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WO2013106672A1 - Methods and compositions for the treatment of neurodegenerative disease - Google Patents

Methods and compositions for the treatment of neurodegenerative disease Download PDF

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Publication number
WO2013106672A1
WO2013106672A1 PCT/US2013/021177 US2013021177W WO2013106672A1 WO 2013106672 A1 WO2013106672 A1 WO 2013106672A1 US 2013021177 W US2013021177 W US 2013021177W WO 2013106672 A1 WO2013106672 A1 WO 2013106672A1
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WIPO (PCT)
Prior art keywords
rnase4
seq
protein
subject
amino acid
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PCT/US2013/021177
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French (fr)
Inventor
Guofu Hu
Shuping Li
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Tufts Medical Center, Inc.
President And Fellow Of Harvard College
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Publication of WO2013106672A1 publication Critical patent/WO2013106672A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/27—Endoribonucleases producing 3'-phosphomonoesters (3.1.27)

Definitions

  • Neurodegenerative diseases are characterized by a progressive neurodegenerative process in which neuron structure and/or function is lost over time.
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's disease Parkinson's disease
  • Huntington's disease genetic characteristics have been linked with some neurodegenerative diseases, such as the
  • ALS also known as Lou Gehrig's disease
  • Lou Gehrig's disease is a neurodegenerative disease characterized by the progressive degradation of motor neurons, which causes the afflicted individual to experience progressive weakness, muscle atrophy and respiratory
  • ALS is always fatal, with the median survival time from symptom onset of ALS being about 20 to 48 months. However, the rate of disease progression can vary greatly, with some patients dying within a year of diagnosis and others surviving for many years.
  • ALS has no cure and the only FDA approved treatment for ALS, the sodium channel blocker Riluzole, only increases patient survival by 3-5 months on average.
  • Described herein are methods of reducing neurodegeneration and/or treating or preventing a neurodegenerative disease in a subject (e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) and/or progressive bulbar palsy (PBP)).
  • a neurodegenerative disease e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) and/or progressive bulbar palsy (PBP)
  • the methods include administering to the subject an a neurodegenerative disease in a subject.
  • RNASE4 protein or a biologically active fragment thereof include administering to the subject an agent that enhances the activity and/or expression of RNASE4.
  • an agent that comprises an RNAS4 protein is administered to the subject.
  • the RNASE4 protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the RNASE4 protein has an amino acid sequence at least 90%> identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40.
  • the RNASE4 protein is a variant RNASE4 protein that has less than 10% of the ribonucleo lytic activity of a wild- type RNASE4 protein having a sequence of SEQ ID NO: 1.
  • an agent that enhances the activity and/or expression of RNASE4 is administered to the subject.
  • the agent is a small molecule, a nucleic acid or a polypeptide.
  • the agent increases the expression of both RNASE4 and ANG.
  • the agent enhances expression of RNASE4 by contacting the ANG/RNASE4 promoter.
  • the method described herein also includes administering to the subject a second agent for the treatment of neurodegenerative disease.
  • the second agent is an ANG protein or biologically active fragment thereof.
  • the second agent is an agent that increases the activity and/or expression of ANG.
  • the subject has or is susceptible to a neurodegenerative disease (e.g. ALS).
  • a neurodegenerative disease e.g. ALS
  • the genome of the subject comprises an T allele at single nucleotide polymorphism rs37484338.
  • the subject has reduced expression or activity of RNASE4.
  • the subject has been identified as having an increased risk of
  • the subject has a mutated SODl gene.
  • methods of identifying a subject as having increased risk of neurodegenerative disease e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP.
  • the methods include the step of detecting in a biological sample from the subject a mutation to a
  • the presence of a mutation to the RNASE4 coding region identifies the subject as having an increased risk of neurodegenerative disease.
  • the mutation to the RNASE4 coding causes reduced RNASE4 expression or activity in the subject.
  • the mutated coding region encodes an RNASE4 signal peptide that comprises a serine at signal peptide position -13.
  • the mutated RNASE4 coding region comprises a thymidine at position -39.
  • RNASE4 protein that includes an amino acid sequence at least 90% identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40. In some embodiments, the amino acid sequence has an alanine at position 40. In some embodiments, the RNASE4 protein is identical to SEQ ID NO: l , except that the amino acid sequence does not have a lysine at position 40. In certain embodiments the amino acid sequence is identical to SEQ ID NO:6. Also described herein are nucleic acid sequences encoding such RNASE4 proteins.
  • RNASE4 RNASE4 -reactive protein semiconductor
  • a test agent that increases the expression of RNASE4 is a candidate therapeutic agent for treating or preventing a neurodegenerative disease.
  • Figure 1 shows the nucleic acid sequence of human RNASE4.
  • the first nucleotide codon encoding the first amino acid in the mature protein is numbered 1.
  • the numbering in the signal peptide region starts at the last nucleotide of the codon ending the last amino acid of the signal peptide and goes backward upstream.
  • the nucleotide changes identified from the sequencing study described herein are given in parentheses.
  • Figure 2 shows the ribonucleolytic activity of recombinant wide type RNASE4 and the K40A variant.
  • A SDS-PAGE and Coomassie blue staining of WT and K40A RNASE4 from one of the four preparations. Recombinant ANG protein was included as a control.
  • B Ribonuclolysis of yeast tR A by WT R ASE4 (squares) and K40A RNASE4 (circles). Data shown are mean ⁇ SEM of four independent preparations with triplicates in each enzyme concentration.
  • RNASE4 ribonucleolytic activity
  • A RNASE4 protein was treated with 5 mM DEPC and its ribonucleolytic activity was examined using a yeast tRNA assay.
  • B DEPC-treated RNASE4 fails to cleave rRNA.
  • C DEPC treatment abolishes angiogenic activity of RNASE4.
  • FIG. 4 shows that RNASE4 induces endothelial cell tube formation.
  • Figure 5 shows the angiogenic activities of various ribonucleases.
  • A Microscopic images of endothelial cell tubes formed in the presence of 1 ⁇ g/ml of the indicated ribonuclease, Bar, 0.1 mm.
  • B Number of circled tubular structures per mm 2 .
  • Figure 6 shows that RNASE4 stimulates endothelial sprouts from mouse aortic explants.
  • A Outward growth of endothelial sprouts from aortic rings that have been flipped inside out. The images are representative of three rings from one of three repeats. Bar, 0.1 mm. ANG and PBS were used as positive and negative controls, respectively.
  • B Image J analysis of the area covered by endothelial sprouts from flipped aortic rings. Data are presented as mean ⁇ SEM from three independent experiments.
  • C Inward growth of endothelial sprouts from unflipped mouse aortic rings. The images are representative of three rings from one of two repeats.
  • Figure 7 shows that RNASE4 induces neovessel growth into Matrigel plug implanted under mouse skin.
  • A IHC staining with vWF antibodies. The images shown are representative of at least four sections from three independent experiments.
  • B ImageJ analysis of vWF-positive spots. Data are presented as mean ⁇ SEM from three independent experiments. Bar, 100 ⁇ . Statistical analysis was performed by two-way ANOVA. *, pO.001.
  • Figure 8 shows the effect of R ASE4 on PI 9 cells.
  • A RNASE4 stimulates neurosphere formation of PI 9 cells.
  • PI 9 cells were cultured on PA6 supporting cell layers in the presence of 0.2 ⁇ g/ml of BSA, R ASE4, or ANG for 24 h. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm.
  • B Numbers of neurosphere counted from the entire 35-mm dish. Data shown are means ⁇ SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. *, p ⁇ 0.001.
  • C Cells were cultured as described in A for 216 h and stained for neurofilaments with an anti-neurofilament medium chain IgG.
  • the images are representative of at least four areas from three independent experiments. Bar, 20 ⁇ (D) Image J analysis of neurofilament length from C. Data shown are means ⁇ SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p ⁇ 2xl0 "9 .
  • Figure 9 shows the effect of RNASE4 on mouse embryonic cortical neurons.
  • Cortical neurons were isolated from E14 mouse embryos and cultured in neurobasal medium in the presence of 0.2 ⁇ g/ml RNASE4 or ANG for 12 days with a medium change on day 6.
  • B27 was used as a positive control and BSA at 0.2 ⁇ g/ml was used as a negative control.
  • the images are representative of at least four areas from three independent experiments. Bar, 0.5 mm.
  • FIG. 10 shows that RNASE4 stimulates mouse embryonic stem cell
  • Figure 11 shows the subcellular localization of RNASE4 in HUVE and P19 cells.
  • HUVE cells were incubated in the absence (top panel) or presence (bottom panel) of 0.5 ⁇ g/ml exogenous RNASE4 protein at 37 °C for 1 h.
  • B P19 cells were incubated in the absence (top panel) or presence (bottom panel) of 1 ⁇ exogenous RNASE4 protein at 37 °C for 1 h.
  • Immunofluorescence was carried out with affinity-purified RNASE4 polyclonal rabbit IgG and Alexa 488-labeld goat anti-rabbit IgG. Nuclei were stained with DAPI. Scale bar, 10 ⁇ .
  • FIG. 12 shows that RNASE4 protects stress-induced neuron degeneration.
  • A Effect on hypothermia-induced neurofilament fragmentation of mouse cortical neurons. Mouse cortical neurons were cultured in the presence of B27 for 12 days. Cells were washed with neurobasal medium, incubated with 0.2 ⁇ g/ml RNASE4 or ANG at 37 °C for 1 hour, and then subjected to hypothermia treatment at 25 °C for 40 min. Cells were returned to incubator and continually cultured for 3 h and stained for neurofilaments. The images are representative of at least four areas from three independent experiments. Bar, 0.4 mm.
  • B Image J analysis of the length of neurofilament.
  • FIG. 13 shows that RNASE4 has no effect on PA6 cell proliferation.
  • PA6 cells were cultured in a-MEM containing 0.1% NEAA and 0.1% NKSR in the presence of 0.2 ⁇ g/ml BSA or RNASE4 with or without 0.5 ⁇ retinoic acid. Cell proliferation was determined by MTT assay.
  • Figure 14 shows decreased RNASE4 mRNA level in the spinal cord motor neurons of ALS patients and in S0D1 G93A mice.
  • A In situ hybridization of human RNASE4 mRNA in the spinal cord of ALS patients and non-ALS control subject. Left panel, representative ISH images from one of the six patients. Bar, 10 ⁇ . Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ⁇ SEM from six patients. Statistical analysis was performed by two-way ANOVA.
  • B In situ hybridization of mouse Rnase4 mRNA in the spinal cord of WT and SODI G93A mice. Left panel, representative ISH images from one of the six mice. Bar, 10 ⁇ . Right panel, ImageJ analysis of photon counts per motor neuron.
  • RNASE4 On SODl ⁇ yiA mice. Starting from 11 weeks of age, mice were treated with weekly i.p. injection of WT RNASE4 protein at 10 ⁇ g per mouse. Three independent experiments were performed with a total of 34 and 31 mice in the RNASE4 treatment group (triangles) and PBS control group (triangles), respectively.
  • A Effect on rotarod performance at 20 rpm without revolving. Two successive measurements were recorded. An upper limit of 1,000 second was used.
  • B Effect on body weight. Data shown are means ⁇ SEM of all survived animals at each data point. Statistical analysis was performed by two-way ANOVA.*, p ⁇ 0.01.
  • Figure 16 provides exemplary amino acid sequences of RNASE4 proteins.
  • SEQ ID NO: 1 is the wild-type RNASE4 protein sequence.
  • SEQ ID NO: 7 is the variant K40A RNASE4 protein sequence. Sequences are for the mature RNASE4 protein and do not include the signal sequence.
  • Figure 17 provides exemplary nucleic acid sequences that encode for RNASE4 proteins.
  • SEQ ID NO: 7 encodes the wild-type RNASE4 protein.
