymes and their interfamily homology.
  • Cyp4V22[NP_997235(human)] revealed that homology within family Cyp4 is >30% for all homologues.
  • Cyp4 class paralogues
  • FIG. 12 also shows a sharp drop in E-value in non-Cyp4 family members.
  • These non-Cyp4 family members are other Cyp classes of enzymes (e.g., Cyp3), which are quite different from Cyp4 in terms of substrates.
  • Cyp4gl polypeptide herein may be any polypeptide having at least about 30% sequence identity to a human Cyp4gl (Cyp4V22) or Drosophila Cyp4gl and having an activity of a Cyp4 enzyme (e.g., omega hydroxylation of a fatty acid).
  • Cyp4 enzyme e.g., omega hydroxylation of a fatty acid
  • the invention features compositions and methods that are useful for suppressing a neurological disease, in particular, symptoms of mutations causing amyotrophic lateral sclerosis (ALS) as well as sporadic cases and related dementias.
  • ALS amyotrophic lateral sclerosis
  • the invention is based, at least in part, on the discovery of genetic sequences in a Drosophila melanogaster model of ALS that suppressed lethal effects of SOD1-G85R (an ALS-causing mutation).
  • the Drosophila melanogaster model is a powerful model for human neurodegenerative diseases such as ALS.
  • ALS neurodegenerative diseases
  • genome-wide genetic screens in yeast have led to the identification of important genetic modifiers of TDP-43 and FUS-mediated ALS toxicity (Elden et al, 2010; Ju et al, 2011; Sun et al, 2011; Couthouis et al., 2011).
  • Drosophila has a very compact genome with -15,000 genes (Adams et al., 2000) including a vast number of genes (197 of 287) in which mutations are known to cause human disease (St Johnston, 2002). As such, Drosophila is an ideal model system for the operation of forward genetic screens to divulge specific genetic mutations that can act as suppressors and modify disease phenotypes.
  • EMS directed mutagenesis method
  • G-to-A transitions Bokel, 2008
  • transposon insertion screens for example, allows for a much greater spectrum of
  • the SOD1 gene is highly conserved between Drosophila and humans.
  • the G85R point mutation was introduced into the endogenous locus of cl So cl I via homologous recombination.
  • the dSodl homozygotes display a distinct phenotype: flies die in the pupal case during the eclosion period.
  • a forward genetic screen described herein revealed four (4) novel missense mutations causing amino acid substitutions in Drosophila melanogaster Cyp4gl that suppressed SOD1 G85R mutant lethality.
  • the suppressor mutations found in Cyp4gl were G92E, D112N, S143N, and M333I.
  • Mapping and molecular studies have revealed the nature of the Cyp4gl gene in flies, a member of the ubiquitous cytochrome P450 (CYP) class of proteins found in all life forms, including bacteria. CYP proteins play two major roles in biology; detoxifying
  • CYP4 family of CYP genes.
  • the CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals.
  • the novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids.
  • the invention provides Cyp4gl polypeptides, or fragments thereof having an activity of a cytochrome P450 (CYP) polypeptide, comprising a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
  • the mutation is G92E, D112N, S143N, or M333I.
  • the invention provides polynucleotides or expression vectors encoding Cyp4gl polypeptides comprising the suppressor mutations described herein.
  • cells or organisms in particular, Drosophila melanogaster mutant lines harboring suppressor Cyp4gl mutations are provided.
  • the suppressor Cyp4gl mutation is a gain-of-function or a loss-of- function mutation.
  • the Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102 (i.e., Drosophila lines harboring Cyp4gl S143N and D112N mutations).
  • Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow.
  • (1) lBb[19] is almost certainly a null allele of Cyp4gl.
  • the (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote.
  • Sodl-G85R lethality weakly suppresses Sodl-G85R lethality as a heterozygote.
  • dCyp4gl The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells.
  • TAG triacylglyceride
  • dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver (Gutierrez et al., Nature (2007) 445:275-280).
  • Fatty acids have been implicated in ALS, as a system-wide metabolic defect called wasting (or hypermetabolism) is seen in patients.
  • wasting or hypermetabolism
  • MNs motor neurons
  • ALS motor neurons
  • Huntington Huntington
  • the invention features methods of modulating fatty acid or lipid metabolism in a subject, comprising administering to the subject a Cyp4gl polypeptide comprising the suppressor mutations described herein (e.g., mutations in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333).
  • a Cyp4gl polypeptide comprising the suppressor mutations described herein (e.g., mutations in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333).
  • the invention provides methods of identifying a modulator of neurode generative diseases (particularly ALS, Parkinson’s, dementia, and Huntington’s), comprising screening of candidate agents that modulate the level or activity of a Cyp4gl polypeptide.
  • This hypermetabolic effect may likely be a result of a chronic induction of innate immunity, through currently unknown toxic insults, and the bodies’ response to mounting a response to a pathogen that is“unreal.” It is motor neurons in particular that rely most on systemic energetics for their function.
  • the CYP4 class of enzymes play a unique biochemical role in regulation of body fat mobilization and utilization.
  • hSODlG85R transgenic mouse model has also been shown to have several metabolic changes consistent with a metabolic switch occurring as an early pathological event (Palamiuc et al., 2015).
  • Cyp4gl a suppressor gene of dSodlG85R lethality and a gene involved in metabolism, is believed to a role in energy balance in cells at a whole-animal level.
  • the Cyp4gl mutants obtained herein define the CYP4 class of enzymes (FIG. 12) as a potential target in regulating energy metabolism in disease, particularly neurodegenerative diseases associated with or characterized by an energy deficit (e.g., ALS, Parksinon’s, dementia, and Huntington’s).
  • an energy deficit e.g., ALS, Parksinon’s, dementia, and Huntington’s.
  • ALS ALS
  • Parksinon’s dementia
  • Huntington Huntington
  • Agents that modulate the level or activity of a Cyp4gl polypeptide are expected to modulate energy balance at a whole-animal level and thereby“correct” the energy deficit in a subject having a neurodegenerative disease such as ALS, Parksinon’s, dementia, or
  • a Cyp4gl polypeptide or polynucleotide comprising the suppressor mutations herein is also expected to ameliorate the energy deficit in a subject having a neurodegenerative disease.
  • the invention additionally provides methods of suppressing or beating a neurodegenerative disease in a subject (particularly a neurodegenerabve disease characterized by an energy deficit, such as ALS, Parkinson’s, dementia, or Huntington’s disease), comprising administering to a subject a Cyp4gl polynucleotide or polypeptide comprising suppressor mutations described herein.
  • the invention further provides Cyp4gl polypeptides in other organisms (e.g., human Cyp4gl polypeptide), comprising the suppressor mutations.
  • the Cyp4gl polypeptide is from an invertebrate or vertebrate.
  • the polypeptide has at least 30%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide.
  • the Cyp4gl polypeptide is human Cyp4gl polypeptide.
  • a human homologue of Cyp4gl is the CYP4V2 gene, mutations in which cause a retinal degenerative disease called Bietti’s Crystalline Dystrophy (BCD).
  • BCD Crystalline Dystrophy
  • Patients with BCD develop a late onset degeneration of the retina with deposits, but more interestingly, BCD patients have a systemic increase in fatty acids/lipids. Many mutations that cause BCD map to very near the suppressor mutations found in Cyp4gl.
  • a mouse model of BCD also recapitulates key feature of the disease, including altered lipid profiles.
  • mutations in CYP4V2 or inhibitors could possibly suppress mouse ALS models, as in the fly system.
  • the present invention provides methods of treating a neurological disease (in particular, ALS) and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation described herein to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human
  • a method of treating a subject suffering from or susceptible to a neurological disease or disorder or symptom thereof includes the step of administering to the mammal a therapeutic amount of an amount of a Cyp4gl polynucleotide or polypeptide sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g.
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the Cyp4gl polynucleotides or polypeptides herein (such as a Cyp4gl polynucleotide or polypeptide (or fragment thereof) comprising a mutation in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333) to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
  • a subject e.g., animal, human
  • Such treatment will be suitably
  • a neurological disease particularly ALS
  • disorder or symptom thereof.
  • Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, ALS causing or ALS associated mutations or misexpression (e.g., a SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression), family history, and the like).
  • ALS causing or ALS associated mutations or misexpression e.g., a SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression), family history, and the like.
  • SOD1 mutation such as SOD1 G85R
  • C9orf72 repeat expansion or TDP-43 misexpression
  • family history and the like.
  • the compounds herein may be also used in the treatment of any other disorders in which ALS causing or ALS associated mutations (e.g
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of a neurological disease marker (e.g.,
  • SOD1 mutation such as SOD1 G85R, C9orf72 repeat expansion, or TDP-43 misexpression
  • diagnostic measurement e.g., screen, assay
  • the level of a neurological disease marker determined in the method can be compared to known levels of the neurological disease marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status.
  • a second level of a neurological disease marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of a neurological disease marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of the neurological disease marker can then be compared to the level of the marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • a subject identified as having increased level of a ALS or neurological disease marker polynucleotide or polypeptide e.g. C9orf72 repeat or TDP-43, or a mutation in SOD1 (particularly, SOD1 G85R)) relative to a reference is administered a therapeutic composition of the invention.
  • a ALS or neurological disease marker polynucleotide or polypeptide e.g. C9orf72 repeat or TDP-43, or a mutation in SOD1 (particularly, SOD1 G85R)
  • polynucleotides or polypeptides are measured in a subject sample and used as an indicator of an ALS or neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide of the invention.
  • Levels of neurological disease marker polynucleotides may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of neurological disease marker polypeptides in a biological sample derived from a subject. Such methods include immunoassay, ELISA, western blotting using an antibody that binds the marker polypeptide, and radioimmunoassay.
  • Methods for detecting a mutation in a marker polypeptide include immunoassay, direct sequencing, and probe hybridization to a polynucleotide encoding the mutant polypeptide. Elevated levels of neurological disease marker polynucleotides or polypeptides and/or a mutation in a neurological disease polynucleotide or polypeptide are considered a positive indicator of ALS or a neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide of the invention.
  • compositions useful for treating a neurological disease or suppressing effects of mutations causing a neurological disease (particularly ALS) in a subject comprises a Cyp4gl polypeptide comprising a suppressor mutation described herein.
  • the composition comprises a polynucleotide encoding an amino acid sequence of the suppressor polypeptide.
  • the composition further comprises a vehicle for intracellular delivery of the polypeptide or polynucleotide (e.g., a liposome).
  • compositions comprising a Cyp4gl polynucleotide or polypeptide herein for the treatment of a neurological disease such as ALS may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject.
  • the composition may be administered systemically, for example, formulated in a pharmaceutically -acceptable buffer such as physiological saline.
  • Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the agent in the patient.
  • the amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neurological disease. Generally, amounts will be in the range of those used for other agents used in the treatment of neurological diseases such as ALS (or other diseases associated with ALS causing mutations, such as SOD1 G85R), although in certain instances lower amounts will be needed because of the increased specificity of the agent.
  • a composition is administered at a dosage that suppresses effects of the neurological disease causing mutation or that decreases effects or symptoms of ALS or neurological disease (e.g., wasting, weakened muscular strength, or shortened lifespan) as determined by a method known to one skilled in the art.
  • the therapeutic suppressor Cyp4gl polynucleotide or polypeptide may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously,
  • compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
  • compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the liver; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a cancer using carriers or chemical derivatives
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.
  • Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
  • the pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants.
  • injection, infusion or implantation subcutaneous, intravenous, intramuscular, intraperitoneal, or the like
  • suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in singledose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • composition may include suspending, solubilizing, stabilizing, pH -adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
  • the composition comprising the active therapeutic is formulated for intravenous delivery.
  • the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection.
  • the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxy benzoate).
  • preservatives e.g., methyl, ethyl or n-propyl p-hydroxy benzoate.
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
  • Cyp4gl polypeptides of the invention comprising mutations suppressing effects of ALS-causing mutations (e.g., SOD1 G85R), are useful for treating or suppressing a neurological disease such as ALS in a subject.
  • Recombinant Cyp4gl polypeptides of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.
  • the invention provides methods of producing a polypeptide of the invention, the method comprising (a) heterologously expressing an expression vector comprising a polynucleotide encoding the polypeptide in a host cell; and (b) isolating the polypeptide from the host cell.
  • a Cyp4gl polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells).
  • a prokaryotic host e.g., E. coli
  • a eukaryotic host e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells.
  • Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997).
  • the method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985,
  • Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
  • the polypeptides of the invention are produced in a bacterial expression system.
  • E. coli pET expression system e.g., pET-28
  • DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
  • pGEX expression system Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia).
  • This system employs a GST gene fusion system that is designed for high- level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products.
  • the protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione.
  • Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain.
  • proteins expressed in pGEX- 2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.
  • recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast.
  • Pichia is capable of metabolizing methanol as the sole carbon source.
  • the first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase.
  • Expression of this enzyme, which is coded for by the AOX1 gene is induced by methanol.
  • the AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.
  • the recombinant Cyp4gl polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography.
  • an antibody e.g., produced as described herein
  • the polypeptide may be attached to a column and used to isolate the recombinant polypeptide.
  • the polypeptide comprises an epitope tag fused to the Cyp4gl polypeptide.
  • the polypeptide is then isolated using an antibody against the epitope tag. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra).
  • polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column.
  • a sequence tag such as a hexahistidine tag
  • the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
  • Polypeptides of the invention particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).
  • Suppressor Cyp4gl polypeptides or polynucleotides of the invention which are useful for suppressing a neurological disease (particularly ALS) in an organism, may be delivered to an organism (particularly liver cells of the organism) in any manner such that the polypeptide is in functional form in the cell.
  • the Cyp4gl polypeptide comprising suppressor mutations may be delivered to cells as a polypeptide.
  • a polynucleotide encoding an amino acid sequence of the Cyp4gl polypeptide may be delivered to cells for heterologous expression of a suppressor Cyp4gl polypeptide in the cells.
  • the present invention features polypeptides delivered to a cell by contacting the cell with a composition comprising the polypeptide or by heterologously expressing the polypeptide in the cell.
  • Polypeptides of the invention may be delivered intracellularly to cells.
  • the polypeptide must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the polypeptide, or fragment thereof, is in functional form in the cells.
  • Methods of intracellular delivery of polypeptides are known to one of skill in the art.
  • Exemplary methods of intracellular delivery of polypeptides include, without limitation, incorporation of the polypeptide into a liposome.
  • Liposomes are phospholipid vesicles with sizes varying from 50 to 1000 nm, which can be loaded with polypeptides or other agents.
  • Liposomal intracellular delivery of polypeptides into cells typically relies on endocytosis of the liposome-encapsulated polypeptide into the cell.
  • suitable liposomes for intracellular delivery of polypeptides may be pH-sensitive liposomes.
  • Such liposomes are made of pH-sensitive components; after being endocytosed in intact form, the liposome fuses with the endovacuolar membrane under lowered pH inside the endosome and destabilizes it, thereby releasing the contents (including the polypeptides encapsulated in the liposome) into the cytoplasm.
  • the liposomes may also be further modified to enhance their stability or lifetime during circulation (e.g., by PEGylated liposomes). Liposomes may also be modified to specifically target antigens (e.g.,“immunoliposomes” or liposomes embedded with antibodies to an antigen).
  • Antibody -bearing liposomes may have the advantages of targetability and facilitated uptake via receptor-mediated endocytosis.
  • a cell penetrating peptide or“CPP” is a protein or peptide that can translocate through cellular membranes.
  • a polypeptide for delivery into a cell is fused with a CPP, thereby enabling or enhancing delivery of the polypeptide fusion into the cell.
  • Cell penetrating peptides include, for example, a trans-activating transcriptional activator (TAT) from HIV-1, Antenapedie (Antp, a transcription factor in Drosophila), and VP22 (a herpes virus protein).
  • TAT trans-activating transcriptional activator
  • Antenapedie Antenapedie
  • VP22 a herpes virus protein
  • Supercharged proteins or supercharged polypeptides are a class of engineered or naturally existing polypeptides having an unusually high positive or negative net theoretical charge. Membranes of cells are typically negatively charged.
  • Superpositively charged polypeptides are able to penetrate cells (particularly mammalian cells), and associating cargo with superpositively charged polypeptides (e.g., polypeptides or polynucleotides) can enable functional delivery of these macromolecules into cells, in vitro or in vivo.