  • SEQ ID NO: 13 encodes for the variant K40A RNASE4 protein. Sequences encode the mature RNASE4 protein and do not include the signal sequence.
  • compositions and methods for reducing neurodegeneration and treating neurodegenerative diseases such as ALS.
  • compositions and methods for identifying subjects at elevated risk of neurodegenerative disease and for identifying agents useful in the treatment of such diseases are also provided herein.
  • RNASEs Secreted ribonucleases
  • RNASE4 is the most conserved gene across the different vertebrate species, with an amino acid homology as high as 94%.
  • RNASE4 has a very strict substrate specificity and generally cleaves RNA immediately 5' of uridine residues.
  • ANG and RNASE4 have 38.7% identity.
  • ANG and RNASE4 genes are located in the same locus and share the same promoters.
  • the ANG1 and RNASE4 genes contain two non-coding exons followed by two distinct exons encoding ANG1 and RNASE4, respectively.
  • the two non-coding exons are preceded by two promoters that control tissue-specific expression of both genes.
  • RNASE4 protein that lacks a lysine at amino acid position 40. Because the lysine at position 40 is part of the catalytic triad that is critical for RNA cleavage of RNASE4, this RNASE4 protein variant has significantly reduced RNA cleaving activity compared to the wild-type RNASE4 protein. As is described herein, both wild-type RNASE4 protein and the variant RNASE4 protein are angiogenic, neurogenic and neuroprotective. Notably, the variant RNASE4 protein actually has higher angiogenic, neurogenic and neuroprotective activity than the wild-type protein, despite having greatly reduced RNASE activity.
  • RNASE protein (either wild-type or variant protein) is useful in the prevention of neurodegeneration and the treatment of neurodegenerative disease.
  • administration of RNASE4 protein in an ALS disease model slowed weight loss and preserved neuromuscular function in the test animal.
  • RNASE4 protein and agents that increase RNASE4 activity and/or expression are therefore promising therapeutic candidates for the treatment of neurodegenerative disease, and individuals with reduced RNASE4 activity and/or expression have an increased risk of developing a neurodegenerative condition.
  • administering means providing a pharmaceutical agent ⁇ e.g., an RNASE4 protein or an agent that increases RNASE4 activity and/or expression) or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • a pharmaceutical agent e.g., an RNASE4 protein or an agent that increases RNASE4 activity and/or expression
  • agent is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds or a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide).
  • Agents may be identified as having a particular activity ⁇ e.g., an RNASE4 enhancing activity) by screening assays described herein below. The activity of such agents may render them suitable as a "therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • binding refers to an association, which may be a stable association, between two molecules, e.g. , between a polypeptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • Bioly active portion ofRNASE4 refers to a portion of R ASE4 protein having a biological activity, such as the ability to induce angiogenesis or inhibit
  • tissue sample each refers to a collection of cells obtained from a tissue of a subject.
  • the source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.
  • control includes any portion of an experimental system designed to demonstrate that the factor being tested is responsible for the observed effect, and is therefore useful to isolate and quantify the effect of one variable on a system.
  • isolated polypeptide refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • isolated nucleic acid refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid” is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • the term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
  • a “patient” or “subject” refers to either a human or a non-human animal.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue ⁇ e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • FAST A FAST A
  • BLAST BLAST
  • ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions described herein.
  • RNASE4-activating compound or "agent that increases RNASE4 activity” refers to an agent that increases the level of RNASE4 protein and/or increases at least one activity of a RNASE4 protein besides RNA cleavage.
  • a RNASE4-activating compound may increase at least one biological activity of a RNASE4 protein by at least about 10%, 25%, 50%, 75%, 100%, or more.
  • Exemplary biological activities of RNASE4 proteins include promotion of angiogenesis and inhibition of neurodegeneration.
  • a "Single Nucleotide Polymorphism” or "SNP” is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).
  • NCBI National Center for Biotechnological Information
  • a SNP allele can be describe based on the sequence of its forward strand or the sequence of its reverse strand. For example, a SNP that has either A or G alleles on its forward strand will have either T or C alleles, respectively, on its reverse strand. The SNP alleles are described herein according to their forward strand sequence.
  • terapéuticaally-effective amount and “effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • "Treating" a disease in a subject or “treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • RNASE4 protein refers to a protein with substantial sequence homology to wild-type RNASE4 (e.g. a protein having the amino acid sequence of SEQ ID NO: 1) that retains at least one activity associated with wild-type RNASE4 (e.g., promotion of angiogenesis and/or inhibition of neurodegeneration).
  • RNASE4 proteins can be wild-type RNASE4 proteins (e.g. proteins having a sequence of SEQ ID NO: 1) or can be variant RNASE4 proteins (e.g. proteins having an amino acid sequence at least 50%,
  • RNASE4 proteins do not need to have RNA cleaving activity.
  • RNASE4 proteins include proteins having amino acid sequences of SEQ ID NO: 1-6. Variants of RNASE4 proteins can be produced by standard means, including site- directed and random mutagenesis .
  • RNASE4 protein As used herein, a "biologically active fragment" of a RNASE4 protein is a polypeptide that retains at least one activity of wild-type RNAS4 protein (e.g., promotion of angiogenesis and/or inhibition of neurodegeneration) and that comprises at least 8 consecutive amino acids, (e.g., at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 amino acids) of SEQ ID NO: 1.
  • wild-type RNAS4 protein e.g., promotion of angiogenesis and/or inhibition of neurodegeneration
  • amino acids e.g., at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 amino acids
  • the RNASE4 protein is a RNASE4 protein that does not have a lysine at position 40 (e.g., such as a RNASE4 protein having an amino acid sequence of SEQ ID NO: 6).
  • the RNASE4 protein is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQID NO: 6 and does not have a lysine at position 40.
  • the RNASE4 proteins described herein have an alanine instead of a lysine at position 40.
  • the lysine at position 40 is part of RNASE4's catalytic triad and plays a role in RNASE4's RNA cleaving activity.
  • Variant RNASE4 proteins that lack a lysine at position 40 therefore have reduced RNASE activity compared to wild-type RNASE4 protein.
  • the variant RNASE4 proteins described herein have less than 50%, 40%, 30%, 20%, or 10% of the RNA cleaving activity of wild-type RNASE4 protein.
  • RNASE4 proteins that lack a lysine at position 40 have elevated angiogenic and neuroprotective activities compared to wild-type RNAS 4 protein.
  • RNASE4 proteins that lack a lysine at position 40 are particularly useful, for example, for the treatment of neurodegenerative diseases and for the prevention of neurodegeneration.
  • R ASE4 proteins that comprise "conservative sequence modifications" of the sequences set forth in SEQ ID NO: 1-6.
  • conservative sequence modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence set forth in the figures by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains ⁇ e.g., lysine, arginine, histidine
  • acidic side chains ⁇ e.g., aspartic acid, glutamic acid
  • uncharged polar side chains ⁇ e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains ⁇ e.g.
  • a protein described herein is further linked to a
  • heterologous polypeptide e.g. , a polypeptide comprising a domain which increases its solubility and/or facilitates its purification, identification, detection, and/or structural characterization.
  • a protein described herein may be linked to at least 2, 3, 4, 5, or more heterologous polypeptides.
  • Polypeptides may be linked to multiple copies of the same heterologous polypeptide or may be linked to two or more heterologous polypeptides.
  • the fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a protein described herein and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein.
  • a protein may be modified so that its rate of traversing the cellular membrane is increased.
  • the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells.
  • the peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment
  • the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof.
  • the 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled.
  • the polypeptide may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al.
  • the transcytosis polypeptide may also be a non-naturally- occurring membrane-translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Patent No. 6,248,558.
  • MTS membrane-translocating sequence
  • RNASE4 proteins ⁇ e.g., acid sequences of SEQ ID NO: 1-6
  • RNAS4 proteins ⁇ e.g., acid sequences of SEQ ID NO: 1-6
  • polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding RNASE4 protein or biologically active fragment thereof.
  • polypeptides can be synthesized by chemical methods.
  • Nucleic acids encoding any of the polypeptides described herein ⁇ e.g., RNASE4 proteins or biologically active fragments thereof) are also provided herein.
  • a nucleic acid may further be linked to a promoter and/or other regulatory sequences, as further described herein.
  • Exemplary nucleic acids are those that are at least about 80%, 85%, 90%>, 95%>, 98%, 99% or 100% identical to SEQ ID NO : 7- 13 or a fragment thereof.
  • Nucleic acids may also hybridize specifically, e.g., under stringent hybridization conditions, to a nucleic acid described herein or a fragment thereof.
  • Arginine AGA, ACG, CGA, CGC, CGG, CGT
  • Glycine Gly, G
  • GGC GGG, GGT
  • Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT
  • nucleotide triplet An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
  • the nucleic acid molecules described herein may differ from nucleotide sequences of SEQ ID NO: 7-13 due to degeneracy of the genetic code and still encode the same polypeptides as those encoded by SEQ ID NO: 7-13.
  • nucleic acid molecules described herein can be isolated using standard molecular biology techniques and the sequence information provided herein (e.g., SEQ ID NO: 7-13).
  • sequence information provided herein e.g., SEQ ID NO: 7-13
  • a nucleic acid molecule encompassing all or a portion of sequences shown in SEQ ID NO: 7-13 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequences provided in SEQ ID NO: 7-13.
  • Oligonucleotides corresponding to nucleic acid sequences described herein can be prepared, for example, by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
  • the nucleic acid sequences described herein hybridize under stringent conditions to a nucleic acid of SEQ ID NO: 7-13.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
  • Nucleic acids e.g., those encoding an RNASE4 protein or biologically active fragment thereof, can be delivered to cells in culture, ex vivo, and in vivo.
  • the cells can be of any type including without limitation neuronal cells, myocytes, and non-neuronal cells.
  • the delivery of nucleic acids can be by any technique known in the art including viral mediated gene transfer, liposome mediated gene transfer, direct injection into a target tissue or organ, injection into vasculature which supplies a target tissue or organ.
  • Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors.
  • viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus).
  • Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
  • a polynucleotide of interest can also be combined with a condensing agent to form a gene delivery vehicle.
  • the condensing agent may be a polycation, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Many suitable methods for making such linkages are known in the art.
  • a polynucleotide of interest is associated with a liposome to form a gene delivery vehicle.
  • Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter. Under appropriate conditions, a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm.
  • the liposome membrane acts as a relatively impermeable barrier that sequesters and protects its contents, for example, from degradative enzymes.
  • a liposome is a synthetic structure, specially designed liposomes can be produced which incorporate desirable features. See Stryker, Biochemistry, pp. 236-240, 1975 (W.H. Freeman, San Francisco, CA); Soak et al, Biochip. Biopsy's. Acta 600: 1, 1980; Bayer et al, Biochip. Biopsy's. Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol. 149: 119, 1987; Wang et al, PROC. NATL. ACAD.
  • Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and expression constructs comprising growth factor polynucleotides such those disclosed in the present invention.
  • Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
  • Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987), niRNA (Malone et al, Proc. Natl. Acad. Sci. USA 86:6077-6081, 1989), and purified transcription factors (Debs et al, J. Biol. Chem.
  • Cationic liposomes are readily available.
  • N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. See also Feigner et al, Proc. Natl. Acad. Sci. USA 91 : 5148-5152.87, 1994.
  • Other commercially available liposomes include Transfectace (DDAB/DOPE) and DOTAP/DOPE
  • cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Soak et al, Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions of the synthesis of DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
  • anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG),
  • dioleoylphoshatidyl ethanolamine DOPE
  • DOPE dioleoylphoshatidyl ethanolamine
  • More than one polypeptides may be encoded by a single nucleic acid.