  • Methods of generating supercharged polypeptides and using supercharged polypeptides for intracellular polypeptide delivery are described in further detail in, for example, Zuris et al. Nat. Biotechnol. (2015) 33:73-80 and Liu et al. Methods Enzymol. 2012, 503: 293-319.
  • the invention provides a suppressor Cyp4gl polypeptide fused to a polypeptide enabling intracellular delivery of the suppressor Cyp4gl polypeptide (e.g., a cell penetrating peptide or supercharged polypeptide).
  • a suppressor Cyp4gl polypeptide e.g., a cell penetrating peptide or supercharged polypeptide.
  • Another therapeutic approach for treating or suppressing a neurological disease is polynucleotide therapy using a polynucleotide encoding a suppressor Cyp4gl polypeptide of the invention, or fragment thereof.
  • isolated polynucleotides encoding a Cyp4gl polypeptide of the invention, or fragment thereof.
  • Expression of such polynucleotides or nucleic acid molecules in a cell or organism is expected to suppress effects of mutations causing neurological disease (particularly, ALS) in the subject.
  • Such nucleic acid molecules can be delivered to cells of a subject having a neurological disease.
  • the nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the Cyp4gl polypeptide, or fragment thereof, can be produced.
  • Transducing viral e.g., retroviral, adenoviral, and adeno-associated viral
  • Transducing viral can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997).
  • a polynucleotide encoding a Cyp4gl polypeptide of the invention, or a fragment thereof can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest.
  • viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet
  • Epstein-Barr Virus also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
  • a viral vector is used to administer a polynucleotide encoding a suppressor Cyp4gl polypeptide (or fragment thereof) systemically.
  • Non-viral approaches can also be employed for the introduction of the therapeutic to a cell of a patient requiring suppression of a neurological disease.
  • a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci.
  • nucleic acids are administered in combination with a liposome and protamine.
  • Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell.
  • Transplantation of genes encoding suppressor Cyp4gl polypeptides into the affected tissues of a patient can also be accomplished by transferring a nucleic acid encoding the suppressor Cyp4gl polypeptide into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.
  • a cultivatable cell type ex vivo e.g., an autologous or heterologous primary cell or progeny thereof
  • cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • metallothionein promoters e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters
  • enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid.
  • the enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.
  • regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
  • Delivery of polynucleotides of the invention may also include or be performed in combination with gene or genome editing methods, such as CRISPR-Cas systems, to introduce polynucleotides encoding suppressor Cyp4gl polypeptides in cells.
  • Gene or genome editing methods such as CRISPR-Cas systems are further described in for example, Sander et al. (2014), Nature Biotechnology 32, 347-355; Hsu et al. (2014), Cell 157(6): 1262-1278.
  • suppressor Cyp4gl polynucleotides or polypeptides of the invention are delivered to a liver cell. It is noted that many systems of delivery of therapeutic polypeptides or polynucleotides (particularly nanoparticulate or liposomal delivery systems) result in high accumulation of the therapeutic polypeptides or polynucleotides in the liver.
  • polynucleotides or polypeptides such as liposomal delivery, may be particularly suitable for delivery of therapeutic Cyp4gl polypeptides or polynucleotides of the invention.
  • the present invention further features methods of identifying modulators of a disease, particularly neurological disease, comprising identifying candidate agents that interact with and/or alter the level or activity of a Cyp4gl polypeptide.
  • the CYP4 class of enzymes plays a unique biochemical role in regulation of body fat mobilization and utilization.
  • the mutants obtained herein define the CYP4 class of enzymes as a potential target in regulating energy metabolism in disease.
  • the CYP4 class of enzymes may, because of their novel active-site chemistry, serve as ideal drug targets to control the pace of lipid metabolism, to achieve some degree of efficacy in treating the whole organism, not just neurons (the current focus of most therapies).
  • the invention provides a method of identifying a modulator of a neurological disease, comprising (a) contacting a polypeptide with a candidate agent, where the polypeptide is a Cyp4gl polypeptide or fragment thereof, and (b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity.
  • the method comprises (a) contacting a cell or organism with a candidate agent, and (b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity. An alteration in the level or activity of the Cyp4gl polypeptide or polynucleotide indicates the candidate agent is a modulator of neurological disease.
  • the activity of the Cyp4gl polypeptide is enzymatic activity or a lipid, combinations of lipids, and/or alteration of fatty acid metabolism.
  • the control activity may be the activity of the polypeptide when the polypeptide is not contacted with the candidate agent, or any agent.
  • the control activity may be activity of the polypeptide contacted with a carrier or solvent that does not contain the candidate agent.
  • the control activity or control level of the polypeptide may be the activity or level of the polypeptide in a cell when the cell is not contacted with the candidate agent (or any agent) or when the cell is contacted with a carrier that does not contain the candidate agent.
  • enzymatic activity of the polypeptide may be measured by measuring levels of substrate(s) modified by the polypeptide.
  • Binding activity of the polypeptide may be measured, for example, by immunoassay methods.
  • Polynucleotide levels may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure polypeptide levels, the methods including without limitation, immunoassay, ELISA, western blotting using an antibody that binds the polypeptide, and radioimmunoassay.
  • the invention provides a method of identifying a modulator of neurological disease, comprising (a) contacting a cell or organism with a candidate agent, and (b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism comprising a Cyp4gl polynucleotide or polypeptide having a mutation.
  • a similarity in the phenotypes indicates the candidate agent is a modulator of neurological disease.
  • the cell or organism also comprises a mutation and/or misexpression associated with ALS (e.g., a SOD1 G85R allele).
  • candidate agents that“phenocopy” a phenotype of an organism having a Cyp4gl mutation are identified as modulators of ALS or neurological disease.
  • the phenotype may be any observable or measurable phenotype, including without limitation, suppression of ALS associated phenotypes (e.g., weakness of muscles, motor neuron death, shortened lifespan), level and/or activity of Cyp4gl polypeptides or polynucleotides, or levels of lipid and/or fatty acid substrates and/or metabolic products.
  • the candidate agent or modulator of neurological activity inhibits an activity of Cyp4gl.
  • the agent or modulator suppresses an ALS phenotype.
  • the agent or modulator is HET0016.
  • HET0016 N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)
  • CYP4V2 human homolog of Cyp4gl.
  • HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122. It is expected that HET0016 will phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media.
  • the invention also provides methods of identifying lipid substrates or products of Cyp4gl -catalyzed reactions, as modulators of ALS or neurological disease.
  • the methods comprise (a) contacting a polypeptide with a substrate, where the polypeptide is a Cyp4gl polypeptide or fragment thereof having enzymatic activity, and (b) detecting a reaction product of the polypeptide contacted with the substrate, where detection of a reaction product indicates the substrate and/or reaction product is a modulator of neurological disease.
  • the activity or enzymatic activity is omega-hydroxylation of a fatty acid. Detection of the substrate and/or reaction product may be performed according to any standard method known in the art (e.g., NMR or mass spectrometry).
  • the invention features methods of treating a neurode generative disease in a subject, the methods comprising administering to the subject an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide having a mutation.
  • the mutant Cyp4gl polypeptide or polynucleotide is administered in combination with an agent that decreases a level or activity of Cyp4gl polypeptide in the subject.
  • An agent that decrease a level or activity of Cyp4gl polypeptide may be, for example, an inhibitory nucleic acid that reduced expression of Cyp4gl polypeptide (e.g., siRNA) or a small molecule compound that inhibits Cyp4gl polypeptide activity (e.g., HET0016).
  • an inhibitory nucleic acid that reduced expression of Cyp4gl polypeptide e.g., siRNA
  • a small molecule compound that inhibits Cyp4gl polypeptide activity e.g., HET0016
  • an anti-neurological disease therapeutic of the invention e.g., a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation as described herein
  • any other standard anti-neurological disease e.g., anti-ALS
  • any other standard anti-neurological disease e.g., anti-ALS
  • kits for the treatment, suppression, or prevention of a neurological disease particularly amyotrophic lateral sclerosis (ALS) associated with SOD1 mutations (e.g., SOD1 G85R), C9orf72 repeat expansion, or TDP-43 overexpression.
  • the kit includes a therapeutic or prophylactic composition containing an effective amount of a suppressor Cyp4gl polypeptide, or fragment thereof (or a polynucleotide encoding such) in unit dosage form.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • the kit further includes reagents detecting a mutation associated with or causing a neurological disease (e.g., ALS) in a subject.
  • the reagents may be primers or hybridization probes for detection of mutation in SOD1 (e.g., SOD1 G85R), a C9orf72 repeat expansion, or TDP-43 overexpression.
  • a composition comprising a therapeutic agent of the invention (e.g., Cyp4gl polypeptide or polynucleotide comprising suppressor mutations described herein) is provided together with instructions for administering the agent to a subject having or at risk of developing a neurological disease.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of neurological disease.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Forward genetic screen in fly model of amyotrophic lateral sclerosis (ALS) revealed mutations suppressing lethal effects of SOD1 G85R.
  • ALS amyotrophic lateral sclerosis
  • dSodlG85R/Tm3,hs- 185 hid,sb line A and line B.
  • Newly eclosed dSodlG85R/Tm3,hs-hid,sb (line A) males were aged for 1-7 days and were then mutagenized.
  • dSodlG85R/Tm3,hs-hid,sb (line A) mutagenized males to minimize the possibility of homozygosing background mutations. Flies were kept at constant 25oC, on standard molasses food, and under 12-h day /night cycles. 6028 dSodlG85R/Tm3,hs-hid (line A) males were starved overnight (12hr) in vials supplied with only water. Surviving males were fed with 25mM EMS (Sigma M-0880) in 5% sucrose solution for 10 hours. The water and sucrose solution containing EMS was administered with Kimwipe wrapped ceaprene stoppers as described by Christian Bokel (Bokel, 2008).
  • dSodlG855RG85R homozygotes and were subsequently screened regularly for survivors over the next two weeks.
  • the surviving progeny were named as EMS1 through EMS145. Flies that survived more than 3 days were crossed with 3 flies (dSodlG85R/Tm3-hsHid) of the opposite sex.
  • the dSodl locus of all the surviving lines were PCR amplified and sequenced with forward primer: 5 '-GC AT GT ATTT CT AAGCT GCTCT GCT ACGGT C AC-3 ' and, reverse primer: 5'- GTCCACTGCTAAGAGCAGCTGCCCTC-3'.
  • the Drosophila line EMS130/35 was found to carry mutation G92E; line EMS102 carried D112N, EMS81 carried S143N, and EMS94 carried mutation M333I.
  • the suppressor alleles Cyp4gl G92E, Cyp4gl D112N, Cyp4gl S143N, and Cyp4gl M333I may be interchangeably referred to herein as“EMS130/35,”“EMS102,”“EMS81,” and“EMS94,” respectively.
  • the suppressor mutations G92E, D112N, S143N, and M333I are interchangeably referred to herein as“EMS130/35 mutation,”“EMS102 mutation,”“EMS81 mutation,” and“EMS94 mutation,” respectively.
  • the EMS 81/102 mutations were first balanced over the Fm7 balancer, as described above, and then heterozygote females were crossed to males of a mapping chromosome stock carrying five recessive visible markers. Non-Fm7 heterozyogous females of the FI (where recombination occurs) were then crossed to wild type males, and phenotypes were scored in the F2.
  • the lethal phenotype of the EMS 81/102 mutations was used to infer the location of these mutations by assessing the lack of particular classes of recombinants. In fact, it was clear that both the EMS 81/102 mutations mapped between two tightly linked (1 cM) visible markers (call them p and q).
  • mapping data (FIG. 3B).
  • the lethal mutation on a wild type background in heterozygous state with a doubly mutant mapping chromosome (p + q, where p, +, and q are the gene order of the p locus, the wild type EMSX locus, and the q locus respectively) will generate males in the FI that are either parental non-recombinant (NR) or recombinant (R) chromosomes (FIG. 3C).
  • NR parental non-recombinant
  • R recombinant
  • Recombination can occur on two intervals that result in viable recombinant offspring (I and II, FIG. 3C).
  • I and II viable recombinant offspring
  • the region of the X chromosome is densely populated with genes, and represents a region where the genetic map is contracted compared to other regions of the X chromosome.
  • the lcM region that the EMSX mutations mapped to covers 2 Mb of physical distance on the X chromosome containing hundreds of genes.
  • EMS 81 hemizygote males could be obtained as viable adults. These males will be referred to herein as pseudo-rescued (EMS 81-PR) males.
  • EMS 81-PR males were found to be fertile and mobile for at least a week. This allowed us to cross these males to molecularly -defined deficiency bearing balanced stocks carrying a series of five deficiencies spanning the 2Mb regions implicated in genetic mapping studies (Cook et al., 2012) (FIG. 3D).
  • One of these deficiencies (BSCdf2) failed to complement EMS 81, giving rise to female progeny that died in exactly the same manner as hemizygous EMS 81 males, namely, as pharate adults during eclosion. All other deficiencies tested complemented the lethal phenotype of EMS 81.
  • a and B A lethal mutation (molecularly uncharacterized) happened to have been attributed to the gene B, which is referred to herein as Su(G85R).
  • This mutation (l(l)x) was obtained as a balanced stock and EMS 81-PR males were crossed to this stock, resulting in failure of complementation of the l(l)x mutation.
  • the Su(G85R) gene was sequenced for EMS 81, EMS 102 and the l(l)x mutations.
  • l(l)x stock a 13nt deletion was found in the open reading frame of Su(G85R), predicted to truncate the protein at about 2/3 its normal length.
  • l(l)x is almost certainly a null mutation for Su(G85R).
  • Mutations were also found in the EMS 81 and 102 mutant stocks, G-to-A mutations, which is consistent with EMS as the mutagen. Both mutations cause missense change in different and highly conserved positions within the Su(G85R) protein, which is known to play a role in metabolism.
  • the Su(G85R) gene is Cyp4gl.
  • EMS 81 and 102 were introgressed into a balanced dSodlH71Y genetic background, using alleles of EMS 81 and 102 that had been recombined with the tightly linked w+ gene, allowing the suppressor alleles to be monitored via eye color. Homozygosity for
  • dSodlH71 Y/H71 Y normally confers a high level of unviability in the pupal stage, and animals only live for about 16 days, with a profound loss of motor function within the first week.
  • FIG. 6 depicts the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene.
  • the dCyp4gl (CG3972) gene in Drosophila is an intronless gene.
  • CYP proteins play two major roles in biology; detoxifying (biodefense) against many xenobiotics and antibiotics, and biosynthesis of endogenous molecules such as steroids and lipids. There are as many as 37 families of CYP genes.
  • the CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals.
  • the novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids.
  • the dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells.
  • TAG triacylglyceride
  • dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver. Gutierrez et al., Nature (2007) 445:275-280.
  • FIG. 7 shows an alignment of dCyp4gl and hCYP4V2, the closest human homologue to dCyp4gl.
  • the amino acid in the position mutated in EMS81 (position 143) is conserved between dCyp4gl and hCYP4V2.
  • the hCYP4V2 gene is mutated in Bietti’s Crystalline Dystrophy (BCD) a retinal degeneration syndrome.
  • BCD Crystalline Dystrophy
  • hCYP4V2 protein has a fairly broad expression pattern including liver. Patients with BCD display a systemic dyslipidemia. A mouse model of BCD recapitulates clinical features of the human disease including systemic dyslipidemia.
  • FIG. 8 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates.
  • Suppressor alleles (EMS35/130, EMS102, and EMS81) isolated as dominant suppressors of SOD1-G85R all mapped to the dCyp4gl gene on the X chromosome.
  • EMS81 and EMS102 alleles were homozygous lethal in Drosophila and were also invariant positions in invertebrate lineages.
  • EMS35/130 and EMS94 were mutations in less conserved positions and were viable in males.
  • EMS35/130 result from the same
  • FIG. 9 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. Suppressor alleles occurred in regions of high conservation, but only EMS81 serine (S) was conserved from flies through mammals.
  • Example 3 Modeling of Cyp4gl structure revealed a suppressor mutation located in substrate binding pocket.
  • FIG. 10 shows an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket.
  • the EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.
  • HET0016 N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6) is a known potent inhibitor of CYP4V2 (FIG. 11).
  • HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122.
  • ALS sporadic ALS
  • Overexpression of the C9orf72 toxic repeat globally in flies was found to lead to a phenotype indistinguishable from the lethality seen with SOD1-G85R expression.
  • TDP-43 Another ALS-causing gene, TDP-43
  • TDP-43 gene normal biological role is currently hotly debated, but it is has been shown to regulate body fat composition.