  • the nucleic acids described herein may encode both and RNASE4 protein or biologically active fragment thereof and an ANG protein or biologically active fragment thereof.
  • the mRNA sequence for ANG is provided, for example, by GI accession number 207113180.
  • the amino acid sequence for ANG is provided, for example, by GI accession number 148277046.
  • the RNASE4 encoding portion of the nucleic acid molecule and the ANG encoding portion of the RNASE4 molecule may both be under the control of a common nucleic acid regulatory element, such as a common promoter.
  • Certain embodiments of the methods described herein relate to methods of preventing neurodegeneration or treating neurodegenerative disease. These methods involve administering an RNASE4 protein, a biologically active fragment thereof, and/or an agent that increases the activity and/or expression of RNASE4.
  • Agents which may be used to increase the activity of RNASE4 include nucleic acids, proteins, peptides and small molecules. Any agent that increases the activity of R ASE4 can be used to practice certain methods described herein. Such agents can be those described herein, those known in the art, or those identified through routine screening assays (e.g. the screening assays described herein).
  • provided herein are methods for determining whether a test agent is a candidate therapeutic agent for treating and/or preventing neurodegenerative disease.
  • the method includes the steps of contacting a cell with a test agent and detecting the activity and/or expression of R ASE4 by the cell.
  • an agent that increases the activity and/or expression of R ASE4 is a candidate therapeutic agent for treating and/or preventing neurodegenerative disease.
  • any cell can be used in the above described screening method.
  • the cell is a human cell.
  • Cells used in the screen can be primary cells or a cell line.
  • Examples of other cell lines useful in the screening assays described herein include, but are not limited to, P19 cells, HUVAC cells, 293-T cells, 3T3 cells, 721 cells, 9L cells, A2780 cells, A172 cells, A253 cells, A431 cells, CHO cells, COS-7 cells, HCA2 cells, HeLa cells, Jurkat cells, NIH-3T3 cells and Vero cells.
  • Enhancers of R ASE4 expression may also be identified, for example, using methods wherein a cell is contacted with a candidate compound and the expression of R ASE4 mRNA or protein is determined. The level of expression of mRNA or protein in the presence of the candidate compound is compared to the level of expression of mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as an enhancer of RNASE4 expression if the expression of RNASE4 is greater in the presence of the candidate compound than in its absence.
  • the expression of the therapeutic targets described herein can be detected using any method known in the art.
  • the expression of the therapeutic target can be detected by detecting therapeutic target mRNA using, e.g., a detectably labeled nucleic acid probe, RT-PCR, and/or microarray technology.
  • the expression of the therapeutic target can also be detected by detecting the therapeutic target protein using, e.g., detectably labeled antibodies that have binding specificity for the therapeutic target.
  • a cell is used in the screening assay that has been genetically engineered to facilitate the performance of the assay.
  • the cell is engineered such that the therapeutic target is expressed as a heterologous protein linked to a detectable moiety (e.g. a fluorescent moiety such as GFP or a luminescent moiety such as luciferase).
  • the cell contains a nucleic acid sequence encoding a detectable moiety operably linked to the promoter of the therapeutic target.
  • the expression of the detectable moiety is detected directly.
  • Such cells can be generated using standard recombinant techniques well known in the art.
  • the endogenous RNASE4 gene and the endogenous ANG gene are under the transcriptional control of a common promoter.
  • the agents describe herein enhance expression of both RNASE4 and ANG.
  • the agent is a transcriptional enhancer that binds to the ANG/RNASE4 promoter.
  • transcriptional enhancers are known in the art and can be targeted to particular genetic loci using, for example, designer TALE effector nucleases, as described in, for example, Moscou et ah, Science 326: 1501 (2009); Boch et ah, Science 326: 1509- 1512 (2009); and Zhang et ah, Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety.
  • Agents useful in the methods of the present invention may be identified, for example, using assays for screening candidate or test compounds which modulate the activity of RNASE4 or a biologically active portion thereof.
  • Exemplary RNASE4 activities include promotion of angiogenesis and inhibition of neurodegeneration.
  • Examples of assays useful in the testing the angiogenic and neuroprotective activities of potential agents are provided in the Examples below and include in vitro endothelial cell tube formation assays, ex vivo aortic ring explant culture assays neurosphere formation assays, neurite outgrowth assays, neurofilament fragmentation assays and serum withdrawal-induced neuronal degeneration assays. Additional assays for angiogenesis promotion and neurodegeneration inhibition are known in the art.
  • agents useful in the methods of the invention may be identified using assays for screening candidate or test compounds which bind to RNASE4 or a biologically active portion thereof. Determining the ability of the test compound to directly bind to RNASE4 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to RNASE4 can be determined by detecting the labeled compound in a complex.
  • compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • Agents useful in the methods of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).
  • compositions of the described herein include R ASE4 proteins, biologically active fragments thereof and/or agents that enhance R ASE4 activity and/or expression ⁇ e.g., any small molecule, protein, polypeptide or polynucleotide that activates the activity or expression of R ASE4) combined with a pharmaceutically acceptable carrier or vehicle.
  • the pharmaceutical compositions described herein may further comprise additional agents useful in the treatment of neurodegenerative diseases, including ANG proteins, biologically active fragments thereof, agents that enhance ANG expression and/or activity, and/or Riluzole.
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.
  • Toxicity and therapeutic efficacy of the agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography. Appropriate dosage agents depends upon a number of factors within the scope of knowledge of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • an R ASE4 protein or biologically active fragment thereof is administered to a subject.
  • an agent that increases the activity or expression of R ASE4 is administered to the subject.
  • an ANG protein, biologically active fragment thereof and/or an agent that increases the activity or expression of ANG is also administered to the subject.
  • the methods described herein can be used to treat any neurodegenerative disease.
  • the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP.
  • the neurodegenerative disease is ALS.
  • a "subject in need thereof includes any subject that has a neurodegenerative disease ⁇ e.g., ALS), and well as any subject with an increased likelihood of acquiring a neurodegenerative disease.
  • the subject in need thereof is identified according to the diagnostic methods described herein below.
  • the subject in need thereof carries a RNASE4 gene mutation.
  • the genome of the subject in need thereof has a T allele at single nucleotide polymorphism rs37484338.
  • the subject in need thereof carries a gene mutation associated with a neurodegenerative disease, such as a mutated SOD1 gene.
  • the subject in need thereof has at least one family member who has a neurodegenerative disease.
  • compositions of the present invention may be delivered by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the pharmaceutical compositions are delivered generally ( e.g., via oral or parenteral administration).
  • the pharmaceutical compositions are delivered locally through direct injection into a specific tissue (e.g., central nervous system tissue and/or peripheral nervous system tissue).
  • the methods of treatment of the present invention comprise administering an R ASE4 protein, biologically active fragment thereof or an agent that increases the activity or expression of RNASE4 in conjunction with a second therapeutic agent to the subject (e.g., an ANG protein, a biologically active fragment thereof and/or an agent that enhances ANG activity and/or expression).
  • a second therapeutic agent e.g., an ANG protein, a biologically active fragment thereof and/or an agent that enhances ANG activity and/or expression.
  • Conjunctive therapy includes sequential, simultaneous and separate, or co-administration of the active compound in a way that the therapeutic effects of the first agent administered have not entirely disappeared when the subsequent agent is administered.
  • the second agent may be co-formulated with the first agent or be formulated in a separate pharmaceutical composition.
  • the dosage of the subject agent may be determined by reference to the plasma concentrations of the agent.
  • the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) may be used.
  • Dosages for the present invention include those that produce the above values for Cmax and AUC (0-4) and other dosages resulting in larger or smaller values for those parameters.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could prescribe and/or administer doses of the agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of an agent described herein will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the methods described herein are used to identify subjects with increased risk of a neurodegenerative disease ⁇ e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP).
  • a subject who carries one or more mutated R ASE4 coding regions is at increased risk for neurodegenerative disease.
  • the method includes the step of detecting in a biological sample a mutation to a R ASE4 coding region.
  • the mutation to the R ASE4 coding region causes reduced RNASE4 activity and/or expression in the subject.
  • the mutated RNASE4 coding region encodes a RNASE4 signal peptide that comprises a serine at position -13.
  • the mutated RNASE4 coding region comprises as thymidine at position -39.
  • the mutation is an allele of a single nucleotide polymorphism that is associated ALS.
  • the mutation is a T allele at single nucleotide polymorphism rs37484338.
  • the subject will be a human child or a human adult. In some embodiments, the subject will be an infant. However, in certain embodiments the subject is not limited to being a fully developed human. Thus, in some embodiments, the subject will be a human fetus, a human embryo and/or a human fertilized cell.
  • the sample is a cell, a body fluid, a swabbing, a tissue sample, a blood sample and/or a germ cell sample.
  • the detecting step includes performing a hybridization assay ⁇ e.g., SNP or gene microarrays, dynamic allele-specific hybridization (DASH), TaqMAN, HP A, scorpion probes and molecular beacons), performing a nucleic acid amplification assay (e.g., PCR, LCR, TMA, SDA, NASBA, BDA, 3SR, RCR, etc.) and/or performing a nucleic acid sequencing assay.
  • a hybridization assay e.g., SNP or gene microarrays, dynamic allele-specific hybridization (DASH), TaqMAN, HP A, scorpion probes and molecular beacons
  • a nucleic acid amplification assay e.g., PCR, LCR, TMA, SDA, NASBA, BDA, 3SR, RCR, etc.
  • analysis of the nucleic acid can be carried out by
  • amplification of the region of interest according to amplification protocols well known in the art (e.g. , polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification, self-sustained sequence replication (3SR), QP replicase protocols, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and boomerang DNA amplification (BDA), etc.).
  • amplification protocols e.g. , polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification, self-sustained sequence replication (3SR), QP replicase protocols, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and boomerang DNA amplification (BDA), etc.
  • NASBA nucleic acid sequence-based amplification
  • RCR repair chain reaction
  • BDA boomerang DNA amplification
  • oligonucleotides for use as primers and/or probes for detecting and/or identifying genetic markers according to the methods described herein.
  • Additional methods for detecting the genetic mutations described herein include sequencing, high performance liquid chromatography (HPLC), restriction enzyme analysis (e.g., restriction fragment length polymorphism or RFLP), hybridization, matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), etc., all of which are well known protocols for analyzing a nucleotide sequence and detecting genetic markers.
  • HPLC high performance liquid chromatography
  • restriction enzyme analysis e.g., restriction fragment length polymorphism or RFLP
  • hybridization e.g., restriction fragment length polymorphism or RFLP
  • MALDI-TOF-MS matrix assisted laser desorption/ionization-time of flight mass spectrometry
  • detecting can be carried out by an amplification reaction and single base extension, and in further embodiments, the product of the amplification reaction and single base extension can be spotted on a silicon chip according to methods well known in the art.
  • the nucleic acid e.g. genomic DNA
  • the nucleic acid may be extracted from the sample using techniques well-established in the art including chemical extraction techniques utilizing phenol-chloroform, guanidine-containing solutions, or CTAB-containing buffers.
  • chemical extraction techniques utilizing phenol-chloroform, guanidine-containing solutions, or CTAB-containing buffers.
  • commercial DNA extraction kits are also widely available from laboratory reagent supply companies, including for example, the QIAamp DNA Blood Minikit available from QIAGEN
  • kits comprising reagents to detect a mutation described herein in a biological sample from a subject.
  • a kit can comprise primers, probes, primer/probe sets, reagents, buffers, etc., as would be known in the art, for the detection of the genetic markers described herein in a biological sample from a subject.
  • a primer or probe can comprise a contiguous nucleotide sequence that is complementary (e.g., fully (100%) complementary or partially (50%, 60%, 70%, 80%, 90%, 95%, etc.) complementary) to a region comprising position -39 of the RNASE4 coding region (e.g., that encodes the R ASE4 signal sequence).
  • Such a kit can further comprise blocking probes, labeling reagents, blocking agents, restriction enzymes, antibodies, sampling devices, positive and negative controls, etc., as would be well known to those of skill in the art.
  • RNASE4 The coding region of RNASE4 gene from 1,575 sporadic ALS patients and 658 controls was sequenced for potential ALS-associated mutations and SNPs. Clinical specimens were obtained under a discarded tissue protocol approved by the institutional review board. Genomic DNA was extracted from peripheral leukocytes using standard protocol. The coding exon of RNASE4 was amplified by PCR with primers located in adjacent intron and non-coding regions, respectively, and the amplicons were sequenced bidirectionally. The sequences of the primers are as follows. Forward. 5'- ACCTTATTTCTCCTGCCCCTTG-3'; reverse: 5 * -AAGCCCAGCCTCATTCATTACAG- 3'. The nucleic acid sequence of RNASE4, including variants identified in this sequencing analysis, is provided in Fig. 1.
  • RNASE4 is angiogenic
  • WT RNASE4 protein and the enzymatically attenuated K40A variant were prepared.
  • K40 was chosen because it is an essential residue constituting the catalytic triad that is conserved across the members of the RNASE superfamily. Mutations at K40 diminish most of the catalytic activity of this family of proteins.
  • WT RNASE 4 gene was cloned from genomic DNA of HeLa cells with the following PCR primers: Forward, 5'- GGAGAT ATC AT ATGC AGGATGGC ATGT AC-3 ' ; Reverse,
  • PCR products were cloned into a pETl la vector between the BamHl and Ndel sites.
  • the resultant plasmid (pETl la-hRNASE4) was sequence confirmed and transformed into BL21 (DE3) cells for protein expression.
  • K40A mutation was generated by QuickChange II Site-Directed Mutagenesis Kit from Stratagene using pETl la-hRNASE4 as the template and the following primers: Forward, 5'- CTTTGTATC ACTGCgcGCGCTTCAACACCTT-3 ' ; Reverse, 5'- AAGGTGTTGAAGCGCgcCCAGTGATACAAAG-3 ' .
  • the resultant plasmid (pETl lcc- hR ASE4-K40A) was sequenced confirmed and cloned into BL21 (DE3) cells.
  • RNASE Ribonucleolytic activities of WT and K40A R ASE4 were examined using yeast. Reactions were initiated by adding the enzyme. Varying amount RNASE were added to a final volume of 300 ⁇ , including 600 ng yeast tRNA, 0.33 M Hepes, 0.33 M NaCl, pH 7.0, and 0.1 mg/ml RNASE-free BSA.
  • RNASE4 had reduced ribonucleolytic activity compared to the wild-type protein. Specifically, the K40A RNASE4 variant retains 5.4% of the activity of WT RNASE4 when yeast tRNA was used as a substrate (Fig. 2B).
  • RNASE4 protein was treated with diethylpyrocarbonate (DEPC), which chemically modifies Lys residues and
  • RNASE4 completely abolished the enzymatic activity of ribonucleases.
  • DEPC-treated RNASE4 fails to cleave both yeast tRNA (Fig. 3A) and HeLa rRNA (Fig. 3B), even with prolonged incubation, indicating that DEPC treatment has completely abolished the enzymatic activity of RNASE4.
  • Fig. 3C shows that DEPC treatment also completely abolished the ability of RNASE4 to induce endothelial tubule formation.
  • RNASE4 ribonucleolytic activity of RNASE4 is essential for angiogenesis.
  • RNASE4 WT and K40A RNASE4 to induce endothelial cell tube formation was analyzed.
  • Each well of the 48-well plate was coated with 50 ⁇ growth factor-reduced Matrigel.
  • HUVEC Cells were seeded on the Matrigel at a density of 1.5 x 10 4 cells per well and cultured in the presence or absence wild-type RNASE4 protein, K40A RNASE4 protein or ANG for 4 h. Cells were fixed in 3.7% paraformaldehyde and photographed. The images were analyzed by ImageJ software to calculate the length or area of capillary-like structures as well as the numbers of circled tubular structure. Experiments were done in triplicate and were repeated at least three times.
  • RNASE4 had about 50% of the activity of ANG in this assay.
  • WT RNASE4 at 1 ⁇ g/ml and ANG at 0.5 ⁇ g/ml had an equivalent activity (Fig. 4B).
  • the K40A variant of RNASE4 actually had a higher angiogenic activity than the WT RNASE4 and is only slightly lower than ANG.
  • No endotoxin was detected in the preparation. Heat inactivation or proteolysis abolished all the activity, indicating that an intact peptide and structure is important.
  • ECP/RNASE3 were detected in the same assay system (Fig. 5).
  • An ex vivo angiogenesis assay was performed using mouse aortic ring explant cultures. Thoracic aortic vessels from 2 month old C57BL/6J mice were dissected and transferred to a dish containing ice-cold MEM. The fibroadipose tissues were removed with microdissecting forceps and scissors. The aorta were cut into 0.5 mm pieces and washed extensively in cold PBS, and placed in Matrigel-coated 48-well plates. Each well was covered with another 50 ⁇ Matrigel and incubated in human endothelial SMF basal growth medium in the presence or absence of testing materials for 15 days. New angiogenic sprouts were stained with 20 ⁇ 1 MTT (5 mg/ml in PBS) for 2 h, photographed and analyzed by ImageJ.
  • RNASE4 elicits an angiogenic response in mouse aorta explant culture.
  • the K40A variant has a higher activity than the WT RNASE4, indicating that a robust ribonucleolytic activity is not necessary for RNASE4 to induce angiogenesis.
  • WT RNASE4 has a slightly lower angiogenic activity as compared with ANG, the activity of K40A RNASE4 variant and ANG are comparable.
  • RNASE4 and its K40A variant are active in in vitro, ex vivo and in vivo angiogenesis assays. Taken together, these results demonstrate that human RNASE4 is angiogenic.
  • RNASE4 stimulates neuronal differentiation and neurite outgrowth
  • RNASE4 The neurogenic activity of RNASE4 examined in P19 mouse embryonal carcinoma cells.
  • PI 9 cells are pluripotent and have stem cell-like property. These cells are able to both self-renew and differentiate into various types of neural cells.
  • P19 cell were seeded on PA6 monolayer and cultured in a-MEM plus 0.1% nonessential amino acids, 0.1% knockout serum replacement, 0.5 ⁇ retinoic acid, and 200 ng/ml testing proteins for 24 h.
  • Embryonal bodies were counted under phase light microscope at 10 X magnification. The numbers of embryonal bodies from the entire well were counted. Experiments were done in triplicate and were repeated three times.
  • ANG and RNASE4 induced the formation of neurospheres or embroynal bodies (Fig. 8A).
  • the number of these neurospheres in a 35 mm dish after 24h incubation was 218 ⁇ 9 and 261 ⁇ 64, respectively, in the presence of RNASE4 and ANG, representing a 7.3- and 8.7-fold increase over that formed in the presence of control protein BSA (30 ⁇ 14) (Fig. 8B).
  • RNASE4 and ANG representing a 7.3- and 8.7-fold increase over that formed in the presence of control protein BSA (30 ⁇ 14)
  • RNASE4 RNASE4 on neurite outgrowth
  • cells were cultures as described above but for 216 h.
  • Cells were fixed successively in 4% paraformaldehyde for 15 min at RT and cold methanol for 5 min at -20 °C, blocked with 0.1% gelatin, 0.5% BSA and 0.1% Tween 20 in TBS or 1 h at RT.
  • Neurofilaments were stained by a rabbit anti- Neurofilament medium chain at 1 :500 dilution at 4°C overnight and Alexa 488-labeled goat anti-rabbit F(ab') at 1 :400 dilution for 1 h at RT.
  • RNASE4 and ANG were able to stimulate neurite outgrowth in a prolonged culture of P19 cell (Fig. 8C). After 9 days culture, the average length of neurofilaments in the presence of RNASE4 and ANG was 20.2 ⁇ 0.8 and 20.7 ⁇ 1.4 ⁇ , respectively, which is 3.0- and 3.1 -fold higher than that in the presence of BSA (6.7 ⁇ 1.2 ⁇ ) (Fig. 8D).
  • ANG and RNASE4 are also able to stimulate neurofilament growth of primary neurons.
  • Brain cortical cells were isolated from C57/B6SJL embryos (E14) and seeded in 6 wells at a density of 1.1 x 10 6 per cm 2 in neurobasal medium plus B27 and 0.5 mM L- glutamine for 36 h. The medium was changed and 10 ⁇ Ara-c was added and cultured for another 6 days with medium change every two days. Ara-c was removed and the cells were cultured in neurobasal medium plus B27 in the presence or absence of testing material for additional 6 days. For hypothermia-induced degeneration, the cells were put at RT temperature for 40 min and returned to the cell culture incubator for additional 3 h.
  • Neurofilaments were stained as described above.
  • FIG. 9A mouse cortical neurons isolated from E14 embryos were able to survive for 12 days in neurobasal medium, but very little neurofilament growth was noted when only BSA was present.
  • B27 a serum-free supplement for growth and long-term viability of primary neurons, an extensive network of neurofilaments was formed.
  • a similar network of neurofilaments was formed in the presence of RNASE4 and ANG.
  • the length of neurofilaments formed in the presence of BSA, B27, RNASE4, and ANG were 295 ⁇ 116, 903 ⁇ 180, 804 ⁇ 135, and 962 ⁇ 122 ⁇ , respectively (Fig. 9B).
  • RNASE4 in stimulating neurofilament growth from the primary culture of the mouse embryonic cortical neuron is comparable to or greater than that of the B27 positive control.
  • RNASE4 The neural differentiation activity of RNASE4 was further examined by looking at e its ability to induce differentiation of mouse ES cells.
  • Fig. 10 shows that both RNASE4 and ANG induced differentiation of mouse ES cells into GFAP -positive progenitor neurons.
  • the length of neurofilaments in the presence of ANG and RNASE4 was 38.8 ⁇ 2.3 and 36.8 ⁇ 0.7 ⁇ , respectively, which is 3.1- and 3.0-fold higher than that in the negative control (BSA: 12.3 ⁇ 0.7), and is 55% and 52% of that in the positive control (Retinoic acid: 70.5 ⁇ 1.5).
  • RNASE4 undergoes nuclear translocation similarly to ANG.
  • RNASE4 protects stress-induced neuronal degeneration
  • RNASE4 possesses neuroprotective activity
  • effect in preventing hypothermia-induced fragmentation of neurofilament derived from mouse embryonic cortical neuron culture was examined.
  • Figure 9 a robust network of neurofilaments formed when mouse embryonic cortical neurons were cultured in the presence of B27. These neurofilaments fragmented into small pieces after being subjected to 25 °C for 40 min in the presence of control protein BSA (Fig. 12A).
  • both RNASE4 or ANG prevented hypothermia-induced neurofilament fragmentation, indicating that both are neuroprotective.
  • RNASE4 and ANG have comparable neuroprotective activity. This is also the first report that ANG prevents stress-induced degeneration of primary neurons in culture.
  • RNASE4 and ANG were compared to serum withdrawal-induced neuronal degeneration of P19-derived neurofilaments.
  • Fig. 12C retinoic acid-induced P19 neurofilaments underwent fragmentation when subjected to serum starvation in the presence of BSA.
  • RNASE4 or ANG no significant fragmentation was observed with the same treatment but in the presence of RNASE4 or ANG.
  • ImageJ analyses indicated that the length of P19 cell-derived neurofilaments after 1 h serum starvation in the presence of BSA, RNASE4, and ANG was 4.2 ⁇ 0.4, 9.7 ⁇ 0.7, and 12.6 ⁇ 1.3 ⁇ , respectively.