  • Cyp4gl whether the suppressor mutations in Cyp4gl also suppress these other seemingly unrelated genetic forms of ALS will be tested. If so, then the work in Drosophila may have revealed an important underlying cause of ALS, and possibly, many age-related cognitive disorders. Suppressors will be tested with the TDP43 model of neurodegeneration in

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Abstract

The present invention features isolated Cyp4gl polypeptides comprising mutations at positions corresponding to amino acid positions 92, 112, 143, and 333 in a Drosophila melanogaster Cyp4gl polypeptide. In some embodiments, the mutations are G92E, D112N, S143N, and M333I. The mutations suppress a lethal effect of a SODl G85R allele. The present invention further features polynucleotides encoding these polypeptides, as well as methods of suppressing or treating a neurological disease such as amyotrophic lateral sclerosis (ALS) using compositions comprising polypeptides and/or polynucleotides of the invention.

Description

COMPOSITIONS AND METHODS FOR SUPPRESSING NEUROLOGICAL DISEASE
RELATED APPLICATION DATA
This application calims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 62/590,902, filed on November 27, 2017, the entire disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Amyotrophic lateral sclerosis (ALS) is a devastating disease that is progressive, invariably fatal and entirely untreatable. Affected patients usually present around age 50 with weakened muscular strength in the periphery, but can also present with difficulty swallowing or breathing, and generally die within 2-5 years of diagnosis due to respiratory failure. All the while, cognitive functions remain nearly intact. Mutations in many genes can cause familial ALS (fALS), and the genes have a wide range of functions and proposed mechanisms. Yet, most cases of ALS (90%) present as sporadic ALS (sALS), meaning no family history and presumably not correlated with genetics. Nevertheless, much evidence exists to support the idea that sALS and fALS have common underlying etiologies. For instance, a common feature of ALS is hypermetabolism, dysregulation of lipids, and wasting. Many mouse models have been developed to help unravel the mechanisms of ALS motor neuron death, but to date, have resulted in candidate gene approaches (educated guesses) that do not extend lifespan of affected mice. Another important aspect of the genetics of ALS, is a strong underlying connection with mutations in some of the same genes as Frontotemporal dementia (FTD). Given these facts, there is a pressing need for fundamental insights into this intractable disease, and treatments.
SUMMARY OF THE INVENTION
As described below, the present invention features compositions and methods useful for reducing or ameliorating an effect of a mutation causing familial amyotrophic lateral sclerosis (fALS) as well as treatment for sporadic ALS (sALS) cases that share many common features of fALS.
In one aspect, the invention provides an isolated polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
In another aspect, the invention provides an isolated Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333. In still another aspect, the invention provides an isolated human Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
In another aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
In yet another aspect, the invention provides an isolated polynucleotide encoding a Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
In still another aspect, the invention provides an isolated polynucleotide encoding a human Cyp4gl polypeptide, where the polypeptide contains a mutation in any one or more of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
In various embodiments of any one of the aspects delineated herein, the Cyp4gl polypeptide is a Cyp4gl polypeptide of an invertebrate or vertebrate. In some embodiments, the vertebrate or invertebrate is Drosophila melanogaster, Homo sapiens, Tenebrio molitor, Aedes aegypti, Diaphorina citri, Blatella germanica, Nasonia vitripennis, Spodoptera frugiperda, Oryctolagus cuniculus, Canis lupus, Gallus gallus, Xenopus tropicalis, or Danio rerio. In some other embodiments, the polypeptide has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide or a human Cyp4gl polypeptide.
In various embodiments of any one of the aspects delineated herein, the mutation is any one or more of mutations G92E, D112N, S143N, and M333I. In some embodiments, the mutation is S138N.
In some embodiments, the polypeptide contains any one of the following sequences:
Drosophila melanogaster Cyp4gl EMS81
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh
121 qhltkaeeyr yfkpwfgdgl lismghhwrh hrkmiaptfh qsilksfvpt fvdhskavva
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva Drosophila melanogaster Cyp4gl EMS102
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp 61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp ssdelilsgh 121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa 181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk 241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa 301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt 361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly 421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma 481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle 541 ngfnvslekr qyatva
Drosophila melanogaster Cyp4gl EMS130/35
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp 61 pelpilgqah vaaglsnaei lavglgylnk yeetmkawlg nvllvfltnp sdielilsgh 121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa 181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk 241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa 301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt 361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly 421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma 481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle 541 ngfnvslekr qyatva
Drosophila melanogaster Cyp4gl EMS 94
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp 61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh 121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa 181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk 241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa 301 skkeglrddl ddidendvga krrlalldam veiaknpdie wnekdimdev ntimfeghdt 361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly 421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma 481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle 541 ngfnvslekr qyatva
Human Cyp4gl (CYP4V2) EMS81
1 maglwlglvw qklllwgaas alslagaslv lsllqrvasy arkwqqmrpi ptvarayplv 61 ghallmkpdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk 121 ssmykflepw lglglltetg nkwrsrrkml tptfhftile dfldimneqa nilvkklekh 181 inqeafncff yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw 241 ldlwylmfke gwehkkslqi lhtftnsvia eranemnane dcrgdgrgsa psknkrrafl 301 dlllsvtdde gnrlshedir eevdtfmfeg hdttaaainw slyllgsnpe vqkkvdheld
361 dvfgksdrpa tvedlkklry lecviketlr lfpsvplfar svsedcevag yrvlkgteav
421 iipyalhrdp ryfpnpeefq perffpenaq grhpyayvpf sagprncigq kfavmeekti
481 lscilrhfwi esnqkreelg legqlilrps ngiwiklkrr nader
In various embodiments , the mutation is a gain-of-function or loss-of-function mutation. In some embodiments, the mutation suppresses a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS). In some other embodiments, the mutation associated with ALS is SOD1-G85R.
In another aspect, the invention provides an expression vector containing the isolated polynucleotide of any one of the aspects delineated herein.
In yet another aspect, the invention provides a therapeutic composition containing the polypeptide, polynucleotide or vector of any one of the aspects delineated herein. In various embodiments, the composition further contains a pharmaceutically acceptable excipient. In some embodiments, the composition further contains a vehicle for intracellular delivery of the polypeptide, polynucleotide, or vector.
In still another aspect, the invention provides a host cell or host organism containing the isolated polynucleotide or expression vector of any one of the aspects delineated herein. In some embodiments, the host cell or host organism is an amyotrophic lateral sclerosis (ALS) model or contains a mutation and/or misexpression associated with ALS. In some other embodiments, the host cell or host organism is mammalian.
In another aspect, the invention provides a Drosophila melanogaster mutant containing a Cyp4gl polynucleotide and/or polypeptide having a mutation in any one or more of amino acid positions 92, 112, 143, and 333. In some embodiments, the mutation is a missense mutation. In some other embodiments, the mutation is any one or more of mutations G92E, D112N, S143N, and M333I. In still other embodiments, the Drosophila melanogaster mutant further contains a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS). In some embodiments, the mutation and/or misexpression associated with ALS has a lethal effect. In various embodiments, the mutation associated with ALS is SOD1-G85R. In some embodiments, the Cyp4gl mutation suppresses the lethal effect of the mutation and/or misexpression associated with ALS. In some other embodiments, the suppression is partial, nearly complete, or complete.
In yet another aspect, the invention provides a method of reducing or ameliorating or preventing an effect of a mutation associated with a neurode generative disease characterized by an energy deficit in a subject. The method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby suppressing an effect of a mutation associated with a neurode generative disease characterized by an energy deficit in the subject.
In still another aspect, the invention provides a method of modulating lipid metabolism in a subject. The method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby modulating lipid metabolism in the subject.
In another aspect, the invention provides a method of treating a neurodegenerative disease characterized by an energy deficit in a subject. The method contains the step of administering to the subject an effective amount of a composition containing a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, where the polypeptide contains a mutation in any one or more positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby treating a neurodegenerative disease characterized by an energy deficit in the subject.
In various embodiments of any one of the aspects delineated herein, the composition is the therapeutic composition of any one of the aspects delineated herein. In various embodiments, the polypeptide or polynucleotide is the polypeptide, polynucleotide, or vector of any one of the aspects delineated herein. In some embodiments, the subject has a mutation associated and/or misexpression with amyotrophic lateral sclerosis (ALS). In some other embodiments, the mutation associated with a neurological disease or the mutation associated with ALS is SOD1-G85R or C9orf72 repeat expansion. In still other embodiments, the subject is human. In various embodiments, the methods of any one of the aspects delineated herein further contain the step of administering to the subject an effective amount of an agent that decreases a level or activity of Cyp4gl polypeptide. In some embodiments, the agent is an inhibitory nucleic acid that inhibits expression of Cyp4gl polypeptide.
In another aspect, the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit. The method contains the steps of (a) contacting a polypeptide with a candidate agent, where the polypeptide is a Cyp4gl polypeptide, and (b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity, where an alteration in activity indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
In yet another aspect, the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit. The method contains the steps of (a) contacting a polypeptide with a substrate, where the polypeptide is a Cyp4gl polypeptide having enzymatic activity, and (b) detecting a reaction product of the polypeptide contacted with the substrate, where detection of a reaction product indicates the substrate and/or reaction product is a modulator a neurodegenerative disease characterized by an energy deficit.
In still another aspect, the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit. The method contains the steps of (a) contacting a cell or organism with a candidate agent, and (b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity, where an alteration in the level or activity indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
In another aspect, the invention provides a method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit. The method contains the steps of (a) contacting a cell or organism with a candidate agent, and (b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism containing a Cyp4gl polynucleotide or polypeptide having a mutation, where a similarity in the phenotypes indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
In various embodiments, the activity is binding of the polypeptide to the candidate agent or enzymatic activity of the polypeptide. In some embodiments, the activity or enzymatic activity is omega-hydroxylation of a fatty acid. In some other embodiments, the substrate is a fatty acid. In still other embodiments, the phenotype is lipid metabolism. In various embodiments, the cell or organism contacted with the candidate agent is an amyotrophic lateral sclerosis (ALS) model or contains a mutation and/or misexpression associated with ALS. In some embodiments, the candidate agent inhibits an activity of Cyp4gl polypeptide. In some other embodiments, the candidate agent suppresses an ALS phenotype. In some embodiments, the cell or organism is the host cell or host organism of any one of the aspects delineated herein.
In various embodiments of any one of the aspects delineated herein, the
neurodegenerative disease characterized by an energy deficit is amyotrophic lateral sclerosis (ALS), dementia, Parkinson’s disease, or Huntington’s disease.
Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By“activity of a cytochrome P450 (CYP) polypeptide” is meant lipid metabolism modulating activity. In some embodiments, the activity is omega-hydroxylation of fatty acids.
By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule including RNA-based molecules that can act as inhibitory molecules including siRNA or antisense RNAs, or polypeptide, or fragments thereof. In some embodiments, an agent that decreases a level or activity of Cyp4gl polypeptide is administered to a subject. In certain embodiments, the agent that decreases a level or activity of Cyp4gl polypeptide is a siRNA. In some other embodiments, the agent that decreases a level or activity of Cyp4gl polypeptide is a small molecule compound (e.g., HET0016).