  • RNASE4 had no effect on PA6 cell proliferation (Fig. 13).
  • Example 5 RNASE4 slows weight loss and enhances neuromuscular function in a mouse model for ALS
  • RNASE4 The beneficial effect of exogenous RNASE4 on S0D1 G93A mice was examined.
  • ISH in situ hybridization
  • Human RNASE4 and mouse Rnase4 DNA were amplified by PCR from HeLa cell DNA and C57B6/SJL mouse tail genomic DNA as templates. The primers were as follows: Human RNASE4, forward, 5'-
  • cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 ⁇ g of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer's protocol .
  • Control cRNA probes were generated from the control DNA template included in the kit.
  • the slides were deparaffmized with Xylene (2 x 10 min), washed in sequence with 100, 75, 50, and 25% ethanol and PBS each for 5min.
  • the slides were then treated with 2 ⁇ g/ml of Proteinase K for 10 min, washed with 2 mg/ml of Lysine and PBS each for 5min, and incubated in acetylation buffer (0.25% acetic acid anhydride in 0.1 M Triethanolamine, pH 8.0) for 20 min. After two washes with 4 x SSC, the slides were prehybridized in
  • Hybridization buffer (5 x SSC, 0.5 mg/ml heparin, 0.8 mg/ml salmon sperm DNA) at 45 °C for 60 min. cRNA probes were added onto the slides and incubated at 45 °C overnight. Hybridization signals were detected and visualized with the digoxigenin Nucleic Acid Detection Kit from Roche following manufacturer's protocol.
  • RNASE4 mRNA in motor neurons was observed in non-ALS spinal cord, but was significantly decreased in ALS spinal cords (Fig. 14A). Quantitative analysis of the ISH images indicated that the total photon counts of RNASE4 mRNA staining per motor neurons of ALS and non-ALS spinal cord were 2.98 ⁇ 0.71 x 10 4 and 9.67 ⁇ 1.21 x 10 4 , respectively, indicating that RNASE4 expression decreased by 69%> in ALS.
  • the mRNA level of mouse Rnase4 is also decreased in the spinal cord motor neurons of S0D1 G93A mice as compared to that of WT mice (Fig. 14B). The photon counts per spinal cord motor neuron in WT and S0D1 G93A mice were 6.26 ⁇ 0.62 x 10 3 and
  • RNASE4 protein The therapeutic activity of RNASE4 protein in ALS treatment was examined.
  • mice ⁇ at 1 1 weeks of age were treated with weekly i.p. injection of PBS or RNASE4 protein at 10 ⁇ g per mouse.
  • the neuromuscular function of the mice was examined by their performance in a rota-rod assay.
  • weekly treatment of RNASE4 increased the time the mice could stay on a rotarod (20 rpm) from week 14 to week 18 to various degrees.
  • the greatest difference was recorded at week 15, when the R ASE4-treated mice were able to stay on the rotarod for 333 ⁇ 106 sec, whereas PBS- treated animals were able to stay on it for only 181 ⁇ 68 sec.
  • R ASE4 treatment also decreased the rate of body weight decrease (Fig. 15B).

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Abstract

Described herein are methods for the prevention of neurodegeneration and the treatment of neurodegenerative disease through the administration of an RNASE4 protein, biologically active fragments thereof or an agent that enhances the activity or expression of RNASE4. Also described herein are methods for identifying candidate therapeutic agents for treating or preventing neurodegenerative disease and for identifying a subject as having an increased risk of neurodegenerative disease.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF
NEURODEGENERATIVE DISEASE RELATED APPLICATIONS
This application claims the benefit of priority to United States Provisional Patent Application serial number 61/586,277, filed January 13, 2012, which is hereby incorporated by reference in its entirety.
BACKGROUND
Neurodegenerative diseases are characterized by a progressive neurodegenerative process in which neuron structure and/or function is lost over time. Among the most common and/or most severe neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease and Huntington's disease. Though genetic characteristics have been linked with some neurodegenerative diseases, such as the
Huntingtin gene in Huntington's disease, for most neurodegenerative diseases the cause remains unclear. What is more, effective treatments have proven elusive for nearly all forms of neurodegenerative disease.
ALS (also known as Lou Gehrig's disease), is a neurodegenerative disease characterized by the progressive degradation of motor neurons, which causes the afflicted individual to experience progressive weakness, muscle atrophy and respiratory
compromise. ALS is always fatal, with the median survival time from symptom onset of ALS being about 20 to 48 months. However, the rate of disease progression can vary greatly, with some patients dying within a year of diagnosis and others surviving for many years.
The cause of ALS remains unknown. Mutation to the gene that encodes superoxide dismutase 1 (SOD1) is present in approximately 20% of familial ALS cases and in approximately 5% of cases of non-familial ALS. There is therefore no definitive test for ALS, and most ALS diagnoses are made based on the progression of physical disease symptoms, as observed by a physician. Currently, ALS has no cure and the only FDA approved treatment for ALS, the sodium channel blocker Riluzole, only increases patient survival by 3-5 months on average.
Thus, there is a need for new and improved compositions and methods for the treatment, prognosis and diagnosis of neurodegenerative diseases, including ALS. SUMMARY
Described herein are methods of reducing neurodegeneration and/or treating or preventing a neurodegenerative disease in a subject (e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) and/or progressive bulbar palsy (PBP)). In some embodiments the methods include administering to the subject an
RNASE4 protein or a biologically active fragment thereof. In some embodiments the methods include administering to the subject an agent that enhances the activity and/or expression of RNASE4.
In some embodiments, an agent that comprises an RNAS4 protein is administered to the subject. In certain embodiments, the RNASE4 protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments the RNASE4 protein has an amino acid sequence at least 90%> identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40. In certain embodiments the RNASE4 protein is a variant RNASE4 protein that has less than 10% of the ribonucleo lytic activity of a wild- type RNASE4 protein having a sequence of SEQ ID NO: 1.
In some embodiments, an agent that enhances the activity and/or expression of RNASE4 is administered to the subject. In some embodiments, the agent is a small molecule, a nucleic acid or a polypeptide. In some embodiments, the agent increases the expression of both RNASE4 and ANG. In certain embodiments the agent enhances expression of RNASE4 by contacting the ANG/RNASE4 promoter.
In some embodiments, the method described herein also includes administering to the subject a second agent for the treatment of neurodegenerative disease. In some embodiments the second agent is an ANG protein or biologically active fragment thereof. In some embodiments, the second agent is an agent that increases the activity and/or expression of ANG.
In certain embodiments the subject has or is susceptible to a neurodegenerative disease (e.g. ALS). For example, in some embodiments the genome of the subject comprises an T allele at single nucleotide polymorphism rs37484338. In certain embodiments the subject has reduced expression or activity of RNASE4. In some embodiments the subject has been identified as having an increased risk of
neurodegenerative disease. In some embodiments, the subject has a mutated SODl gene. Also described herein are methods of identifying a subject as having increased risk of neurodegenerative disease (e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP). In some embodiments the methods include the step of detecting in a biological sample from the subject a mutation to a
R ASE4 coding region. In general, the presence of a mutation to the RNASE4 coding region identifies the subject as having an increased risk of neurodegenerative disease. In some embodiments the mutation to the RNASE4 coding causes reduced RNASE4 expression or activity in the subject. In some embodiments the mutated coding region encodes an RNASE4 signal peptide that comprises a serine at signal peptide position -13.
In certain embodiments the mutated RNASE4 coding region comprises a thymidine at position -39.
Also described herein is an RNASE4 protein that includes an amino acid sequence at least 90% identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40. In some embodiments, the amino acid sequence has an alanine at position 40. In some embodiments, the RNASE4 protein is identical to SEQ ID NO: l , except that the amino acid sequence does not have a lysine at position 40. In certain embodiments the amino acid sequence is identical to SEQ ID NO:6. Also described herein are nucleic acid sequences encoding such RNASE4 proteins.
Also described herein are methods for determining whether a test agent is a candidate therapeutic agent for treating or preventing a neurodegenerative disease. In certain embodiments, the methods include the steps of contacting a cell with the test agent and detecting the expression of RNASE4 by the cell. In general, a test agent that increases the expression of RNASE4 is a candidate therapeutic agent for treating or preventing a neurodegenerative disease. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleic acid sequence of human RNASE4. The first nucleotide codon encoding the first amino acid in the mature protein is numbered 1. The numbering in the signal peptide region starts at the last nucleotide of the codon ending the last amino acid of the signal peptide and goes backward upstream. The nucleotide changes identified from the sequencing study described herein are given in parentheses.
Figure 2 shows the ribonucleolytic activity of recombinant wide type RNASE4 and the K40A variant. (A) SDS-PAGE and Coomassie blue staining of WT and K40A RNASE4 from one of the four preparations. Recombinant ANG protein was included as a control. (B) Ribonuclolysis of yeast tR A by WT R ASE4 (squares) and K40A RNASE4 (circles). Data shown are mean ± SEM of four independent preparations with triplicates in each enzyme concentration.
Figure 3 that the ribonucleolytic activity of RNASE4 is essential for its angiogenic activity. (A) RNASE4 protein was treated with 5 mM DEPC and its ribonucleolytic activity was examined using a yeast tRNA assay. (B) DEPC-treated RNASE4 fails to cleave rRNA. (C) DEPC treatment abolishes angiogenic activity of RNASE4.
Figure 4 shows that RNASE4 induces endothelial cell tube formation. (A)
Microscopic images of endothelial cell tubes formed in various concentration of WT and K40A RNASE4. ANG and PBS were used as positive and negative controls, respectively. The images shown are representative of at least four sections from three independent experiments. Bar, 0.1 mm. (B) Number of circled tubular structures per mm2. Values are mean ± SEM of three independent experiments. Statistical analysis was performed by two- way ANOVA. *, p<0.05; **, p<0.01.
Figure 5 shows the angiogenic activities of various ribonucleases. (A) Microscopic images of endothelial cell tubes formed in the presence of 1 μg/ml of the indicated ribonuclease, Bar, 0.1 mm. (B) Number of circled tubular structures per mm2.
Figure 6 shows that RNASE4 stimulates endothelial sprouts from mouse aortic explants. (A) Outward growth of endothelial sprouts from aortic rings that have been flipped inside out. The images are representative of three rings from one of three repeats. Bar, 0.1 mm. ANG and PBS were used as positive and negative controls, respectively. (B) Image J analysis of the area covered by endothelial sprouts from flipped aortic rings. Data are presented as mean ± SEM from three independent experiments. (C) Inward growth of endothelial sprouts from unflipped mouse aortic rings. The images are representative of three rings from one of two repeats. (D) ImageJ analysis the area covered by endothelial sprouts from unflipped aortic rings. Data are presented as mean ± SEM from two independent experiments. Bar, 1.0 mm. Statistical analysis was performed by two-way ANOVA. *, p<0.001.
Figure 7 shows that RNASE4 induces neovessel growth into Matrigel plug implanted under mouse skin. (A) IHC staining with vWF antibodies. The images shown are representative of at least four sections from three independent experiments. (B) ImageJ analysis of vWF-positive spots. Data are presented as mean ± SEM from three independent experiments. Bar, 100 μιη. Statistical analysis was performed by two-way ANOVA. *, pO.001.
Figure 8 shows the effect of R ASE4 on PI 9 cells. (A) RNASE4 stimulates neurosphere formation of PI 9 cells. PI 9 cells were cultured on PA6 supporting cell layers in the presence of 0.2 μg/ml of BSA, R ASE4, or ANG for 24 h. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. (B) Numbers of neurosphere counted from the entire 35-mm dish. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. *, p<0.001. (C) Cells were cultured as described in A for 216 h and stained for neurofilaments with an anti-neurofilament medium chain IgG. The images are representative of at least four areas from three independent experiments. Bar, 20 μιη (D) Image J analysis of neurofilament length from C. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p<2xl0"9.