By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally -occurring polypeptide, while having certain biochemical modifications that enhance or change the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
By“biological sample” is meant any liquid, cell, or tissue obtained from a subject.
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and 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. By“Cyp4gl polypeptide” is meant a polypeptide or fragment thereof having at least 30% amino acid sequence identity to NCBI Accession No. NP 525031 (Drosophila melanogaster) or NCBI Accession No. NP 997235 (human) and having an activity of a cytochrome P450 (CYP) polypeptide. Exemplary Cyp4gl polypeptide sequences at NCBI Accession No. NP 525031 and NP 997235 are provided below:
NP 525031 (Drosophila melanogaster)
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh
121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 mhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva
NP 997235 (human)
1 maglwlglvw qklllwgaas alslagaslv lsllqrvasy arkwqqmrpi ptvarayplv
61 ghallmkpdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk
121 ssmykflepw lglglltstg nkwrsrrkml tptfhftile dfldimneqa nilvkklekh
181 inqeafncff yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw
241 ldlwylmfke gwehkkslqi lhtftnsvia eranemnane dcrgdgrgsa psknkrrafl
301 dlllsvtdde gnrlshedir eevdtfmfeg hdttaaainw slyllgsnpe vqkkvdheld
361 dvfgksdrpa tvedlkklry lecviketlr lfpsvplfar svsedcevag yrvlkgteav
421 iipyalhrdp ryfpnpeefq perffpenaq grhpyayvpf sagprncigq kfavmeekti
481 lscilrhfwi esnqkreelg legqlilrps ngiwiklkrr nader
By“Cyp4gl nucleic acid, gene or gene-containing fragment” or“Cyp4gl polynucleotide” is meant a nucleic acid molecule encoding a Cyp4gl polypeptide. Exemplary Cyp4gl polynucleotide sequences are provided at NCBI Accession No. NM 080292 (Drosophila melanogaster) and No. NM 207352 (human), which are reproduced below:
NM 080292 (Drosophila melanogaster)
1 tcacagttga gcactggcgg ctgatatagc aacagtgcca tcttcagaag acaaaaagga
61 tttgcaccag aggaccaggg atcaggagca aagaagcaac agcaaccatg gcagtggaag
121 tagttcagga gacgctgcaa caagcggcgt ccagttcgtc gacgacggtc ctgggattca
181 gtcctatgtt aaccacctta gtgggcaccc tggtggccat ggcattgtac gagtattggc 241 gcaggaatag ccgggaatac cgcatggttg ccaatatacc atccccaccg gagttgccta 301 ttttgggaca ggctcatgtg gccgccggct tgagcaatgc cgagatcctg gccgttggct 361 tgggttacct caacaagtac ggagaaacca tgaaggcctg gttgggcaac gtcctgtgg 421 tgtttctaac caatcccagt gacatcgagt tgatcctgag tgggcaccag cactgacca 481 aggcggagga gtatcgctac ttcaagccct ggttcggtga tggtctactg atcagcaatg 541 gacatcatg gcgtcatcat cgtaagatga ttgcccccac cttccaccag agcatctga 601 agagcttcgt gcctacattt gtggatcact caaaggcggt agtgccagg atgggcttag 661 aagcgggcaa atccttgat gttcatgact atatgtcgca gaccacggtt gacatcctgt 721 tgtctaccgc catgggtgtg aagaagcttc cggagggtaa caagagtttc gaatacgccc 781 aagccgtcgt cgacatgtgt gatatcatac ataagaggca ggttaaatta ctgtaccgcc 841 tggattccat ctacaagttt actaagcttc gcgagaaggg cgatcgcatg atgaacatca 901 tcttgggtat gaccagcaag gtggtcaagg atcgtaagga gaacttccaa gaggagtcac 961 gtgcgatgt tgaggagatt tctacacctg ttgccagcac tcccgcttcc aagaaggagg 1021 gtcttcgcga tgatctggat gatatcgatg aaaatgatgt gggcgccaag aggcgatgg 1081 ctcttctaga tgccatggtg gaaatggcta agaaccccga tatcgagtgg aacgagaagg 1141 acatcatgga tgaggtgaat acaattatgt tgagggcca cgataccacc tcggcgggat 1201 ctagtttcgc cctctgcatg atgggaatcc acaaggacat ccaggctaaa gtcttcgccg 1261 aacagaaggc catcttcggg gataatatgc tgagggattg caccttgcc gataccatgg 1321 agatgaaata tttggagcgc gtaatttag agacttgag gtgtaccca ccagtaccac 1381 ttatcgccag gcgtctggac tacgacctga agtggccag tggtccgtac acggttccca 1441 agggcactac ggtcatcgtg ctgcagtact gcgtgcacag acgtccagac atctacccca 1501 atcccaccaa attcgatccg gacaacttcc tacccgagag gatggccaac aggcattact 1561 actccttcat tcccttagc gctggaccca gaagctgtgt gggccgcaag tacgccatgc 1621 tgaagctaaa ggtcctgcta tccaccatcg tgaggaacta tattgtccac tccaccgaca 1681 cggaggcaga tttcaagctg caggctgaca tcatcctaaa gctgagaat ggattcaatg 1741 tctcgttgga gaagcgtcag tacgccacgg tggcctagaa tccagaaatc taggaccccg 1801 actacacaca cgcaaccccg aacccgaaac cggaatccag ccctgtatat agatgatgaa 1861 taccgatgaa tatcccaaac cgaaaacttg atgacgaact tataaatcta aaacaccgaa 1921 taagaaccca acgcacaagc cagccagaga gtcaattaat ttttctttcg ttttttaact 1981 cgttactttt atatttgatt aatacctttt tgttgtgg tcttagcga gtggtgcccc
2041 tatataatgt atacgtatat actatatatc cttttaacca actattcaac gcaactgttt
2101 gtgctcttca cctttttagt actcctactt taccactat ctatactttt ttttcgtagc
2161 catgtagtgt gatttttttt cttattcta gtattata agtcaaatgg tttaaacgaa
2221 acccaaaaaa tatgaaaaat acacgtatgc gaggcacgta gccgatagag ctgcaaaac
NM_207352 (human)
1 gggcctccag tgcaatcact acgccctggg gcccggaaac cgttttccgg tctttcgctt 61 tcggctgggg cgtggaggcc gcggtgctgc gtaggccggg ccgggcgcag gaacagcccc 121 gtggcgccct ctctggccgc cgcccgggcg ggaaacgtcg ttccggggac cgggcgaccc 181 cgcagcgggg agcgcgccag gtccgcgcgg ggaagtgggc ggtgtgcggc cggcaccgcc 241 tcgcaccacg cccccgcggg cccgcacttt cccggagtgc accccgcggc cgccagccgg 301 ggcgatggcg gggctctggc tggggctcgt gtggcagaag ctgctgctgt ggggcgcggc 361 gagtgccctt tccctggccg gcgccagtct ggtcctgagc ctgctgcaga gggtggcgag 421 ctacgcgcgg aaatggcagc agatgcggcc catccccacg gtggcccgcg cctacccact 481 ggtgggccac gcgctgctga tgaagccgga cgggcgagaa ttttttcagc agatcattga 541 gtacacagag gaataccgcc acatgccgct gctgaagctc tgggtcgggc cagtgcccat 601 ggtggccctt tataatgcag aaaatgtgga ggtaatttta actagttcaa agcaaattga 661 caaatcctct atgtacaagt tttagaacc atggctggc ctaggacttc tacaagtac 721 tggaaacaaa tggcgctcca ggagaaagat gttaacaccc actttccatt taccattct 781 ggaagatttc tagatatca tgaatgaaca agcaaatata tggtaaga aacttgaaaa 841 acacataac caagaagcat taactgctt tttttacatc actcttgtg cctagatat 901 catctgtgaa acagctatgg ggaagaatat tggtgctcaa agtaatgatg attccgagta 961 tgtccgtgca gttatagaa tgagtgagat gatatttcga agaataaaga tgccctggct 1021 ttggcttgat ctctggtacc ttatgtttaa agaaggatgg gaacacaaaa agagccttca 1081 gatcctacat acttttacca acagtgtcat cgctgaacgg gccaatgaaa tgaacgccaa 1141 tgaagactgt agaggtgatg gcaggggctc tgccccctcc aaaaataaac gcagggcctt 1201 tcttgacttg ctttaagtg tgactgatga cgaagggaac aggctaagtc atgaagatat 1261 tcgagaagaa gttgacacct tcatgttga ggggcacgat acaactgcag ctgcaataaa 1321 ctggtcctta tacctgtgg gttctaaccc agaagtccag aaaaaagtgg atcatgaatt 1381 ggatgacgtg tttgggaagt ctgaccgtcc cgctacagta gaagacctga agaaacttcg 1441 gtatctggaa tgtgttatta aggagaccct tcgccttttt ccttctgttc cttattgc 1501 ccgtagtgtt agtgaagatt gtgaagtggc aggtacaga gttctaaaag gcactgaagc 1561 cgtcatcatt ccctatgcat tgcacagaga tccgagatac ttccccaacc ccgaggagtt 1621 ccagcctgag cggttcttcc ccgagaatgc acaagggcgc catccatatg cctacgtgcc 1681 cttctctgct ggccccagga actgtatagg tcaaaagttt gctgtgatgg aagaaaagac 1741 cattctttcg tgcatcctga ggcactttg gatagaatcc aaccagaaaa gagaagagct 1801 tggtctagaa ggacagttga ttcttcgtcc aagtaatggc atctggatca agtgaagag 1861 gagaaatgca gatgaacgct aactatatta tgggtgtg ccttatcat gagaaaggtc 1921 ttatttaa gagatcctg tcattacaa tttacagatc atgagttcaa tatgctgaa 1981 tcccctagac ctaatttttc ctgatccca ctgatctga catcaagtct aacaaagaaa 2041 aagttttgag tttgtattt tcttttttct tttttctta tttttttttt tgaaaccgt
2101 gtctcactct gtcgcccagg ctggaggagt gcagtggtgt gatctcagct cactgcaacc 2161 tccacctccc aggttcaagc aattcttctg cctcagcctc ccaagtagct gggattacag 2221 gtgcctgcca ccatgcctgg ctaatttttt tgtattttta gtagaaacag ggtgtcacca 2281 tgtggccag actggtctca aactcctgac ctcaagtgat ccacctgcct cagcctccca 2341 aagtgctggg atatagtcg tgagccacca cgcctggcca gagtttttta ttttatcac 2401 caccatagat gtacagtg gctgtggtca caaaagtagt taatgtgtc agcacccaaa 2461 taaacatcta acaggtttct caacagagga atccacagtc caattccact tcaattgata 2521 gacccaaaaa atataatta atcaaagttc tagagttttt gttgttgt tgagatgga 2581 gtcttgctct gtcgcccagg ctggaacgca gtggtgacat ctcggctcac tgcaacctcc 2641 acctcccagg ttcaagtgat tctcctgcct cagcctcctg agtagctggg actacaggcg 2701 cctgccacca cgcccagcta atttttgtat tttagtaga gatggggttt caccatgtg 2761 gccaggatgg tctgatctc ttgacctcgt gatctgcctg cctcggcctc ccaaagtgct 2821 ggcattacag gcatgagcca ccatgcctgg cccaaagttc tagaattttt taaaggtatt 2881 catggtgact caggaataca cacatacaca cacacacaca cacacacaca cacacacata 2941 cacacatata atttgaaaga ggtgagtatg tactctgact tcagctctca ggttttaaaa 3001 attatattag tgggaccagt tatgacaaga ataatcata tagtactttt cagatttat 3061 aacctggagc agatatttt aagttgatta gtaggttctg tacagtttt tctttgatc 3121 gtgcacttat agtcttcatt taattcctca tagaatccca gtcacctta tatatcatat 3181 tattggaaga gattcatctt cataatctcc agttttttca cagtgcctca cagagtaat 3241 catgcctttt ggagctagaa ggactttaga actatctag tatgctcct tatattata 3301 agtaagggaa tagaatcaat aagacagttt ctgcccaaag tcatgttacc agtggtgac 3361 agagctggaa atacgtagag atctataccc ttaaatctct ccactcacat gctgatatac 3421 tttctactac aatatgctat agcttatgg aactcagggt gatgatcaga cgtgtcatta 3481 gaacatgagt cctctgcttc tgattcaggc atacttttgg gattcttcca tcttaaagg 3541 aaaaaggaag ccattcatct atatttagta acccagtaat atctcacta gttagggtt 3601 agatctttag taattcaac ctatagatc atactatga aggtgataac tgacacgtgt 3661 tcactgaatt taattgat aggcaataca tctacccact ccattatttt taaaacttc 3721 atttaatagt taaacaaga tggtttgt tttcaatttt tattcactct tcatagaatc
3781 acaattacct tatatatca tatgtatg gaagagattc ctcagtaatc tccaatctct 3841 catagtgcct cacagggttg gtcaatggct ttggaactg gaaggacctt agaactatc 3901 tgttatgctc ctgatagcca atagcagata gaagctgca atcaagaggg taggacatgt 3961 gttcttcaat ggatatcaaa ggaagaggtt gcaaaccaaa gccattggc aagccctgta 4021 gcctgggcca tttaagacag gggcggtctc agccaaatg cacccatta actatcccaa 4081 agagccacag tgcctacaac ccaggcccta agttgatgaa gaaaaagtca aggaaggagg 4141 tgatacaatt ggaaatattc ccatcaaatg gtaatcta ttagaaaat gggcatata 4201 gaaaaagtcc ttccaagatg atttggata ataaaagtg tatttgtgga aatggtatt 4261 atctctgttt tatgcacta catttatccc tacatttg ttttagtga ccctacatga 4321 cattaaattt aaagtaaaac atgttaat gtacctttt ggctgagaa tgtctttcag 4381 ctccagaatt atgtactc atatttaat cagtaagtca tttaagctat gacagagtag 4441 gaatgagaa attatttcat atgctacagt atgaaatgt ggatgctgcc tgtttata 4501 agaagatgat caaggtttgt gtgcccatta cctttcctct gcctgaaaga cgtgtctcaa 4561 gaaaaataaa ttctattta gatgcaggta ctgcattta ttctaagaat tgatatcaat 4621 tcaaaacata gaaaactgta aaagataaat caggagatgg ctgattcata atgggtaata 4681 aaataaatag cactttcgag ctgaaaaaaa aaa By“C9orf72 repeat expansion” is meant an expansion of the hexanucleotide
(GGGGCC) repeat in the C9orf72 gene, which encodes the C9orf72 polypeptide. In a healthy subject, or a subject that does not have ALS or a neurological disease, there are usually a few repeats of the hexanucleotide. However, in subject having fALS or sALS, there may be up to hundreds or thousands of repeats of the hexanucleotide. An exemplary sequence of the C9orf72 gene is provided at NCBI Accession No. NG 031977 (Homo sapiens).
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neurological diseases, including neurodegenerative diseases associated with or characterized by an energy deficit such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD), Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease.
By "effective amount" is meant the amount that is required to ameliorate the symptoms and/or progression of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, stage of disease, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
The invention provides a number of targets that are useful for the development of highly specific drugs to beat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Puri y" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally -occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally -occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By“marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By“misexpression” is meant an alteration in expression level of a polypeptide or polynucleotide in a cell relative to expression level in a control (e.g., cells from a healthy subject or cells from a subject that does not have amyotrophic lateral sclerosis (ALS) or a neurological disease). Misexpression may be an overexpression (i.e., positive alteration in expression level) or underexpression (i.e., negative alteration in expression level) of a polynucleotide or polypeptide. In some embodiments, overexpression of TDP-43 is a misexpression associated with ALS.
By“mutation” is meant a change in a polypeptide or polynucleotide sequence relative to a wild-type reference sequence. Exemplary mutations include point mutations, missense mutations, amino acid substitutions, and frameshift mutations. In some embodiments, a missense mutation in a Cyp4gl polynucleotide causes an amino acid substitution in a Cyp4gl polypeptide (e.g., a human Cyp4gl polypeptide) . In other embodiments, a mutation in Cyp4gl polypeptide or polynucleotide suppresses an effect of a mutation causing or associated with amyotrophic lateral sclerosis (ALS) (in particular, an effect of a SOD1 G85R allele). A“loss-of- fimction mutation” is a mutation that decreases or abolishes an activity or function of a polypeptide. A“gain-of-function mutation” is a mutation that enhances or increases an activity or function of a polypeptide. For example, in some embodiments, a gain-of-function mutation in a Cyp4gl polypeptide increases enzymatic activity of the Cyp4gl polypeptide (e.g., increases its ability to perform omega-hydroxy lation of fatty acids) or causes an alternative product lipid to be synthesized as opposed to the normal product.
By“mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS)” or“mutation associated with a neurological disease” is meant any mutation causing or correlated with ALS or a neurological disease in a subject. Exemplary mutations associated with ALS or a neurological disease include, without limitation, mutations in SOD1 (e.g., SOD1 G85R, SOD1 H71Y) or expansion of C9orf72 repeat.
By“neurodegenerative disease characterized by an energy deficit” is meant a disease characterized by or associated with progressive loss of function or structure of neurons, including death of neurons. In some embodiments, the neuron is a motor neuron. Examples of neurode generative disease characterized by an energy deficit include, without limitation, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
Exemplary neurodegenerative diseases amenable to treatment with a composition delineated herein include, but are not limited to, ALS, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.
As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
By“phenotype associated with amyotrophic lateral sclerosis (ALS)” or“ALS phenotype” is meant any observable or measurable characteristic exhibited by an organism having ALS. Examples of ALS phenotypes include, without limitation, weakened muscles, motor neuron death, metabolic wasting and shortened lifespan.
By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By“reference” is meant a standard or control condition.
A "reference sequence" is a defined sequence used as a basis for sequence comparison.
A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. Lor polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. Lor nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21,
22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.