Figure 9 shows the effect of RNASE4 on mouse embryonic cortical neurons. (A)
Cortical neurons were isolated from E14 mouse embryos and cultured in neurobasal medium in the presence of 0.2 μg/ml RNASE4 or ANG for 12 days with a medium change on day 6. B27 was used as a positive control and BSA at 0.2 μg/ml was used as a negative control. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. (B) ImageJ analysis of the length of the neurofilaments. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p<0.005.
Figure 10 shows that RNASE4 stimulates mouse embryonic stem cell
differentiation. (A) Mouse ES cells were induced by 0.5 mM retinoic acid, 0.2 μg/ml ANG, or 0.2 μg/ml RNASE4 for 6 days. The top panel is the morphology of differentiated progenitor cells derived from the embryoid bodies. Bar, 15 μιη. The bottom panel is GFAP- positive neuroprogenitor cells. Bar, 10 μιη. (B) ImageJ analysis of the length GFP-positive neurofilaments. *, p<0.01. **, p<0.001.
Figure 11 shows the subcellular localization of RNASE4 in HUVE and P19 cells. (A) HUVE cells were incubated in the absence (top panel) or presence (bottom panel) of 0.5 μg/ml exogenous RNASE4 protein at 37 °C for 1 h. (B) P19 cells were incubated in the absence (top panel) or presence (bottom panel) of 1 μ^ιηΐ exogenous RNASE4 protein at 37 °C for 1 h. Immunofluorescence was carried out with affinity-purified RNASE4 polyclonal rabbit IgG and Alexa 488-labeld goat anti-rabbit IgG. Nuclei were stained with DAPI. Scale bar, 10 μηι.
Figure 12 shows that RNASE4 protects stress-induced neuron degeneration. (A) Effect on hypothermia-induced neurofilament fragmentation of mouse cortical neurons. Mouse cortical neurons were cultured in the presence of B27 for 12 days. Cells were washed with neurobasal medium, incubated with 0.2 μg/ml RNASE4 or ANG at 37 °C for 1 hour, and then subjected to hypothermia treatment at 25 °C for 40 min. Cells were returned to incubator and continually cultured for 3 h and stained for neurofilaments. The images are representative of at least four areas from three independent experiments. Bar, 0.4 mm. (B) Image J analysis of the length of neurofilament. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p<0.006. (C) Effect of serum starvation-induced degeneration of P19 neurofilaments. PI 9 cells were cultured on PA6 supporting cell layers in the presence of 0.5 μΜ retinoic acid for 216 h, subjected to serum starvation for 1 h in the absence or presence of 0.2 μg/ml RNASE4 or ANG, and stained with neurofilament antibody. The images are representative of at least four areas from three independent experiments. Bar, 10 μιη (D) ImageJ analysis of the length of neurofilament. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA.**, p<3xl0~6.
Figure 13 shows that RNASE4 has no effect on PA6 cell proliferation. PA6 cells were cultured in a-MEM containing 0.1% NEAA and 0.1% NKSR in the presence of 0.2 μg/ml BSA or RNASE4 with or without 0.5 μΜ retinoic acid. Cell proliferation was determined by MTT assay.
Figure 14 shows decreased RNASE4 mRNA level in the spinal cord motor neurons of ALS patients and in S0D1G93A mice. (A) In situ hybridization of human RNASE4 mRNA in the spinal cord of ALS patients and non-ALS control subject. Left panel, representative ISH images from one of the six patients. Bar, 10 μιη. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA. (B) In situ hybridization of mouse Rnase4 mRNA in the spinal cord of WT and SODIG93A mice. Left panel, representative ISH images from one of the six mice. Bar, 10 μιη. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA. Figure 15 shows the effect of RNASE4 on SODl^yiA mice. Starting from 11 weeks of age, mice were treated with weekly i.p. injection of WT RNASE4 protein at 10 μg per mouse. Three independent experiments were performed with a total of 34 and 31 mice in the RNASE4 treatment group (triangles) and PBS control group (triangles), respectively. (A) Effect on rotarod performance at 20 rpm without revolving. Two successive measurements were recorded. An upper limit of 1,000 second was used. (B) Effect on body weight. Data shown are means ± SEM of all survived animals at each data point. Statistical analysis was performed by two-way ANOVA.*, p<0.01.
Figure 16 provides exemplary amino acid sequences of RNASE4 proteins. SEQ ID NO: 1 is the wild-type RNASE4 protein sequence. SEQ ID NO: 7 is the variant K40A RNASE4 protein sequence. Sequences are for the mature RNASE4 protein and do not include the signal sequence.
Figure 17 provides exemplary nucleic acid sequences that encode for RNASE4 proteins. SEQ ID NO: 7 encodes the wild-type RNASE4 protein. SEQ ID NO: 13 encodes for the variant K40A RNASE4 protein. Sequences encode the mature RNASE4 protein and do not include the signal sequence.
DETAILED DESCRIPTION
General
Provided herein are compositions and methods for reducing neurodegeneration and treating neurodegenerative diseases, such as ALS. Also provided herein are compositions and methods for identifying subjects at elevated risk of neurodegenerative disease and for identifying agents useful in the treatment of such diseases.
Secreted ribonucleases (RNASEs) are a vertebrate-specific enzyme family that is composed of 13 distinct paralogs. Among the 13 members of this superfamily, RNASE4 is the most conserved gene across the different vertebrate species, with an amino acid homology as high as 94%. RNASE4 has a very strict substrate specificity and generally cleaves RNA immediately 5' of uridine residues.
At the protein level, human ANG and RNASE4 have 38.7% identity. ANG and RNASE4 genes are located in the same locus and share the same promoters. The ANG1 and RNASE4 genes contain two non-coding exons followed by two distinct exons encoding ANG1 and RNASE4, respectively. The two non-coding exons are preceded by two promoters that control tissue-specific expression of both genes. As described herein, when the coding exon (exon IV) of RNASE4 was sequenced for potential ALS-associated mutations and SNPs, SNP rs3748338 A/T(-39), which results in an amino acid change from Thr to Ser at position -13 of the RNASE4 signal peptide, showed a significant association with ALS (p=0.042).
Also described herein is a variant RNASE4 protein that lacks a lysine at amino acid position 40. Because the lysine at position 40 is part of the catalytic triad that is critical for RNA cleavage of RNASE4, this RNASE4 protein variant has significantly reduced RNA cleaving activity compared to the wild-type RNASE4 protein. As is described herein, both wild-type RNASE4 protein and the variant RNASE4 protein are angiogenic, neurogenic and neuroprotective. Notably, the variant RNASE4 protein actually has higher angiogenic, neurogenic and neuroprotective activity than the wild-type protein, despite having greatly reduced RNASE activity.
Thus, as described herein, administration of RNASE protein (either wild-type or variant protein) is useful in the prevention of neurodegeneration and the treatment of neurodegenerative disease. Indeed, administration of RNASE4 protein in an ALS disease model slowed weight loss and preserved neuromuscular function in the test animal.
RNASE4 protein and agents that increase RNASE4 activity and/or expression are therefore promising therapeutic candidates for the treatment of neurodegenerative disease, and individuals with reduced RNASE4 activity and/or expression have an increased risk of developing a neurodegenerative condition.
Definitions
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "administering" means providing a pharmaceutical agent {e.g., an RNASE4 protein or an agent that increases RNASE4 activity and/or expression) or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
The terms "agent" is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds or a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide). Agents may be identified as having a particular activity {e.g., an RNASE4 enhancing activity) by screening assays described herein below. The activity of such agents may render them suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
The term "binding" or "interacting" refers to an association, which may be a stable association, between two molecules, e.g. , between a polypeptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
"Biologically active portion ofRNASE4" refers to a portion of R ASE4 protein having a biological activity, such as the ability to induce angiogenesis or inhibit
neurodegeneration.
The term "biological sample," "tissue sample," or simply "sample" each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.
The term "control" includes any portion of an experimental system designed to demonstrate that the factor being tested is responsible for the observed effect, and is therefore useful to isolate and quantify the effect of one variable on a system.
The term "isolated polypeptide" refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
The term "isolated nucleic acid" refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid" is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
The terms "polynucleotide", and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non- limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. The term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
A "patient" or "subject" refers to either a human or a non-human animal.
The term "percent identical" refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue {e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FAST A, BLAST, or ENTREZ. FAST A and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term "pharmaceutically-acceptable salts" is art-recognized and refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions described herein.
The term "RNASE4-activating compound" or "agent that increases RNASE4 activity" refers to an agent that increases the level of RNASE4 protein and/or increases at least one activity of a RNASE4 protein besides RNA cleavage. In an exemplary
embodiment, a RNASE4-activating compound may increase at least one biological activity of a RNASE4 protein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplary biological activities of RNASE4 proteins include promotion of angiogenesis and inhibition of neurodegeneration.
A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides). The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI). A SNP allele can be describe based on the sequence of its forward strand or the sequence of its reverse strand. For example, a SNP that has either A or G alleles on its forward strand will have either T or C alleles, respectively, on its reverse strand. The SNP alleles are described herein according to their forward strand sequence.
The phrases "therapeutically-effective amount" and "effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. "Treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
RNASE 4 Proteins
As used herein, the term "RNASE4 protein" refers to a protein with substantial sequence homology to wild-type RNASE4 (e.g. a protein having the amino acid sequence of SEQ ID NO: 1) that retains at least one activity associated with wild-type RNASE4 (e.g., promotion of angiogenesis and/or inhibition of neurodegeneration). RNASE4 proteins can be wild-type RNASE4 proteins (e.g. proteins having a sequence of SEQ ID NO: 1) or can be variant RNASE4 proteins (e.g. proteins having an amino acid sequence at least 50%,
60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1). RNASE4 proteins do not need to have RNA cleaving activity.
Examples of RNASE4 proteins include proteins having amino acid sequences of SEQ ID NO: 1-6. Variants of RNASE4 proteins can be produced by standard means, including site- directed and random mutagenesis .
As used herein, a "biologically active fragment" of a RNASE4 protein is a polypeptide that retains at least one activity of wild-type RNAS4 protein (e.g., promotion of angiogenesis and/or inhibition of neurodegeneration) and that comprises at least 8 consecutive amino acids, (e.g., at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 amino acids) of SEQ ID NO: 1.
In some embodiments, the RNASE4 protein is a RNASE4 protein that does not have a lysine at position 40 (e.g., such as a RNASE4 protein having an amino acid sequence of SEQ ID NO: 6). In some embodiments the RNASE4 protein is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQID NO: 6 and does not have a lysine at position 40. In certain embodiments, the RNASE4 proteins described herein have an alanine instead of a lysine at position 40.
The lysine at position 40 is part of RNASE4's catalytic triad and plays a role in RNASE4's RNA cleaving activity. Variant RNASE4 proteins that lack a lysine at position 40 therefore have reduced RNASE activity compared to wild-type RNASE4 protein. Thus, in some embodiments, the variant RNASE4 proteins described herein have less than 50%, 40%, 30%, 20%, or 10% of the RNA cleaving activity of wild-type RNASE4 protein.
However, RNASE4 proteins that lack a lysine at position 40 have elevated angiogenic and neuroprotective activities compared to wild-type RNAS 4 protein. Thus, RNASE4 proteins that lack a lysine at position 40 are particularly useful, for example, for the treatment of neurodegenerative diseases and for the prevention of neurodegeneration.
Also described herein are R ASE4 proteins that comprise "conservative sequence modifications" of the sequences set forth in SEQ ID NO: 1-6. Such conservative sequence modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence set forth in the figures by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains {e.g., lysine, arginine, histidine), acidic side chains {e.g., aspartic acid, glutamic acid), uncharged polar side chains {e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains {e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains {e.g., threonine, valine, isoleucine) and aromatic side chains {e.g., tyrosine, phenylalanine, tryptophan, histidine).