By“SOD1 polypeptide” is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to GenBank Accession No. CAG46542 (Homo sapiens), NCBI Accession No. NP 001261700, or NCBI Accession No. NP 476735 (various isoforms in Drosophila melanogaster), and having an activity of superoxide dismutase (e.g., antioxidant activitiy or catalysis of dismutation of a superoxide radical). The exemplary SOD1 polypeptide sequence at GenBank Accession No. CAG46542 is provided below: 1 matkavcvlk gdgpvqgiin feqkesngpv kvwgsikglt eglhgfhvhe fgdntagcts
61 agphfnplsr khggpkdeer hvgdlgnvta dkdgvadvsi edsvislsgd hciigrtlvv
121 hekaddlgkg gneestktgn agsrlacgvi giaq
By“S0D1 polynucleotide” is meant a nucleic acid molecule encoding a SOD1 polypeptide. An exemplary SOD1 polynucleotide sequence is provided at GenBank Accession No. CR541742, which is reproduced below:
1 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat
61 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact
121 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt
181 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg
241 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt
301 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc
361 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac
421 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa
By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT,
GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
By“TDP-43 polypeptide” is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to NCBI Accession No. NP 031401.1 and DNA-binding activity. The exemplary TDP-43 polypeptide sequence at NCBI Accession No. NP 031401.1 is provided below:
1 mseyirvted endepieips eddgtvllst vtaqfpgacg lrympvsqc mrgvrlvegi
61 lhapdagwgn lvywnypkd nkrkmdetda ssavkvkrav qktsdlivlg lpwktteqdl
121 keyfstfgev lmvqvkkdlk tghskgfgfv rfteyetqvk vmsqrhmidg rwcdcklpns
181 kqsqdeplrs rkvfvgrcte dmtedelref fsqygdvmdv fipkpfrafa fvtfaddqia
241 qslcgedlii kgisvhisna epkhnsnrql ersgrfggnp ggfgnqggfg nsrgggaglg
301 nnqgsnmggg mnfgafsinp ammaaaqaal qsswgmmgml asqqnqsgps gnnqnqgnmq
361 repnqafgsg nnsysgsnsg aaigwgsasn agsgsgfngg fgssmdskss gwgm
By“TDP-43 polynucleotide” is meant a nucleic acid molecule encoding a TDP-43 polypeptide. An exemplary TDP-43 polynucleotide sequence is provided at NCBI Accession No. NM 007375.3, which is reproduced below:
1 ggtgggcggg gggaggaggc ggccctagcg ccattttgtg ggagcgaagc ggtggctggg
61 ctgcgcttgg gtccgtcgct gcttcggtgt ccctgtcggg cttcccagca gcggcctagc
121 gggaaaagta aaagatgtct gaatatattc gggtaaccga agatgagaac gatgagccca
181 ttgaaatacc atcggaagac gatgggacgg tgctgctctc cacggttaca gcccagtttc
241 caggggcgtg tgggcttcgc tacaggaatc cagtgtctca gtgtatgaga ggtgtccggc
301 tggtagaagg aattctgcat gccccagatg ctggctgggg aaatctggtg tatgttgtca 361 actatccaaa agataacaaa agaaaaatgg atgagacaga tgcttcatca gcagtgaaag 421 tgaaaagagc agtccagaaa acatccgatt taatagtgtt gggtctccca tggaaaacaa 481 ccgaacagga cctgaaagag tattttagta cctttggaga agttcttatg gtgcaggtca 541 agaaagatct taagactggt cattcaaagg ggttggctt tgttcgtttt acggaatatg 601 aaacacaagt gaaagtaatg tcacagcgac atatgataga tggacgatgg tgtgactgca 661 aacttcctaa ttctaagcaa agccaagatg agcctttgag aagcagaaaa gtgttgtgg 721 ggcgctgtac agaggacatg actgaggatg agctgcggga gttcttctct cagtacgggg 781 atgtgatgga tgtcttcatc cccaagccat tcagggcctt tgccttgtt acattgcag 841 atgatcagat tgcgcagtct ctttgtggag aggactgat cataaagga atcagcgttc 901 atatatccaa tgccgaacct aagcacaata gcaatagaca gttagaaaga agtggaagat 961 ttggtggtaa tccaggtggc tttgggaatc agggtggatt tggtaatagc agagggggtg 1021 gagctggttt gggaaacaat caaggtagta atatgggtgg tgggatgaac ttggtgcgt 1081 tcagcataa tccagccatg atggctgccg cccaggcagc actacagagc agttggggta 1141 tgatgggcat gtagccagc cagcagaacc agtcaggccc atcgggtaat aaccaaaacc 1201 aaggcaacat gcagagggag ccaaaccagg ccttcggttc tggaaataac tcttatagtg 1261 gctctaattc tggtgcagca atggtggg gatcagcatc caatgcaggg tcgggcagtg 1321 gttttaatgg aggctttggc tcaagcatgg attctaagtc ttctggctgg ggaatgtaga 1381 cagtggggtt gtggtggtt ggtatagaat ggtgggaatt caaatttttc taaactcatg 1441 gtaagtatat tgtaaaatac atatgtacta agaattttca aaatggttt gttcagtgtg 1501 gagtatattc agcagtattt tgacatttt tcttagaaa aaggaagagc taaaggaatt 1561 tataagttt tgtacatga aaggtgaaa tattgagtgg tgaaagtga actgctgttt 1621 gcctgatgg taaaccaaca cactacaatt gatatcaaaa ggtttctcct gtaatatttt 1681 atccctggac ttgtcaagtg aattcttgc atgttcaaaa cggaaaccat tgattagaac 1741 tacattcttt acccctgtt ttaatttgaa ccccaccata tggatttttt tcctaagaa 1801 aatctccttt taggagatca tggtgtcaca gtgttggtt ctttgtttt gttttttaac 1861 actgtctcc cctcatacac aaaagtacaa tatgaagcct tcatttaatc tctgcagttc 1921 atctcatttc aaatgtttat ggaagaagca cttcatgaa agtagtgctg taaatattct 1981 gccataggaa tactgtctac atgctttctc attcaagaat tcgtcatcac gcatcacagg 2041 ccgcgtcttt gacggtgggt gtcccatttt tatccgctac tctttatttc atggagtcgt 2101 atcaacgcta tgaacgcaag gctgtgatat ggaaccagaa ggctgtctga actttgaaa 2161 ccttgtgtgg gatgatggt ggtgccgagg catgaaaggc tagtatgagc gagaaaagga 2221 gagagcgcgt gcagagactt ggtggtgcat aatggatatt tttaactg gcgagatgtg 2281 tctctcaatc ctgtggcttt ggtgagagag tgtgcagaga gcaatgatag caaataatgt 2341 acgaatgttt ttgcattca aaggacatcc acatctgtg gaagactttt aagtgagttt 2401 tgttctag ataacccaca ttagatgaat gtgtaagtg aaatgatact tgtactcccc 2461 ctaccccttt gtcaactgct gtgaatgctg tatggtgtgt gttctcttct gttactgata 2521 tgtaagtgtg gcaatgtgaa ctgaagctga tgggctgaga acatggactg agcttgtggt 2581 gtgcttgca ggaggacttg aagcagagtt caccagtgag ctcaggtgtc tcaaagaagg 2641 gtggaagttc taatgtctgt tagctaccca taagaatgct gtttgctgca gttctgtgtc 2701 ctgtgctgg atgctttta taagagttgt catgtgga aattctaaa taaaactgat 2761 ttaaataata tgtgtcttg tttgcagcc ctgaatgcaa agaattcata gcagttaatt 2821 cccctttttt gacccttg agatggaact ttcataaagt ttctggcag tagttattt 2881 tgcttcaaat aaacttattt gaaaagtgt ctcaagtcaa atggattcat cacctgtcat 2941 gcattgacac ctgataccca gactaatg gtattgttc ttgcattggc caaagtgaaa 3001 attttttttt ttcttttgaa atctagtttt gaataagtct gggtgaccgc acctaaaatg
3061 gtaagcagta ccctccggct ttttcttagt gcctctgtgc atttgggtga tgttctattt
3121 acatggcctg tgtaaatctc catgggaag tcatgccttc taaaaagatt ctatttggg
3181 ggagtgggca aaatgtgat tattttctaa tgcttgtag caaagcatat caatgaaaa
3241 gggaatatca gcaccttcct agttgggat tgaaaagtg gaataatg cagtagggat
3301 aaagtagaag aaaccacaaa tatctgtg cctgaaatcc attaagaggc ctgatagctt
3361 taagaatag ggtgggttgt ctgtctggaa gtgtaagtg gaatgggctt tgtcctccag
3421 gaggtggggg aatgtggtaa catgaatac agtgaataa aatcgcttac aaaactcaca
3481 ctctcacaat gcatgtaa gtatgtaaaa gcaataacat tgattctctg tgtactttt
3541 ttgtaactaa ttctgtgaga gtgagctca ttttctagtt ggaagaatgt gatatttgtt
3601 gtgtggtag ttacctaat gccctacct aattagatta tgataaatag gttgtcatt
3661 tgcaagta cataaacatt tatcaatgaa gtcatccttt agactgtaa tcgccacatt
3721 gtttcattat tcagtttcct ctgtaaaggg atctgagtt gttttaattt tttttttctg
3781 catctgaatc tgcatgattt ccaaaccctg taccatctga attttgcatt tagcactg
3841 cactattact cagcagcagt aacatggtaa cactaaaat ggtactcggg gacctccaaa
3901 gactaaactg acaagccttc aaggagccca ggggtaagtt aactgtcaa cggcatggtt
3961 taatcccttc tttacacttg tgtaaatttc agtactggt catagaaggc tttcaatgtt
4021 gagtggcctt tataacat gttatggta ctgcatagat acgggtattt atttaccct
4081 aagaagattt tgaagttaa aagtacttaa actattggc aaagattgt tttaaaaat
4141 ctatttggtc aatctaaatg cattcattct aaaaaatttt tgaaccaga taaataaaat
4201 ttttttttga caccacaaaa aaaaaaaaaa aaaaaa
By "subject" is meant a multicellular organism, including, but not limited to, a vertebrate (e.g. a human or a non-human mammal such as bovine, equine, canine, ovine, feline) or invertebrate organism (e.g., a fly or worm). In some embodiments, the subject is a vertebrate or invertebrate. In other embodiments, the subject is human. In particular embodiments, the subject is Drosophila melanogaster.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of an unbiased forward genetic screen described herein. Forward genetics is the process of identifying a phenotype with a genotype. A fly (Drosophila melanogaster) model of amyotrophic lateral sclerosis (ALS) was generated by introduction of SOD1 ALS-causing mutations into the fly genome. Such human rapid- progressing mutations are lethal in the fly, in particular, the G85R SOD1 allele. Flies die late in development or early in adult life with a profound nerve degeneration. A forward genetic screen using random chemical mutagenesis was performed. The goal was to identify second-site mutations in genes that would restore viability of G85R when mutated.
FIGS. 2A-2C are schematic representations of a forward genetic screen designed to identify suppressors for SODf lethality. Shown in FIG. 2A is a screen designed such that all progeny of the mutagenesis cross have a genotype that is ultimately lethal, unless the progeny carries a mutation in another gene that suppresses the lethal effect of the SOD1 G85R allele. In FIG. 2B, males heterozygous for G85R (line A) are starved, fed 25mM EMS, and mated to another line of heterozygous G85R (line B) unmutagenized females. The mated females are allowed to lay eggs, then the progeny larvae are exposed to 37°C heat shock to kill any offspring carrying the TM3,hs-hid balancer to yield homozygous G85R flies. FIG. 2C shows representative progeny as an outcome of this cross. G85R homozygotes will only survive this treatment if they carry a potential dominant suppressor mutation (asterisk). The balancer homozygous flies will die due to the recessive lethal gene on the TM3,hs-hid balancer chromosome, and G85R homozygous flies will die due to the lethality of the G85R allele. The survivors are mated back to the original balanced stock of generation 1 (Line B), to show that the suppressor behaves in a Mendelian fashion and to generate a stock. FIG. 3A-3E are schematics and diagrams depicting results of the screen performed and mapping of the mutations as described herein. FIG. 3A is diagram showing a summary of the results of the screen performed. From the screen, five (5) true-breeding suppressed stocks of G85R/G85R were obtained. All five rescuing mutations mapped to the same gene (Cyp4gl). FIGS. 3B-3C show genetic mapping of the lethality of EMS 81 and EMS 102. Genetic crosses were performed to combine the suppressor mutants with a standard mapping chromosome in females. In FIG. 3B, since it was clear that the lethal mutants both map to between the p and q loci (where p and q are the recessive alleles, and + indicated wild type), recombinants on the p-q interval were obtained by recombination on either interval I or interval II. The lethal suppressor mutations are indicated by let for mutant, or + for wild type alleles. In FIG. 3C, only three classes of viable males (non-let containing) chromosomes could be obtained from this configuration in recombinogenic females, and all are distinguishable by visible markers. The relative distance on the p-q interval was obtained by [RI/(RI+RII)] x lcM. FIGS. 3D-3E show molecular mapping of the lethality of EMS 81 and EMS 102. In FIG. 3D, EMS 81- PR males were crossed to females of five balanced deficiency stocks for the region determined by genetic mapping (BSCdfl-5). Deficiencies that complement EMS 81 are shown in green. Only BSCdf2 (red) failed to complement EMS 81 lethality, mapping it to a ~260kb region. In FIG. 3E, both EMS 81 and EMS 102 balanced females were crossed to males of seven stocks carrying the w+-marked duplications on the third chromsome. Duplications that failed to rescue male lethality are shown in pink. Duplications 2-4 rescued male lethality of both EMS 81 and EMS 102, and limit the molecular interval where they are located. The refined interval contained two genes, A and B (shown at the bottom of FIG. 3E). Gene B had a previously molecularly uncharacterized lethal mutation, l(l)x, which was sequence verified as a 13bp deletion in coding sequence, and EMS 81 failed to complement l(l)x, thus confirming the identity of the gene. Sequence analysis also revealed mutations in both alleles EMS 81 and EMS 102 of the Su(G85R) gene. The Su(G85R) gene is Cyp4gl.
FIG. 4 is a sequence diagram showing the mutations in the protein sequence of Cyp4gl. The mutated amino acids are in bold. The amino acid substitutions are listed at the bottom of FIG. 4.
FIG. 5 is a sequence diagram showing the mutations in the nucleotide sequence of Cyp4gl. The nucleotide sequence encoding the protein sequence of Cyp4gl is in bold. The mutated codons are underlined. The nucleotide mutations are indicated at the bottom of FIG. 5.
FIG. 6 is a diagram depicting the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene. The dCyp4gl (CG3972) gene in Drosophila is an intronless gene. FIG. 7 is an alignment of dCyp4gl and hCYP4V2 (closest human homologue). The box labeled“EMS81” indicates a position of mutation in the mutant line EMS81 obtained in a screen performed herein. The mutation suppressed SOD1 is G85R lethality. The amino acid in this position is conserved between dCyp4gl and hCYP4V2.
FIG. 8 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates. The boxes labeled“EMS35/130,”“EMS102,” and“EMS81” indicate positions of suppressor mutations in mutant lines EMS35/130, EMS102, and EMS81 obtained in the screen performed herein, which suppressed SOD1 G85R lethality. Alignment was performed using T-COFFEE, Version_11.00.8cbe486.
The Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102. Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow. (1) lBb[19] is almost certainly a null allele of Cyp4gl. The (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote. Thus, without being bound by theory, it is believed that there are loss-of-function and gain-of- function components to the EMS35/130, EMS102, and EMS81 suppressor alleles, which make them novel.
FIG. 9 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. The alignment was performed using T-COFFEE,
Version_11.00.8cbe486. “Dmel” is Drosophila. All other sequences are vertebrate sequences. Suppressor alleles occurred in regions of high conservation, but only EMS81 serine(S) was conserved from flies through mammals
FIG. 10 is an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket. Modeling of the active site of the CYP4A11 protein (related to CYP4V2) showed residues lining the substrate binding pocket and active site involved in omega-hydroxy lation. The EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.
FIG. 11 is a schematic showing a structure of the compound HET0016. HET0016 (N- hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a potent inhibitor of CYP4V2.
FIG. 12 is a table showing the Cyp4 class of enzymes and their interfamily homology.
A Blast search with Cyp4V22[NP_997235(human)] revealed that homology within family Cyp4 is >30% for all homologues. Thus, the Cyp4 class (paralogues) in humans and flies (across families) is conserved at a level of about >30%. FIG. 12 also shows a sharp drop in E-value in non-Cyp4 family members. These non-Cyp4 family members are other Cyp classes of enzymes (e.g., Cyp3), which are quite different from Cyp4 in terms of substrates. Thus, a Cyp4gl polypeptide herein may be any polypeptide having at least about 30% sequence identity to a human Cyp4gl (Cyp4V22) or Drosophila Cyp4gl and having an activity of a Cyp4 enzyme (e.g., omega hydroxylation of a fatty acid).
DETAILED DESCRIPTION OF THE INVENTION
The invention features compositions and methods that are useful for suppressing a neurological disease, in particular, symptoms of mutations causing amyotrophic lateral sclerosis (ALS) as well as sporadic cases and related dementias. The invention is based, at least in part, on the discovery of genetic sequences in a Drosophila melanogaster model of ALS that suppressed lethal effects of SOD1-G85R (an ALS-causing mutation).
The Drosophila melanogaster model is a powerful model for human neurodegenerative diseases such as ALS. Over the last decade, genome-wide genetic screens in yeast have led to the identification of important genetic modifiers of TDP-43 and FUS-mediated ALS toxicity (Elden et al, 2010; Ju et al, 2011; Sun et al, 2011; Couthouis et al., 2011). In addition to yeast screens, for decades, genetic screens carried out in Drosophila have proven extremely powerful in identifying new genes and/or modifiers (enhancers and suppressors) of genes, especially when it comes to the nervous system (Arrizabalaga & Lehmann, 1999; St Johnston, 2002; Somalinga et al, 2012; Deivasigamani et al, 2014). The sequencing of the Drosophila genome using next generation sequencing technologies has revealed the extent of similarity between the Drosophila and human nervous system regarding gene context (Adams et al., 2000). Drosophila has a very compact genome with -15,000 genes (Adams et al., 2000) including a vast number of genes (197 of 287) in which mutations are known to cause human disease (St Johnston, 2002). As such, Drosophila is an ideal model system for the operation of forward genetic screens to divulge specific genetic mutations that can act as suppressors and modify disease phenotypes.
The most common directed mutagenesis method used in Drosophila, EMS, provides a rapid methodology for the introduction of largely unbiased mutations into the entire genome. Though EMS is capable of causing different types of mutations, it is mainly an alkylating agent that induces missense mutations through G-to-A transitions (Bokel, 2008). Introduction of missense mutations, in contrast to commonly used
transposon insertion screens for example, allows for a much greater spectrum of
mutations including novel dominant gain of function mutations, contrary to the next generation screening kits in Drosophila that mostly introduce loss of function mutations (Wolf & Rockman, 2011).
The SOD1 gene is highly conserved between Drosophila and humans. Previously, the G85R point mutation was introduced into the endogenous locus of cl So cl I via homologous recombination. Among other ALS-like phenotypes such as locomotion G85R/G85R defects and muscle denervation, the dSodl homozygotes display a distinct phenotype: flies die in the pupal case during the eclosion period. After using the power of Drosophila genetics to model ALS, the unbiased power of Drosophila forward genetic screens was used to identify potential gene products that, when appropriately modified, eliminate the deleterious effect of the dSodlG85R allele, namely unconditional late (near adult eclosion) lethality. Without wishing to be bound by theory, it is believed that genetic suppressors that reverse G85R/G85R the homozygous lethal phenotype of dSodl in Drosophila may be genes whose human counterparts are involved in the etiology of ALS and are potential targets for drugs.
CYP4 proteins and fatty acid metabolism in neurodegenerative disease
A forward genetic screen described herein revealed four (4) novel missense mutations causing amino acid substitutions in Drosophila melanogaster Cyp4gl that suppressed SOD1 G85R mutant lethality. The suppressor mutations found in Cyp4gl were G92E, D112N, S143N, and M333I. Mapping and molecular studies have revealed the nature of the Cyp4gl gene in flies, a member of the ubiquitous cytochrome P450 (CYP) class of proteins found in all life forms, including bacteria. CYP proteins play two major roles in biology; detoxifying
(biodefense) against many xenobiotics and antibiotics, and biosynthesis of endogenous molecules such as steroids and lipids. There are as many as 37 families of CYP genes. The CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals.
The novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids.
Accordingly, in some aspects, the invention provides Cyp4gl polypeptides, or fragments thereof having an activity of a cytochrome P450 (CYP) polypeptide, comprising a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333. In some embodiments, the mutation is G92E, D112N, S143N, or M333I. In some aspects, the invention provides polynucleotides or expression vectors encoding Cyp4gl polypeptides comprising the suppressor mutations described herein. In other aspects, cells or organisms (in particular, Drosophila melanogaster mutant lines) harboring suppressor Cyp4gl mutations are provided.
In some aspects, the suppressor Cyp4gl mutation is a gain-of-function or a loss-of- function mutation. The Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102 (i.e., Drosophila lines harboring Cyp4gl S143N and D112N mutations). Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow. (1) lBb[19] is almost certainly a null allele of Cyp4gl. The (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote. Thus, without being bound by theory, it is believed that there are loss-of-function and gain-of- function components to the EMS35/130, EMS102, and EMS81 suppressor alleles, which make them novel.
The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells. Interestingly, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver (Gutierrez et al., Nature (2007) 445:275-280). Fatty acids have been implicated in ALS, as a system-wide metabolic defect called wasting (or hypermetabolism) is seen in patients. In addition, a very early event in disease progression has been demonstrated in mice, namely, a switch of muscles to utilizing lipids versus glucose. In addition, motor neurons (MNs), in particular those involved in voluntary motion, are the most energy consumptive cells in an organism. Modeling studies and other experiments have strongly suggested that a key determinant of MN health may likely be the ability of the cell to keep up with energy demands. Thus, there is ample precedent in the ALS, as well as dementia, Huntington’s disease,
Parkinson’s disease, and Alzheimer’s disease fields, that energy metabolism, in particular lipid metabolism may be a key pathological target. Without intending to be bound by theory, it is hypothesized, based on phenotypes and RNAseq data indicating systemic metabolic dysregulation (especially lipid metabolism), that SOD-based ALS model animals die because MNs are not sufficiently“powered” by the animal’s metabolism. The suppressor mutations in the Cyp4gl gene found in the forward genetic screen described herein point to key and conserved changes in protein sequence in this CYP4 family member leading to nearly complete suppression of effects of these SOD1 ALS-causing mutations. In all, 4 novel missense mutations causing amino acid substitutions in Cyp4gl were found. It is worth emphasizing that all genes of the genome were mutagenized without bias and at random, and four independent changes in Cyp4gl that all suppress ALS-mutant lethality were obtained. It is proposed that these Cyp4gl suppressor mutations alter the ability of flies harboring these mutations to mobilize or utilize fat in such a way that the“wasting” of the fly is suppressed or greatly alleviated. This benefits MNs in the fly most, as like most animals, because MNs demand the most energy. Thus, in some aspects, the invention features methods of modulating fatty acid or lipid metabolism in a subject, comprising administering to the subject a Cyp4gl polypeptide comprising the suppressor mutations described herein (e.g., mutations in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333).
In some other aspects, the invention provides methods of identifying a modulator of neurode generative diseases (particularly ALS, Parkinson’s, dementia, and Huntington’s), comprising screening of candidate agents that modulate the level or activity of a Cyp4gl polypeptide. Without being bound by theory, given current understanding of results obtained in the studies described herein, which are quite novel, it is foreseen that ALS and other age-related cognitive, neuromuscular, or neurodegenerative disorders may result from the aberrant “wasting” of resources, especially stored fats. This hypermetabolic effect may likely be a result of a chronic induction of innate immunity, through currently unknown toxic insults, and the bodies’ response to mounting a response to a pathogen that is“unreal.” It is motor neurons in particular that rely most on systemic energetics for their function. The CYP4 class of enzymes play a unique biochemical role in regulation of body fat mobilization and utilization.
It was not expected that the Cyp4gl protein would be involved in the metabolic pathway, considering that motor neuron specific genes were the first candidates expected from an ALS-related suppressor screen. Previous human studies have shown that ALS patients show an early and persistent hypermetabolic signature associated with energy wasting (Desport et al., 2001; Bouteloup et al., 2009). Moreover, a strong transcriptomic signature was observed that indicated that metabolism was altered in the ALS flies. Furthermore, a hSODlG93A transgenic mouse model of ALS shows defective energy homeostasis that benefits from a high energy diet (Dupuis et al., 2004) or lowered amounts of leptin, a regulator of whole-animal energy expenditure (Lim et al., 2014). In addition, the hSODlG85R transgenic mouse model has also been shown to have several metabolic changes consistent with a metabolic switch occurring as an early pathological event (Palamiuc et al., 2015). Thus, without being bound by theory, Cyp4gl a suppressor gene of dSodlG85R lethality and a gene involved in metabolism, is believed to a role in energy balance in cells at a whole-animal level.
Thus, the Cyp4gl mutants obtained herein define the CYP4 class of enzymes (FIG. 12) as a potential target in regulating energy metabolism in disease, particularly neurodegenerative diseases associated with or characterized by an energy deficit (e.g., ALS, Parksinon’s, dementia, and Huntington’s). Without being bound by theory, it is believed common cellular mechanisms underlie the motor neuron death that links all of ALS and other neurodegenerative diseases characterized by an energy deficit, such as Parksinon’s, dementia, or Huntington’s disease. Agents that modulate the level or activity of a Cyp4gl polypeptide, therefore, are expected to modulate energy balance at a whole-animal level and thereby“correct” the energy deficit in a subject having a neurodegenerative disease such as ALS, Parksinon’s, dementia, or
Huntington’s disease.
In addition to agents that modulate the level or activity of a Cyp4gl polypeptide, a Cyp4gl polypeptide or polynucleotide comprising the suppressor mutations herein is also expected to ameliorate the energy deficit in a subject having a neurodegenerative disease.
Accordingly, in some aspects, the invention additionally provides methods of suppressing or beating a neurodegenerative disease in a subject (particularly a neurodegenerabve disease characterized by an energy deficit, such as ALS, Parkinson’s, dementia, or Huntington’s disease), comprising administering to a subject a Cyp4gl polynucleotide or polypeptide comprising suppressor mutations described herein.
In other aspects, the invention further provides Cyp4gl polypeptides in other organisms (e.g., human Cyp4gl polypeptide), comprising the suppressor mutations. In some embodiments, the Cyp4gl polypeptide is from an invertebrate or vertebrate. In some other embodiments, the polypeptide has at least 30%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide. In particular
embodiments, the Cyp4gl polypeptide is human Cyp4gl polypeptide. Interestingly, a human homologue of Cyp4gl is the CYP4V2 gene, mutations in which cause a retinal degenerative disease called Bietti’s Crystalline Dystrophy (BCD). Patients with BCD develop a late onset degeneration of the retina with deposits, but more interestingly, BCD patients have a systemic increase in fatty acids/lipids. Many mutations that cause BCD map to very near the suppressor mutations found in Cyp4gl. A mouse model of BCD also recapitulates key feature of the disease, including altered lipid profiles. Thus, without intending to be bound by theory, it is hypothesized that mutations in CYP4V2 or inhibitors could possibly suppress mouse ALS models, as in the fly system.
Methods of treatment
The present invention provides methods of treating a neurological disease (in particular, ALS) and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation described herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a neurological disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a Cyp4gl polynucleotide or polypeptide sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g.
measurable by a test or diagnostic method).
As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the Cyp4gl polynucleotides or polypeptides herein (such as a Cyp4gl polynucleotide or polypeptide (or fragment thereof) comprising a mutation in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333) to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably
administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a neurological disease (particularly ALS), disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, ALS causing or ALS associated mutations or misexpression (e.g., a SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which ALS causing or ALS associated mutations (e.g. mutations in SOD1) may be implicated. For instance, many sALS patients have been shown to possess expanded C90rf72 repeats even though there is no history in the family, thus, repeats may expand de novo. Likewise, even though only a small proportion of fALS patients have mutations in the TDP-43 gene, almost all sALS patients display pathological aggregation of TDP-43 protein upon analyses of post mortem tissue.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of a neurological disease marker (e.g.,
SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a neurological disease, in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of a neurological disease marker determined in the method can be compared to known levels of the neurological disease marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status. In particular embodiments, a second level of a neurological disease marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre-treatment level of a neurological disease marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of the neurological disease marker can then be compared to the level of the marker in the subject after the treatment commences, to determine the efficacy of the treatment.
In some embodiments, a subject identified as having increased level of a ALS or neurological disease marker polynucleotide or polypeptide (e.g. C9orf72 repeat or TDP-43, or a mutation in SOD1 (particularly, SOD1 G85R)) relative to a reference is administered a therapeutic composition of the invention. Levels of neurological disease marker
polynucleotides or polypeptides are measured in a subject sample and used as an indicator of an ALS or neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide of the invention. Levels of neurological disease marker polynucleotides may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of neurological disease marker polypeptides in a biological sample derived from a subject. Such methods include immunoassay, ELISA, western blotting using an antibody that binds the marker polypeptide, and radioimmunoassay. Methods for detecting a mutation in a marker polypeptide (e.g., SOD1 mutation) include immunoassay, direct sequencing, and probe hybridization to a polynucleotide encoding the mutant polypeptide. Elevated levels of neurological disease marker polynucleotides or polypeptides and/or a mutation in a neurological disease polynucleotide or polypeptide are considered a positive indicator of ALS or a neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide of the invention.
Pharmaceutical compositions
The present invention features compositions useful for treating a neurological disease or suppressing effects of mutations causing a neurological disease (particularly ALS) in a subject. In some embodiments, the composition comprises a Cyp4gl polypeptide comprising a suppressor mutation described herein. In some other embodiments, the composition comprises a polynucleotide encoding an amino acid sequence of the suppressor polypeptide. In particular embodiments, the composition further comprises a vehicle for intracellular delivery of the polypeptide or polynucleotide (e.g., a liposome).
The administration of a composition comprising a Cyp4gl polynucleotide or polypeptide herein for the treatment of a neurological disease such as ALS may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically -acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the agent in the patient.
The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neurological disease. Generally, amounts will be in the range of those used for other agents used in the treatment of neurological diseases such as ALS (or other diseases associated with ALS causing mutations, such as SOD1 G85R), although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that suppresses effects of the neurological disease causing mutation or that decreases effects or symptoms of ALS or neurological disease (e.g., wasting, weakened muscular strength, or shortened lifespan) as determined by a method known to one skilled in the art.
The therapeutic suppressor Cyp4gl polynucleotide or polypeptide may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously,
intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the liver; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a cancer using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., liver cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.
Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in singledose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neurological disease, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., a Cyp4gl polypeptide or polynucleotide described herein) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
Furthermore, the composition may include suspending, solubilizing, stabilizing, pH -adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
In some embodiments, the composition comprising the active therapeutic (i.e., a Cyp4gl polypeptide or polynucleotide herein) is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxy benzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Recombinant polypeptide expression
Cyp4gl polypeptides of the invention, comprising mutations suppressing effects of ALS-causing mutations (e.g., SOD1 G85R), are useful for treating or suppressing a neurological disease such as ALS in a subject. Recombinant Cyp4gl polypeptides of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle. Accordingly, the invention provides methods of producing a polypeptide of the invention, the method comprising (a) heterologously expressing an expression vector comprising a polynucleotide encoding the polypeptide in a host cell; and (b) isolating the polypeptide from the host cell.
Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A Cyp4gl polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985,
Supp. 1987).
A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof. In some embodiments, the polypeptides of the invention are produced in a bacterial expression system. One particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high- level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX- 2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.
Alternatively, recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOX1 gene is induced by methanol. The AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.
Once the recombinant Cyp4gl polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. In some embodiments, to facilitate purification of the recombinant polypeptide, the polypeptide comprises an epitope tag fused to the Cyp4gl polypeptide. The polypeptide is then isolated using an antibody against the epitope tag. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).
Methods of Delivery
Suppressor Cyp4gl polypeptides or polynucleotides of the invention, which are useful for suppressing a neurological disease (particularly ALS) in an organism, may be delivered to an organism (particularly liver cells of the organism) in any manner such that the polypeptide is in functional form in the cell. The Cyp4gl polypeptide comprising suppressor mutations may be delivered to cells as a polypeptide. Alternatively, a polynucleotide encoding an amino acid sequence of the Cyp4gl polypeptide may be delivered to cells for heterologous expression of a suppressor Cyp4gl polypeptide in the cells. Thus, the present invention features polypeptides delivered to a cell by contacting the cell with a composition comprising the polypeptide or by heterologously expressing the polypeptide in the cell.
Intracellular Delivery of Polypeptides
Polypeptides of the invention, such as Cyp4gl polypeptides comprising mutations suppressing ALS, may be delivered intracellularly to cells. The polypeptide must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the polypeptide, or fragment thereof, is in functional form in the cells.
Methods of intracellular delivery of polypeptides are known to one of skill in the art. Exemplary methods of intracellular delivery of polypeptides include, without limitation, incorporation of the polypeptide into a liposome. Liposomes are phospholipid vesicles with sizes varying from 50 to 1000 nm, which can be loaded with polypeptides or other agents. Liposomal intracellular delivery of polypeptides into cells typically relies on endocytosis of the liposome-encapsulated polypeptide into the cell. Examples of suitable liposomes for intracellular delivery of polypeptides may be pH-sensitive liposomes. Such liposomes are made of pH-sensitive components; after being endocytosed in intact form, the liposome fuses with the endovacuolar membrane under lowered pH inside the endosome and destabilizes it, thereby releasing the contents (including the polypeptides encapsulated in the liposome) into the cytoplasm. The liposomes may also be further modified to enhance their stability or lifetime during circulation (e.g., by PEGylated liposomes). Liposomes may also be modified to specifically target antigens (e.g.,“immunoliposomes” or liposomes embedded with antibodies to an antigen). Antibody -bearing liposomes may have the advantages of targetability and facilitated uptake via receptor-mediated endocytosis.
Other methods of intracellular delivery of polypeptides include, without limitation, use of cell penetrating peptides (CPPs). A cell penetrating peptide or“CPP” is a protein or peptide that can translocate through cellular membranes. A polypeptide for delivery into a cell is fused with a CPP, thereby enabling or enhancing delivery of the polypeptide fusion into the cell. Cell penetrating peptides include, for example, a trans-activating transcriptional activator (TAT) from HIV-1, Antenapedie (Antp, a transcription factor in Drosophila), and VP22 (a herpes virus protein).
Another exemplary method for intracellular delivery of polypeptides of the invention is the use of supercharged proteins. Supercharged proteins or supercharged polypeptides are a class of engineered or naturally existing polypeptides having an unusually high positive or negative net theoretical charge. Membranes of cells are typically negatively charged.
Superpositively charged polypeptides are able to penetrate cells (particularly mammalian cells), and associating cargo with superpositively charged polypeptides (e.g., polypeptides or polynucleotides) can enable functional delivery of these macromolecules into cells, in vitro or in vivo. Methods of generating supercharged polypeptides and using supercharged polypeptides for intracellular polypeptide delivery are described in further detail in, for example, Zuris et al. Nat. Biotechnol. (2015) 33:73-80 and Liu et al. Methods Enzymol. 2012, 503: 293-319.
Accordingly, in some aspects, the invention provides a suppressor Cyp4gl polypeptide fused to a polypeptide enabling intracellular delivery of the suppressor Cyp4gl polypeptide (e.g., a cell penetrating peptide or supercharged polypeptide).
Another therapeutic approach for treating or suppressing a neurological disease is polynucleotide therapy using a polynucleotide encoding a suppressor Cyp4gl polypeptide of the invention, or fragment thereof. Thus, provided herein are isolated polynucleotides encoding a Cyp4gl polypeptide of the invention, or fragment thereof. Expression of such polynucleotides or nucleic acid molecules in a cell or organism is expected to suppress effects of mutations causing neurological disease (particularly, ALS) in the subject. Such nucleic acid molecules can be delivered to cells of a subject having a neurological disease. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the Cyp4gl polypeptide, or fragment thereof, can be produced.
Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a polynucleotide encoding a Cyp4gl polypeptide of the invention, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet
337: 1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311- 322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In some embodiments, a viral vector is used to administer a polynucleotide encoding a suppressor Cyp4gl polypeptide (or fragment thereof) systemically.
Non-viral approaches can also be employed for the introduction of the therapeutic to a cell of a patient requiring suppression of a neurological disease. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.
Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of genes encoding suppressor Cyp4gl polypeptides into the affected tissues of a patient can also be accomplished by transferring a nucleic acid encoding the suppressor Cyp4gl polypeptide into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.
For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.
Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
Delivery of polynucleotides of the invention may also include or be performed in combination with gene or genome editing methods, such as CRISPR-Cas systems, to introduce polynucleotides encoding suppressor Cyp4gl polypeptides in cells. Gene or genome editing methods such as CRISPR-Cas systems are further described in for example, Sander et al. (2014), Nature Biotechnology 32, 347-355; Hsu et al. (2014), Cell 157(6): 1262-1278.
In Drosophila melanogaster, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver. Gutierrez et al., Nature (2007) 445:275-280. Accordingly, in particular embodiments, suppressor Cyp4gl polynucleotides or polypeptides of the invention are delivered to a liver cell. It is noted that many systems of delivery of therapeutic polypeptides or polynucleotides (particularly nanoparticulate or liposomal delivery systems) result in high accumulation of the therapeutic polypeptides or polynucleotides in the liver. It is also noted that, contrary to other polynucleotide therapies for ALS or neurological disease, the therapeutic Cyp4gl polynucleotides or polypeptides of the invention likely do not need to be delivered to neurons. Thus, currently available systems for delivery of
polynucleotides or polypeptides, such as liposomal delivery, may be particularly suitable for delivery of therapeutic Cyp4gl polypeptides or polynucleotides of the invention.
Screening Assays
The present invention further features methods of identifying modulators of a disease, particularly neurological disease, comprising identifying candidate agents that interact with and/or alter the level or activity of a Cyp4gl polypeptide. The CYP4 class of enzymes plays a unique biochemical role in regulation of body fat mobilization and utilization. The mutants obtained herein define the CYP4 class of enzymes as a potential target in regulating energy metabolism in disease. The CYP4 class of enzymes may, because of their novel active-site chemistry, serve as ideal drug targets to control the pace of lipid metabolism, to achieve some degree of efficacy in treating the whole organism, not just neurons (the current focus of most therapies).
Thus, in some aspects, the invention provides a method of identifying a modulator of a neurological disease, comprising (a) contacting a polypeptide with a candidate agent, where the polypeptide is a Cyp4gl polypeptide or fragment thereof, and (b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity. In other aspects, the method comprises (a) contacting a cell or organism with a candidate agent, and (b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity. An alteration in the level or activity of the Cyp4gl polypeptide or polynucleotide indicates the candidate agent is a modulator of neurological disease.
In some embodiments, the activity of the Cyp4gl polypeptide is enzymatic activity or a lipid, combinations of lipids, and/or alteration of fatty acid metabolism. The control activity may be the activity of the polypeptide when the polypeptide is not contacted with the candidate agent, or any agent. Alternatively, the control activity may be activity of the polypeptide contacted with a carrier or solvent that does not contain the candidate agent. Likewise, the control activity or control level of the polypeptide may be the activity or level of the polypeptide in a cell when the cell is not contacted with the candidate agent (or any agent) or when the cell is contacted with a carrier that does not contain the candidate agent. Methods of measuring or detecting activity and/or levels of the polypeptide or polynucleotide are known to one skilled in the art. For example, enzymatic activity of the polypeptide may be measured by measuring levels of substrate(s) modified by the polypeptide. Binding activity of the polypeptide may be measured, for example, by immunoassay methods. Polynucleotide levels may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure polypeptide levels, the methods including without limitation, immunoassay, ELISA, western blotting using an antibody that binds the polypeptide, and radioimmunoassay.
In still other aspects, the invention provides a method of identifying a modulator of neurological disease, comprising (a) contacting a cell or organism with a candidate agent, and (b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism comprising a Cyp4gl polynucleotide or polypeptide having a mutation. A similarity in the phenotypes indicates the candidate agent is a modulator of neurological disease. In some embodiments, the cell or organism also comprises a mutation and/or misexpression associated with ALS (e.g., a SOD1 G85R allele). In the method provided, candidate agents that“phenocopy” a phenotype of an organism having a Cyp4gl mutation (in particular, agents that phenocopy suppression of ALS mutations by a suppressor Cyp4gl mutation in an organism) are identified as modulators of ALS or neurological disease. The phenotype may be any observable or measurable phenotype, including without limitation, suppression of ALS associated phenotypes (e.g., weakness of muscles, motor neuron death, shortened lifespan), level and/or activity of Cyp4gl polypeptides or polynucleotides, or levels of lipid and/or fatty acid substrates and/or metabolic products. In particular embodiments, the candidate agent or modulator of neurological activity inhibits an activity of Cyp4gl. In certain embodiments, the agent or modulator suppresses an ALS phenotype. For example, in some embodiments, the agent or modulator is HET0016. HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a potent inhibitor of CYP4V2 (human homolog of Cyp4gl). In particular, HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122. It is expected that HET0016 will phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media.
If the current strategy to treat dying motor neurons (MNs) specifically are misguided, due to the true nature of these diseases in defective metabolism, there are even more tantalizing potential avenues to pursue based on the current invention. Without wishing to be bound by theory, it is believed the Cyp4gl mutations could identify a class of lipids which, when increased, or decreased, would themselves provide an ideal therapeutic index. Some research has been directed towards dietary interventions in ALS, but specific metabolites have not been forthcoming. Perhaps, these studies in Drosophila, extended further, could reveal a specifically balanced dietary intervention (particular mixtures of complex natural lipids and fats) that could prolong the pathologic wasting seen in human neurological disease, especially ALS and dementia. It has been documented that hypercaloric dietary intervention has efficacy, even when begun after symptom onset. Likewise, it has been suggested that intervening during the presymptomatic phase may be more effective. However, these studies are not specific as to the source of calories. Thus, it could be that the current invention could identify tailored mixtures of specific metabolic intermediates (lipid), or a pharmacological method to alter lipid profiles that would optimize the energy balance of motor neurons, providing protection against a“deadly loop” of energy depletion of motor neurons.
Accordingly, the invention also provides methods of identifying lipid substrates or products of Cyp4gl -catalyzed reactions, as modulators of ALS or neurological disease. The methods comprise (a) contacting a polypeptide with a substrate, where the polypeptide is a Cyp4gl polypeptide or fragment thereof having enzymatic activity, and (b) detecting a reaction product of the polypeptide contacted with the substrate, where detection of a reaction product indicates the substrate and/or reaction product is a modulator of neurological disease. In particular embodiments, the activity or enzymatic activity is omega-hydroxylation of a fatty acid. Detection of the substrate and/or reaction product may be performed according to any standard method known in the art (e.g., NMR or mass spectrometry).
Combination Therapies In some aspects, the invention features methods of treating a neurode generative disease in a subject, the methods comprising administering to the subject an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide having a mutation. In some embodiments, the mutant Cyp4gl polypeptide or polynucleotide is administered in combination with an agent that decreases a level or activity of Cyp4gl polypeptide in the subject. An agent that decrease a level or activity of Cyp4gl polypeptide may be, for example, an inhibitory nucleic acid that reduced expression of Cyp4gl polypeptide (e.g., siRNA) or a small molecule compound that inhibits Cyp4gl polypeptide activity (e.g., HET0016).
Optionally, an anti-neurological disease therapeutic of the invention (e.g., a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation as described herein) may be administered in combination with any other standard anti-neurological disease (e.g., anti-ALS) therapy; such methods are known to the skilled artisan and described in Remington's
Pharmaceutical Sciences by E. W. Martin.
Kits
The invention provides kits for the treatment, suppression, or prevention of a neurological disease, particularly amyotrophic lateral sclerosis (ALS) associated with SOD1 mutations (e.g., SOD1 G85R), C9orf72 repeat expansion, or TDP-43 overexpression. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a suppressor Cyp4gl polypeptide, or fragment thereof (or a polynucleotide encoding such) in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. In some other embodiments, the kit further includes reagents detecting a mutation associated with or causing a neurological disease (e.g., ALS) in a subject. For example, the reagents may be primers or hybridization probes for detection of mutation in SOD1 (e.g., SOD1 G85R), a C9orf72 repeat expansion, or TDP-43 overexpression.
If desired a composition comprising a therapeutic agent of the invention (e.g., Cyp4gl polypeptide or polynucleotide comprising suppressor mutations described herein) is provided together with instructions for administering the agent to a subject having or at risk of developing a neurological disease. The instructions will generally include information about the use of the composition for the treatment or prevention of neurological disease. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
Example 1: Forward genetic screen in fly model of amyotrophic lateral sclerosis (ALS) revealed mutations suppressing lethal effects of SOD1 G85R.
Genetic models of human neurological disease were developed, where human disease- causing mutations are introduced into the cognate genes of the facile genetic organism, Drosophila melanogaster. Initial studies revealed that introduced human mutations could very effectively and accurately unravel important details about hereditary disease, such as pediatric epilepsy. This led to modeling ALS in the fly. First, mutations were introduced into one of the first and most common fALS genes, superoxide dismutase (SOD1). Flies carrying human disease mutations (H71Y and G85R) in SOD1 have a range of phenotypes from adult onset loss of motor coordination and early death (H71Y), to an even earlier death during metamorphosis (G85R). Significant efforts were spent in characterizing these mutants as to their expression of mutant SOD proteins, the dosage effects as they relate to disease progression, and genome-wide transcriptomics. All of these studies validated that the Drosophila ALS model recapitulated important aspects of the human disease, including motor neurons’ death. To make a leap forward in the understanding of motor neuron diseases, the ALS model described above was used, coupled with the power of forward genetics (FIG. 1). In short, forward genetics associates phenotypes with genes in large-scale unbiased screens usually involving mutagenesis of the entire genome of a model system. In this study, the genomes of flies were mutagenized is such a way as to obtain, if possible, suppressor genes that reversed the lethal effects of the SOD1-G85R mutation, an aggressive and fast progressing mutation in human ALS, and likewise completely lethal in flies (FIG. 2A). About 500,000 mutagenized flies or fly strains that would otherwise die of ALS were screened, and five (5) lines of Drosophila carrying mutations which almost completely suppressed, or cured, the deleterious effects of the SOD1 mutation were obtained (FIG. 3 A). The mutations are shown in FIG. 4 and FIG. 5.
The results described in the Examples herein were obtained with the following materials and methods.
Materials and Methods
Drosophila strains, EMS mutagenesis and survival screen
Two independently derived homologous recombinant lines of dSodlG85R/Tm3, hshid, sb (Balancer chromosome, Bloomington Drosophila Stock Center number: 1558)
derived from independent targeting transgene stocks were used: dSodlG85R/Tm3,hs- 185 hid,sb, line A and line B. Newly eclosed dSodlG85R/Tm3,hs-hid,sb (line A) males were aged for 1-7 days and were then mutagenized. The stock of dSodlG85R/Tm3,hs-hid,sb
(line B) was used for the crosses with the possible suppressor mutation bearing
dSodlG85R/Tm3,hs-hid,sb (line A) mutagenized males to minimize the possibility of homozygosing background mutations. Flies were kept at constant 25oC, on standard molasses food, and under 12-h day /night cycles. 6028 dSodlG85R/Tm3,hs-hid (line A) males were starved overnight (12hr) in vials supplied with only water. Surviving males were fed with 25mM EMS (Sigma M-0880) in 5% sucrose solution for 10 hours. The water and sucrose solution containing EMS was administered with Kimwipe wrapped ceaprene stoppers as described by Christian Bokel (Bokel, 2008). Surviving males were allowed to recover on regular food for 14 hours to allow for the clearance of any excess EMS from the digestive system. Then, the surviving 4800 males were mated with 6400 virgin females dSodlG85R/Tm3,hs-hid (line B). The crosses were set up in bottles (Genesee 32-130) with each bottle containing 15 males and 20 virgin females. (FIGS. 2B-2C) The parents in the bottle stock were passed after 5 days. The progeny in the bottles were heat shocked on days 5 and 6 for 2 hours at 37oC in order to induce hs-hid expression, thus killing any animals containing the Tm3 chromosome. Development was allowed to continue for the remaining cohort of flies, which should be exclusively
dSodlG855RG85R homozygotes and were subsequently screened regularly for survivors over the next two weeks. The surviving progeny were named as EMS1 through EMS145. Flies that survived more than 3 days were crossed with 3 flies (dSodlG85R/Tm3-hsHid) of the opposite sex. The dSodl locus of all the surviving lines were PCR amplified and sequenced with forward primer: 5 '-GC AT GT ATTT CT AAGCT GCTCT GCT ACGGT C AC-3 ' and, reverse primer: 5'- GTCCACTGCTAAGAGCAGCTGCCCTC-3'.
Example 2: Characterization of suppressor mutations from genetic screen
From the forward genetic screen described above, mutations in Cyp4gl of Drosophila, a member of the CYP4 family that has novel catalytic and biological roles, was obtained. In all, four (4) novel missense mutations causing amino acid substitutions in Cyp4gl were found (FIGS. 4 and 5). It is worth emphasizing, all genes of the genome were mutagenized without bias and at random, and four independent changes in only the Cyp4gl that all suppressed ALS- mutant lethality were obtained. The mutations in Cyp4gl were G92E, D112N, S143N, and M333I. The Drosophila line EMS130/35 was found to carry mutation G92E; line EMS102 carried D112N, EMS81 carried S143N, and EMS94 carried mutation M333I. The suppressor alleles Cyp4gl G92E, Cyp4gl D112N, Cyp4gl S143N, and Cyp4gl M333I may be interchangeably referred to herein as“EMS130/35,”“EMS102,”“EMS81,” and“EMS94,” respectively. Likewise, the suppressor mutations G92E, D112N, S143N, and M333I are interchangeably referred to herein as“EMS130/35 mutation,”“EMS102 mutation,”“EMS81 mutation,” and“EMS94 mutation,” respectively.
Genetic and molecular mapping of mutations EMS 81 and EMS 102 revealed that they are dominant gain-of-function alleles ofCyp4gl.
The EMS 81/102 mutations were first balanced over the Fm7 balancer, as described above, and then heterozygote females were crossed to males of a mapping chromosome stock carrying five recessive visible markers. Non-Fm7 heterozyogous females of the FI (where recombination occurs) were then crossed to wild type males, and phenotypes were scored in the F2. The lethal phenotype of the EMS 81/102 mutations was used to infer the location of these mutations by assessing the lack of particular classes of recombinants. In fact, it was clear that both the EMS 81/102 mutations mapped between two tightly linked (1 cM) visible markers (call them p and q). Thus, recombinants on this interval alone could be used to generate mapping data (FIG. 3B). Briefly, the lethal mutation on a wild type background in heterozygous state with a doubly mutant mapping chromosome (p + q, where p, +, and q are the gene order of the p locus, the wild type EMSX locus, and the q locus respectively) will generate males in the FI that are either parental non-recombinant (NR) or recombinant (R) chromosomes (FIG. 3C). In any case where the resultant chromosome retains the lethal (1) EMSX allele, no male progeny will be recovered. Recombination can occur on two intervals that result in viable recombinant offspring (I and II, FIG. 3C). By measuring the ratios of recombinants on this interval (I/(I+II)), one obtains the distance (after multiplying this fraction by lcM) from the p locus. This allowed the mapping of the EMSX mutations to within a O. lcM of one another, strongly suggesting that these were mutations in the same gene.
While the genetic mapping was highly informative, the region of the X chromosome is densely populated with genes, and represents a region where the genetic map is contracted compared to other regions of the X chromosome. In fact, the lcM region that the EMSX mutations mapped to covers 2 Mb of physical distance on the X chromosome containing hundreds of genes. In order to increase mapping accuracy, an interesting and equally surprising observation was exploited. For the EMS 81 allele, it was noticed that when animals were reared in fresh food, complete lethality was always seen with 100% penetrance. However, if the parental animals (and some FI) were carried over in vials to a second generation, and the food was more than two weeks old, and "conditioned" by overcrowding, death, and waste material, EMS 81 hemizygote males could be obtained as viable adults. These males will be referred to herein as pseudo-rescued (EMS 81-PR) males.
EMS 81-PR males were found to be fertile and mobile for at least a week. This allowed us to cross these males to molecularly -defined deficiency bearing balanced stocks carrying a series of five deficiencies spanning the 2Mb regions implicated in genetic mapping studies (Cook et al., 2012) (FIG. 3D). One of these deficiencies (BSCdf2) failed to complement EMS 81, giving rise to female progeny that died in exactly the same manner as hemizygous EMS 81 males, namely, as pharate adults during eclosion. All other deficiencies tested complemented the lethal phenotype of EMS 81. Thus, we were able to localize the EMS 81 mutation to a ~260kb deficiency, however, this region still contained around 35 annotated transcription units.