In certain embodiments, a protein described herein is further linked to a
heterologous polypeptide, e.g. , a polypeptide comprising a domain which increases its solubility and/or facilitates its purification, identification, detection, and/or structural characterization. A protein described herein may be linked to at least 2, 3, 4, 5, or more heterologous polypeptides. Polypeptides may be linked to multiple copies of the same heterologous polypeptide or may be linked to two or more heterologous polypeptides. The fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a protein described herein and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein.
In another embodiment, a protein may be modified so that its rate of traversing the cellular membrane is increased. For example, the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells. The peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment
corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989) Cell 55: 1179-1188). Alternatively, the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof. The 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. Thus, the polypeptide may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al. (1996) J Biol Chem 271 : 18188-18193; Derossi et al. (1994) J Biol Chem 269: 10444-10450; and Perez et al. (1992) J Cell Sci 102:717-722). The transcytosis polypeptide may also be a non-naturally- occurring membrane-translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Patent No. 6,248,558.
The amino acid sequences of RNASE4 proteins {e.g., acid sequences of SEQ ID NO: 1-6) will enable those of skill in the art to produce RNAS4 proteins and biologically active fragments thereof. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding RNASE4 protein or biologically active fragment thereof. Alternatively, such polypeptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif; Merrifield, J. (1969) J. Am. Chem. Soc. 91 :501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11 :255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference). RNASE 4 Nucleic Acids
Nucleic acids encoding any of the polypeptides described herein {e.g., RNASE4 proteins or biologically active fragments thereof) are also provided herein. A nucleic acid may further be linked to a promoter and/or other regulatory sequences, as further described herein. Exemplary nucleic acids are those that are at least about 80%, 85%, 90%>, 95%>, 98%, 99% or 100% identical to SEQ ID NO : 7- 13 or a fragment thereof. Nucleic acids may also hybridize specifically, e.g., under stringent hybridization conditions, to a nucleic acid described herein or a fragment thereof. There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
GENETIC CODE
Alanine (Ala, A) GCA, GCC, GCG, GCT
Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT
Asparagine (Asn, N) AAC, AAT
Aspartic acid (Asp, D) GAC, GAT
Cysteine (Cys, C) TGC, TGT
Glutamic acid (Glu, E) GAA, GAG
Glutamine (Gin, Q) CAA, CAG
Glycine (Gly, G) GGA, GGC, GGG, GGT
Histidine (His, H) CAC, CAT
Isoleucine (He, I) ATA, ATC, ATT
Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG
Lysine (Lys, K) AAA, AAG
Methionine (Met, M) ATG
Phenylalanine (Phe, F) TTC, TTT
Proline (Pro, P) CCA, CCC, CCG, CCT
Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT
Threonine (Thr, T) ACA, ACC, ACG, ACT
Tryptophan (Trp, W) TGG
Tyrosine (Tyr, Y) TAC, TAT
Valine (Val, V) GTA, GTC, GTG, GTT
Termination signal (end) TAA, TAG, TGA
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid. Thus, in some embodiments, the nucleic acid molecules described herein may differ from nucleotide sequences of SEQ ID NO: 7-13 due to degeneracy of the genetic code and still encode the same polypeptides as those encoded by SEQ ID NO: 7-13.
The nucleic acid molecules described herein can be isolated using standard molecular biology techniques and the sequence information provided herein (e.g., SEQ ID NO: 7-13). For example, a nucleic acid molecule encompassing all or a portion of sequences shown in SEQ ID NO: 7-13 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequences provided in SEQ ID NO: 7-13. Oligonucleotides corresponding to nucleic acid sequences described herein can be prepared, for example, by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
In some embodiments, the nucleic acid sequences described herein hybridize under stringent conditions to a nucleic acid of SEQ ID NO: 7-13. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11.
Nucleic acids, e.g., those encoding an RNASE4 protein or biologically active fragment thereof, can be delivered to cells in culture, ex vivo, and in vivo. The cells can be of any type including without limitation neuronal cells, myocytes, and non-neuronal cells. The delivery of nucleic acids can be by any technique known in the art including viral mediated gene transfer, liposome mediated gene transfer, direct injection into a target tissue or organ, injection into vasculature which supplies a target tissue or organ. Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
A polynucleotide of interest can also be combined with a condensing agent to form a gene delivery vehicle. The condensing agent may be a polycation, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Many suitable methods for making such linkages are known in the art.
In an alternative embodiment, a polynucleotide of interest is associated with a liposome to form a gene delivery vehicle. Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter. Under appropriate conditions, a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm. Prior to interaction with the surface of a cell, however, the liposome membrane acts as a relatively impermeable barrier that sequesters and protects its contents, for example, from degradative enzymes. Additionally, because a liposome is a synthetic structure, specially designed liposomes can be produced which incorporate desirable features. See Stryker, Biochemistry, pp. 236-240, 1975 (W.H. Freeman, San Francisco, CA); Soak et al, Biochip. Biopsy's. Acta 600: 1, 1980; Bayer et al, Biochip. Biopsy's. Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol. 149: 119, 1987; Wang et al, PROC. NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et al, Anal. Biochem. 176:420, 1989, and U.S. Patent 4,762,915. Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and expression constructs comprising growth factor polynucleotides such those disclosed in the present invention.
Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987), niRNA (Malone et al, Proc. Natl. Acad. Sci. USA 86:6077-6081, 1989), and purified transcription factors (Debs et al, J. Biol. Chem.
265: 10189-10192, 1990), in functional form. Cationic liposomes are readily available. For example, N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. See also Feigner et al, Proc. Natl. Acad. Sci. USA 91 : 5148-5152.87, 1994. Other commercially available liposomes include Transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Soak et al, Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions of the synthesis of DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG),
dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
More than one polypeptides may be encoded by a single nucleic acid. In certain embodiments, the nucleic acids described herein may encode both and RNASE4 protein or biologically active fragment thereof and an ANG protein or biologically active fragment thereof. The mRNA sequence for ANG is provided, for example, by GI accession number 207113180. The amino acid sequence for ANG is provided, for example, by GI accession number 148277046. In some embodiments, the RNASE4 encoding portion of the nucleic acid molecule and the ANG encoding portion of the RNASE4 molecule may both be under the control of a common nucleic acid regulatory element, such as a common promoter.
Enhancers of RNASE4 Activity and/or Expression
Certain embodiments of the methods described herein relate to methods of preventing neurodegeneration or treating neurodegenerative disease. These methods involve administering an RNASE4 protein, a biologically active fragment thereof, and/or an agent that increases the activity and/or expression of RNASE4. Agents which may be used to increase the activity of RNASE4 include nucleic acids, proteins, peptides and small molecules. Any agent that increases the activity of R ASE4 can be used to practice certain methods described herein. Such agents can be those described herein, those known in the art, or those identified through routine screening assays (e.g. the screening assays described herein).
In some embodiments, provided herein are methods for determining whether a test agent is a candidate therapeutic agent for treating and/or preventing neurodegenerative disease. In some embodiments the method includes the steps of contacting a cell with a test agent and detecting the activity and/or expression of R ASE4 by the cell. In general, an agent that increases the activity and/or expression of R ASE4 is a candidate therapeutic agent for treating and/or preventing neurodegenerative disease.
Any cell can be used in the above described screening method. For example, in some embodiments the cell is a human cell. Cells used in the screen can be primary cells or a cell line. Examples of other cell lines useful in the screening assays described herein include, but are not limited to, P19 cells, HUVAC cells, 293-T cells, 3T3 cells, 721 cells, 9L cells, A2780 cells, A172 cells, A253 cells, A431 cells, CHO cells, COS-7 cells, HCA2 cells, HeLa cells, Jurkat cells, NIH-3T3 cells and Vero cells.
Enhancers of R ASE4 expression may also be identified, for example, using methods wherein a cell is contacted with a candidate compound and the expression of R ASE4 mRNA or protein is determined. The level of expression of mRNA or protein in the presence of the candidate compound is compared to the level of expression of mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as an enhancer of RNASE4 expression if the expression of RNASE4 is greater in the presence of the candidate compound than in its absence.
The expression of the therapeutic targets described herein can be detected using any method known in the art. For example, the expression of the therapeutic target can be detected by detecting therapeutic target mRNA using, e.g., a detectably labeled nucleic acid probe, RT-PCR, and/or microarray technology. The expression of the therapeutic target can also be detected by detecting the therapeutic target protein using, e.g., detectably labeled antibodies that have binding specificity for the therapeutic target.
In some embodiments, a cell is used in the screening assay that has been genetically engineered to facilitate the performance of the assay. For example, in some embodiments, the cell is engineered such that the therapeutic target is expressed as a heterologous protein linked to a detectable moiety (e.g. a fluorescent moiety such as GFP or a luminescent moiety such as luciferase). In other embodiments, the cell contains a nucleic acid sequence encoding a detectable moiety operably linked to the promoter of the therapeutic target. In such embodiments, rather than detecting expression of the therapeutic target, the expression of the detectable moiety is detected directly. Such cells can be generated using standard recombinant techniques well known in the art.
As described above, the endogenous RNASE4 gene and the endogenous ANG gene are under the transcriptional control of a common promoter. Thus, in certain embodiments, the agents describe herein enhance expression of both RNASE4 and ANG. In some embodiments, the agent is a transcriptional enhancer that binds to the ANG/RNASE4 promoter. Such transcriptional enhancers are known in the art and can be targeted to particular genetic loci using, for example, designer TALE effector nucleases, as described in, for example, Moscou et ah, Science 326: 1501 (2009); Boch et ah, Science 326: 1509- 1512 (2009); and Zhang et ah, Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety.
Agents useful in the methods of the present invention may be identified, for example, using assays for screening candidate or test compounds which modulate the activity of RNASE4 or a biologically active portion thereof. Exemplary RNASE4 activities include promotion of angiogenesis and inhibition of neurodegeneration. Examples of assays useful in the testing the angiogenic and neuroprotective activities of potential agents are provided in the Examples below and include in vitro endothelial cell tube formation assays, ex vivo aortic ring explant culture assays neurosphere formation assays, neurite outgrowth assays, neurofilament fragmentation assays and serum withdrawal-induced neuronal degeneration assays. Additional assays for angiogenesis promotion and neurodegeneration inhibition are known in the art.
In another embodiment, agents useful in the methods of the invention may be identified using assays for screening candidate or test compounds which bind to RNASE4 or a biologically active portion thereof. Determining the ability of the test compound to directly bind to RNASE4 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to RNASE4 can be determined by detecting the labeled compound in a complex. For example, compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
Agents useful in the methods of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al.
(1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37: 1233.
Libraries of agents may be presented in solution (e.g., Houghten, 1992,
Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor,
1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith,
1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc.
Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).
Pharmaceutical Compositions
Pharmaceutical compositions of the described herein include R ASE4 proteins, biologically active fragments thereof and/or agents that enhance R ASE4 activity and/or expression {e.g., any small molecule, protein, polypeptide or polynucleotide that activates the activity or expression of R ASE4) combined with a pharmaceutically acceptable carrier or vehicle. The pharmaceutical compositions described herein may further comprise additional agents useful in the treatment of neurodegenerative diseases, including ANG proteins, biologically active fragments thereof, agents that enhance ANG expression and/or activity, and/or Riluzole.
As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.
Toxicity and therapeutic efficacy of the agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Appropriate dosage agents depends upon a number of factors within the scope of knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
Therapeutic Methods
Provided herein are methods for the treatment of neurodegenerative disease and/or the prevention of neurodegeneration. In certain embodiments an R ASE4 protein or biologically active fragment thereof is administered to a subject. In some embodiments, an agent that increases the activity or expression of R ASE4 is administered to the subject. In certain embodiments, an ANG protein, biologically active fragment thereof and/or an agent that increases the activity or expression of ANG is also administered to the subject.