To further refine the mapping, a series of molecularly defined duplication stocks carrying ~80kb duplications inserted into a common site on the third chromosome, and covering almost completely the entire X chromosome (Venken et al, 2010) (FIG. 3E) was utilized. Females balanced for EMS 81 or 102 (EMSX/Fm7) were crossed to seven duplication-bearing lines. Duplications could be followed by the presence of eye color, as the EMS stocks were in a white-eyed background. Three duplications (DP2, DP3, DP4) rescued the lethality of EMS 81 and 102. The combination of rescue by these deletions allowed the delimitation of a much smaller genetic interval containing only two genes (here labeled A and B). A lethal mutation (molecularly uncharacterized) happened to have been attributed to the gene B, which is referred to herein as Su(G85R). This mutation (l(l)x) was obtained as a balanced stock and EMS 81-PR males were crossed to this stock, resulting in failure of complementation of the l(l)x mutation.
The Su(G85R) gene was sequenced for EMS 81, EMS 102 and the l(l)x mutations. In the case of the l(l)x stock, a 13nt deletion was found in the open reading frame of Su(G85R), predicted to truncate the protein at about 2/3 its normal length. Thus, l(l)x is almost certainly a null mutation for Su(G85R). Mutations were also found in the EMS 81 and 102 mutant stocks, G-to-A mutations, which is consistent with EMS as the mutagen. Both mutations cause missense change in different and highly conserved positions within the Su(G85R) protein, which is known to play a role in metabolism. The Su(G85R) gene is Cyp4gl.
Mapping suppressor lines EMS 35, EMS 94 and EMS 130
The mutations EMS 35, 94 and 130 were backcrossed in the balanced dSodlG85R background through females for >10 generations and it was observed that suppressed males were obtained in the expected ratios. In order to test initial linkage, suppressed G85R/G85R dSodl homozygote males were crossed to the balanced G85R stock. In this case, only suppressed G85R homozygous females were obtained. Such females, when crossed to balanced G85R males gave rise to suppressed males and females in the expected ratios. Thus, these suppressor mutations, whose only phenotype appeared to be suppression of G85R homozygosity (in contrast to EMS 81 and 102) also appeared to map to the X chromosome.
Suppressor Lines Also Suppresses Eclosion Defect and Short Life Span of
dSodlH71 Y/H71 Y
EMS 81 and 102 were introgressed into a balanced dSodlH71Y genetic background, using alleles of EMS 81 and 102 that had been recombined with the tightly linked w+ gene, allowing the suppressor alleles to be monitored via eye color. Homozygosity for
dSodlH71 Y/H71 Y normally confers a high level of unviability in the pupal stage, and animals only live for about 16 days, with a profound loss of motor function within the first week.
Females homozygous for dSodlH71Y/H71Y but also containing either EMS 81 or EMS 102 appeared in much greater numbers from the doubly balanced stock, and initial experiments showed that they live for at least one month with no apparent loss of locomotor function, compared to controls lacking the suppressor alleles which behaved as previously described.
These experiments strongly support the notion that EMS 81 and 102, despite being selected for suppression of G85R homozygous lethality, also suppress unviability and locomotor defects in a less severe ALS model.
Nature of Cyp4gl and CYP4 class of proteins
Mapping and molecular studies have revealed the nature of the Cyp4gl gene in flies, a member of the ubiquitous cytochrome P450 (CYP) class of proteins found in all life forms, including bacteria. FIG. 6 depicts the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene. The dCyp4gl (CG3972) gene in Drosophila is an intronless gene. CYP proteins play two major roles in biology; detoxifying (biodefense) against many xenobiotics and antibiotics, and biosynthesis of endogenous molecules such as steroids and lipids. There are as many as 37 families of CYP genes. The CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals. The novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids. The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells. Interestingly, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver. Gutierrez et al., Nature (2007) 445:275-280.
FIG. 7 shows an alignment of dCyp4gl and hCYP4V2, the closest human homologue to dCyp4gl. The amino acid in the position mutated in EMS81 (position 143) is conserved between dCyp4gl and hCYP4V2. The hCYP4V2 gene is mutated in Bietti’s Crystalline Dystrophy (BCD) a retinal degeneration syndrome. hCYP4V2 protein has a fairly broad expression pattern including liver. Patients with BCD display a systemic dyslipidemia. A mouse model of BCD recapitulates clinical features of the human disease including systemic dyslipidemia.
FIG. 8 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates. Suppressor alleles (EMS35/130, EMS102, and EMS81) isolated as dominant suppressors of SOD1-G85R all mapped to the dCyp4gl gene on the X chromosome. EMS81 and EMS102 alleles were homozygous lethal in Drosophila and were also invariant positions in invertebrate lineages. EMS35/130 and EMS94 were mutations in less conserved positions and were viable in males. EMS35/130 result from the same
nucleotide/amino acid change.
FIG. 9 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. Suppressor alleles occurred in regions of high conservation, but only EMS81 serine (S) was conserved from flies through mammals.
Example 3: Modeling of Cyp4gl structure revealed a suppressor mutation located in substrate binding pocket.
Modeling of the active site of the CYP4A11 protein (related to CYP4V2) revealed residues lining the substrate binding pocket and active site involved in omega-hydroxylation. Chang et al., Proteins (1999) 34(3):403-415. FIG. 10 shows an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket. The EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.
Without intending to be bound by theory, it is believed at least some of the suppressor mutations obtained in the genetic screen described herein alter the activity of Cyp4gl, particularly enzymatic activity or fatty acid / lipid metabolism activities of Cyp4gl. HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a known potent inhibitor of CYP4V2 (FIG. 11). In particular, HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122. To further investigate the relationship between Cyp4gl activity and ALS phenotype, the ability of HET0016 to phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media is assessed.
Example 4: Mutations in Cyp4gl suppressed additional ALS causing mutations.
There are other genetic models of ALS in Drosophila based on very different causative gene mutations, or misexpression models. One of these is the C9orf72 hexanucleotide repeat expansion model that is the most common form of familial ALS (fALS) and sporadic ALS (sALS). It is proposed that a hexanucleotide which is expanded (GGGGCC)n in the disease causes ALS or dementia when the repeat number expands (n= 1000s) through a toxic RNA- based mechanism. Overexpression of the C9orf72 toxic repeat globally in flies was found to lead to a phenotype indistinguishable from the lethality seen with SOD1-G85R expression. Preliminary data revealed that the suppressor mutations (G92E, D112N, S143N, and M333I in Cyp4gl) obtained from the forward genetic described above also suppressed the lethal phenotype of C9orf72 repeat overexpression in Drosophila.
In addition, it has been reported for another ALS-causing gene, TDP-43, that motor neuron overexpression of TDP-43 leads to a lethal phenotype that appears very similar to SOD1- G85R. The TDP-43 gene’s normal biological role is currently hotly debated, but it is has been shown to regulate body fat composition. Given the commonalities of phenotype of these other ALS models, whether the suppressor mutations in Cyp4gl also suppress these other seemingly unrelated genetic forms of ALS will be tested. If so, then the work in Drosophila may have revealed an important underlying cause of ALS, and possibly, many age-related cognitive disorders. Suppressors will be tested with the TDP43 model of neurodegeneration in
Drosophila.
Other Embodiments
Prom the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:
1. An isolated polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
2. An isolated Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
3. An isolated human Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
4. An isolated polynucleotide encoding a polypeptide having at least 85% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
5. An isolated polynucleotide encoding a Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333.
6. An isolated polynucleotide encoding a human Cyp4gl polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306.
7. The polypeptide or polynucleotide of any one of claims 1-2 or 4-5, wherein the Cyp4gl polypeptide is a Cyp4gl polypeptide of an invertebrate or vertebrate.
8. The polypeptide or polynucleotide of claim 7, wherein the vertebrate or invertebrate is selected from the group consisting of: Drosophila melanogaster, Homo sapiens, Tenebrio molitor, Aedes aegypti, Diaphorina citri, Blatella germanica, Nasonia vitripennis, Spodoptera frugiperda, Oryctolagus cuniculus, Canis lupus, Gallus gallus, Xenopus tropicalis, and Danio rerio.
9. The polypeptide or polynucleotide of any one of claims 2-3 or 5-8, wherein the polypeptide has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide or a human Cyp4gl polypeptide.
10. The polypeptide or polynucleotide of any one of claims 1-2, 4-5, or 7-9, wherein the mutation is selected from the group consisting of mutations G92E, D112N, S143N, and M333I.
11. The polypeptide or polynucleotide of any one of claims 3 or 6, wherein the mutation is S138N.
12. The polypeptide or polynucleotide of any one of claims 1-11, wherein the polypeptide comprises any one of the following sequences:
Drosophila melanogaster Cyp4gl EMS81
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh
121 qhltkaeeyr yfkpwfgdgl l nghhwrh hrkmiaptfh qsilksfvpt fvdhskavva
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva Drosophila melanogaster Cyp4gl EMS102
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp ssdelilsgh
121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskavva
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva Drosophila melanogaster Cyp4gl EMS130/35
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk yeetmkawlg nvllvfltnp sdielilsgh
121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva
Drosophila melanogaster Cyp4gl EMS 94
1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrmsre yrmvanipsp
61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh
121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa
181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk
241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa
301 skkeglrddl ddidendvga krrlalldam veiaknpdie wnekdimdev ntimfeghdt
361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly
421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma
481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle
541 ngfnvslekr qyatva
Human Cyp4gl (CYP4V2) EMS81
1 maglwlglvw qklllwgaas alslagaslv lsllqrvasy arkwqqmrpi ptvarayplv
61 ghallmkpdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk
121 ssmykflepw lglglltetg nkwrsrrkml tptfhftile dfldimneqa nilvkklekh
181 inqeafncff yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw
241 ldlwylmfke gwehkkslqi lhtftnsvia eranemnane dcrgdgrgsa psknkrrafl
301 dlllsvtdde gnrlshedir eevdtfmfeg hdttaaainw slyllgsnpe vqkkvdheld
361 dvfgksdrpa tvedlkklry lecviketlr lfpsvplfar svsedcevag yrvlkgteav
421 iipyalhrdp ryfpnpeefq perffpenaq grhpyayvpf sagprncigq kfavmeekti
481 lscilrhfwi esnqkreelg legqlilrps ngiwiklkrr nader
13. The polypeptide or polynucleotide of any one of claims 1-12, wherein the mutation is a gain-of-function or loss-of-function mutation.
14. The polypeptide or polynucleotide of any one of claims 1-13, wherein the mutation suppresses a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS).
15. The polypeptide or polynucleotide of any one of claims 1-14, wherein the mutation associated with ALS is SOD1-G85R.
16. An expression vector comprising the isolated polynucleotide of any one of claims 3-15.
17. A therapeutic composition comprising the polypeptide, polynucleotide or vector of any one of claims 1-16.
18. The therapeutic composition of claim 17, further comprising a pharmaceutically acceptable excipient.
19. The therapeutic composition of any one of claims 17-18, further comprising a vehicle for intracellular delivery of the polypeptide, polynucleotide, or vector.
20. A host cell or host organism comprising the isolated polynucleotide or expression vector of any one of claims 4-16.
21. The host cell or host organism of claim 20, wherein the host cell or host organism is an amyotrophic lateral sclerosis (ALS) model or comprises a mutation and/or misexpression associated with ALS.
22. The host cell or host organism of any one of claims 20-21, wherein the host cell or host organism is mammalian.
23. A Drosophila melanogaster mutant comprising a Cyp4gl polynucleotide and/or polypeptide having a mutation in at least one amino acid position selected from the group consisting of positions 92, 112, 143, and 333.
24. The Drosophila melanogaster mutant of claim 23, wherein the mutation is a missense mutation.
25. The Drosophila melanogaster mutant of any one of claims 23-24, wherein the mutation is selected from the group consisting of mutations G92E, D112N, S143N, and M333I.
26. The Drosophila melanogaster mutant of any one of claims 23-25, further comprising a mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS).
27. The Drosophila melanogaster mutant of claim 26, wherein the mutation and/or misexpression associated with ALS has a lethal effect .
28. The Drosophila melanogaster mutant of claim 27, wherein the mutation associated with ALS is SOD1-G85R.
29. The Drosophila melanogaster mutant of any one of claims 27-28, wherein the Cyp4gl mutation suppresses the lethal effect of the mutation and/or misexpression associated with ALS.
30. The Drosophila melanogaster mutant of claim 29, wherein the suppression is partial, nearly complete, or complete.
31. A method of reducing or ameliorating or preventing an effect of a mutation associated with a neurodegenerative disease characterized by an energy deficit in a subject, the method comprising administering to the subject an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide,
wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby suppressing an effect of a mutation associated with a neurodegenerative disease characterized by an energy deficit in the subject.
32. A method of modulating lipid metabolism in a subject, the method comprising administering to the subject an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide,
wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby modulating lipid metabolism in the subject.
33. A method of beating a neurodegenerative disease characterized by an energy deficit in a subject, the method comprising administering to the subject an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide encoding the polypeptide, wherein the polypeptide comprises a mutation in at least one amino acid position selected from the group consisting of positions corresponding to human Cyp4gl polypeptide amino acid positions 85, 107, 138, and 306, thereby treating a neurodegenerative disease characterized by an energy deficit in the subject.
34. The method of any one of claims 31-33, wherein the composition is the therapeutic composition of any one of claims 17-19.
35. The method of any one of claims 31-34, wherein the polypeptide or polynucleotide is the polypeptide, polynucleotide, or vector of any one of claims 1-16.
36. The method of any one of claims 31-35, wherein the subject has a mutation associated and/or misexpression with amyotrophic lateral sclerosis (ALS).
37. The method of any one of claims 31-36, wherein the mutation associated with a neurological disease or the mutation associated with ALS is SOD1-G85R or C9orf72 repeat expansion.
38. The method of any one of claims 31-37, wherein the subject is human.
39. A method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit, the method comprising
(a) contacting a polypeptide with a candidate agent, wherein the polypeptide is a Cyp4gl polypeptide, and
(b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity,
wherein an alteration in activity indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
40. A method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit, the method comprising
(a) contacting a polypeptide with a substrate, wherein the polypeptide is a Cyp4gl polypeptide having enzymatic activity, and
(b) detecting a reaction product of the polypeptide contacted with the substrate, wherein detection of a reaction product indicates the substrate and/or reaction product is a modulator a neurodegenerative disease characterized by an energy deficit.
41. A method of identifying a modulator of a neurode generative disease characterized by an energy deficit, the method comprising
(a) contacting a cell or organism with a candidate agent, and
(b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity, wherein an alteration in the level or activity indicates the candidate agent is a modulator of a neurode generative disease characterized by an energy deficit.
42. A method of identifying a modulator of a neurodegenerative disease characterized by an energy deficit, the method comprising
(a) contacting a cell or organism with a candidate agent, and
(b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism comprising a Cyp4gl polynucleotide or polypeptide having a mutation, wherein a similarity in the phenotypes indicates the candidate agent is a modulator of a neurodegenerative disease characterized by an energy deficit.
43. The method of claim 39, wherein the activity is binding of the polypeptide to the candidate agent or enzymatic activity of the polypeptide.
44. The method of any one of claims 39-41 or 43, wherein the activity or enzymatic activity is omega-hydroxy lation of a fatty acid.
45. The method of claim 40, wherein the substrate is a fatty acid.
46. The method of claim 42, wherein the phenotype is lipid metabolism.
47. The method of any one of claims 41-42, wherein the cell or organism contacted with the candidate agent is an amyotrophic lateral sclerosis (ALS) model or comprises a mutation and/or misexpression associated with ALS.
48. The method of any one of claims 39 or 41-42, wherein the candidate agent inhibits an activity of Cyp4gl polypeptide.
49. The method of any one of claims 39 or 41-42, wherein the candidate agent suppresses an ALS phenotype.
50. The method of any one of claims 41-42, wherein the cell or organism is the host cell or host organism of any one of claims 20-22.
51. The method of any one of claims 31, 33, and 39-42, wherein the neurodegenerative disease characterized by an energy deficit is selected from the group consisting of amyotrophic lateral sclerosis (ALS), dementia, Parkinson’s disease, and Huntington’s disease.
52. The method of any one of claims 31-33, further comprising administering to the subject an effective amount of an agent that decreases a level or activity of Cyp4gl polypeptide.
53. The method of claim 52, wherein the agent is an inhibitory nucleic acid that inhibits expression of Cyp4gl polypeptide.
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