The methods described herein can be used to treat any neurodegenerative disease. For example, in some embodiments the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP. In certain embodiments the neurodegenerative disease is ALS.
The methods described herein can be used to treat any subject in need thereof. As used herein, a "subject in need thereof includes any subject that has a neurodegenerative disease {e.g., ALS), and well as any subject with an increased likelihood of acquiring a neurodegenerative disease. In certain embodiments, the subject in need thereof is identified according to the diagnostic methods described herein below. In some embodiments the subject in need thereof carries a RNASE4 gene mutation. In some embodiments, the genome of the subject in need thereof has a T allele at single nucleotide polymorphism rs37484338. In certain embodiments, the subject in need thereof carries a gene mutation associated with a neurodegenerative disease, such as a mutated SOD1 gene. In some embodiments the subject in need thereof has at least one family member who has a neurodegenerative disease.
The pharmaceutical compositions of the present invention may be delivered by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. In certain embodiments the pharmaceutical compositions are delivered generally ( e.g., via oral or parenteral administration). In certain other embodiments the pharmaceutical compositions are delivered locally through direct injection into a specific tissue (e.g., central nervous system tissue and/or peripheral nervous system tissue).
In certain embodiments, the methods of treatment of the present invention comprise administering an R ASE4 protein, biologically active fragment thereof or an agent that increases the activity or expression of RNASE4 in conjunction with a second therapeutic agent to the subject (e.g., an ANG protein, a biologically active fragment thereof and/or an agent that enhances ANG activity and/or expression). Conjunctive therapy includes sequential, simultaneous and separate, or co-administration of the active compound in a way that the therapeutic effects of the first agent administered have not entirely disappeared when the subsequent agent is administered. In certain embodiments, the second agent may be co-formulated with the first agent or be formulated in a separate pharmaceutical composition.
The dosage of the subject agent may be determined by reference to the plasma concentrations of the agent. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) may be used. Dosages for the present invention include those that produce the above values for Cmax and AUC (0-4) and other dosages resulting in larger or smaller values for those parameters.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of an agent described herein will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
Identification of Subjects with Increased Risk of Neurodegenerative Disease
In some embodiments, the methods described herein are used to identify subjects with increased risk of a neurodegenerative disease {e.g., ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, SMA, PLS, PMA, and/or PBP). As described herein, a subject who carries one or more mutated R ASE4 coding regions is at increased risk for neurodegenerative disease.
Described herein are methods of identifying a subject who has an increased risk of neurodegenerative disease. In certain embodiments, the method includes the step of detecting in a biological sample a mutation to a R ASE4 coding region. In certain embodiments, the mutation to the R ASE4 coding region causes reduced RNASE4 activity and/or expression in the subject. In certain embodiments, the mutated RNASE4 coding region encodes a RNASE4 signal peptide that comprises a serine at position -13. In some embodiments, the mutated RNASE4 coding region comprises as thymidine at position -39.
In some embodiments, the mutation is an allele of a single nucleotide polymorphism that is associated ALS. For example, in some embodiments the mutation is a T allele at single nucleotide polymorphism rs37484338.
In some embodiments, of the methods described herein, the subject will be a human child or a human adult. In some embodiments, the subject will be an infant. However, in certain embodiments the subject is not limited to being a fully developed human. Thus, in some embodiments, the subject will be a human fetus, a human embryo and/or a human fertilized cell.
Any type of biological sample that contains genetic material can be used in the methods described herein. Thus, for example, in some embodiments the sample is a cell, a body fluid, a swabbing, a tissue sample, a blood sample and/or a germ cell sample.
Any method known in the art can be used to detect the mutations described herein. Thus, in certain embodiments, the detecting step includes performing a hybridization assay {e.g., SNP or gene microarrays, dynamic allele-specific hybridization (DASH), TaqMAN, HP A, scorpion probes and molecular beacons), performing a nucleic acid amplification assay (e.g., PCR, LCR, TMA, SDA, NASBA, BDA, 3SR, RCR, etc.) and/or performing a nucleic acid sequencing assay.
In some embodiments, analysis of the nucleic acid can be carried out by
amplification of the region of interest according to amplification protocols well known in the art (e.g. , polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification, self-sustained sequence replication (3SR), QP replicase protocols, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and boomerang DNA amplification (BDA), etc.). The amplification product can then be visualized directly in a gel by staining or the product can be detected by hybridization with a detectable probe. When amplification conditions allow for
amplification of all allelic types of a genetic marker, the types can be distinguished by a variety of well-known methods, such as hybridization with an allele-specific probe, secondary amplification with allele-specific primers, by restriction endonuclease digestion, and/or by electrophoresis. Thus, also provided herein are oligonucleotides for use as primers and/or probes for detecting and/or identifying genetic markers according to the methods described herein.
Additional methods for detecting the genetic mutations described herein include sequencing, high performance liquid chromatography (HPLC), restriction enzyme analysis (e.g., restriction fragment length polymorphism or RFLP), hybridization, matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), etc., all of which are well known protocols for analyzing a nucleotide sequence and detecting genetic markers. The methods described herein can be carried out by using any assay or procedure that can interrogate a nucleic acid sequence.
In some embodiments, detecting can be carried out by an amplification reaction and single base extension, and in further embodiments, the product of the amplification reaction and single base extension can be spotted on a silicon chip according to methods well known in the art.
Prior to analyzing the sample, it may be necessary to process the sample to yield a form acceptable for analysis. For example, the nucleic acid (e.g. genomic DNA) may be extracted from the sample using techniques well-established in the art including chemical extraction techniques utilizing phenol-chloroform, guanidine-containing solutions, or CTAB-containing buffers. As well, as a matter of convenience, commercial DNA extraction kits are also widely available from laboratory reagent supply companies, including for example, the QIAamp DNA Blood Minikit available from QIAGEN
(Chatsworth, CA), or the Extract-N-Amp blood kit available from Sigma (St. Louis, MO).
Also provided herein is a kit comprising reagents to detect a mutation described herein in a biological sample from a subject. Such a kit can comprise primers, probes, primer/probe sets, reagents, buffers, etc., as would be known in the art, for the detection of the genetic markers described herein in a biological sample from a subject. For example, a primer or probe can comprise a contiguous nucleotide sequence that is complementary (e.g., fully (100%) complementary or partially (50%, 60%, 70%, 80%, 90%, 95%, etc.) complementary) to a region comprising position -39 of the RNASE4 coding region (e.g., that encodes the R ASE4 signal sequence). Such a kit can further comprise blocking probes, labeling reagents, blocking agents, restriction enzymes, antibodies, sampling devices, positive and negative controls, etc., as would be well known to those of skill in the art.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference.
EXAMPLES Example 1: Polymorphism of human RNASE4 gene in ALS patients
The coding region of RNASE4 gene from 1,575 sporadic ALS patients and 658 controls was sequenced for potential ALS-associated mutations and SNPs. Clinical specimens were obtained under a discarded tissue protocol approved by the institutional review board. Genomic DNA was extracted from peripheral leukocytes using standard protocol. The coding exon of RNASE4 was amplified by PCR with primers located in adjacent intron and non-coding regions, respectively, and the amplicons were sequenced bidirectionally. The sequences of the primers are as follows. Forward. 5'- ACCTTATTTCTCCTGCCCCTTG-3'; reverse: 5*-AAGCCCAGCCTCATTCATTACAG- 3'. The nucleic acid sequence of RNASE4, including variants identified in this sequencing analysis, is provided in Fig. 1.
A total of 6 SNPs were found in these 2,233 subjects (Table 1). The most abundant SNP found was the nucleotide change from A to T at position -39, which corresponds to SNP rs37484338 in the SNP data base. This change results in a change of amino acid from Thr to Ser in the signal peptide position -13. A total of 11 and 413 homozygous and heterozygous T alleles were observed in the 1,575 ALS subjects, representing a minor allele frequency of 13.81%. However, in the 658 control subjects, there were 152 heterozygous but no homozygous T alleles, representing a minor allele frequency of 11.55%. Hardy- Weinberg equilibrium and tests of association showed that the p value for the allele frequency difference is 0.042, and the p value for the common odds ratio from Armitage's trend test is 0.031. None of the other 5 SNPs reached a statistically significant difference between ALS and control subjects. These genetic data indicate that there is an association between the presence of a serine at position -13 in RNASE4 and ALS susceptibility.
Table 1. Relative frequencies of RNase4 SNPs in the SALS and control population
Figure imgf000030_0001
1 The numbering of amino acid and nucleotide started from the first amino acid in the mature protein and the first nucleotide of the corresponding codon.
2 the number in parenthesis is the cases of homozygous changes. Example 2: RNASE4 is angiogenic
Recombinant WT RNASE4 protein and the enzymatically attenuated K40A variant were prepared. K40 was chosen because it is an essential residue constituting the catalytic triad that is conserved across the members of the RNASE superfamily. Mutations at K40 diminish most of the catalytic activity of this family of proteins. WT RNASE 4 gene was cloned from genomic DNA of HeLa cells with the following PCR primers: Forward, 5'- GGAGAT ATC AT ATGC AGGATGGC ATGT AC-3 ' ; Reverse,
5 'CCGGGATCCCTAACCGTCAAAGTGC-3 ' . PCR products were cloned into a pETl la vector between the BamHl and Ndel sites. The resultant plasmid (pETl la-hRNASE4) was sequence confirmed and transformed into BL21 (DE3) cells for protein expression. K40A mutation was generated by QuickChange II Site-Directed Mutagenesis Kit from Stratagene using pETl la-hRNASE4 as the template and the following primers: Forward, 5'- CTTTGTATC ACTGCgcGCGCTTCAACACCTT-3 ' ; Reverse, 5'- AAGGTGTTGAAGCGCgcCCAGTGATACAAAG-3 ' . The resultant plasmid (pETl lcc- hR ASE4-K40A) was sequenced confirmed and cloned into BL21 (DE3) cells.
Pure and active R ASE4 proteins were obtained, as shown by SDS-PAGE (Fig. 2A) and yeast tR A ribonucleolytic assay (Fig. 2B). Ribonucleolytic activities of WT and K40A R ASE4 were examined using yeast. Reactions were initiated by adding the enzyme. Varying amount RNASE were added to a final volume of 300 μΐ, including 600 ng yeast tRNA, 0.33 M Hepes, 0.33 M NaCl, pH 7.0, and 0.1 mg/ml RNASE-free BSA. After incubation at 37 °C for 120 min, 700 μΐ of ice-cold 3.4% perchloric acid was added and incubated on ice for 10 min, centrifuged at 14,000 X g for 10 min at 4 °C, and the absorbance at 260 nm of the supernatants was recorded. All buffers and water used above was filtered through Sep-Pak cartridge (Waters) to ensure that the system is RNASE-free. Experiments were done in triplicate. The result of the assay demonstrated that the K40A variant of RNASE4 had reduced ribonucleolytic activity compared to the wild-type protein. Specifically, the K40A RNASE4 variant retains 5.4% of the activity of WT RNASE4 when yeast tRNA was used as a substrate (Fig. 2B).
Even though the enzymatic activity of the K40A RNASE4 variant is only 5.4% of that of wild-type RNASE4, the retained enzymatic activity of the variant is still about 10 times higher than that of wild-type ANG. To determine whether the ribonucleolytic activity of the RNASE4 variant is essential for its angiogenic activity, RNASE4 protein was treated with diethylpyrocarbonate (DEPC), which chemically modifies Lys residues and
completely abolished the enzymatic activity of ribonucleases. DEPC-treated RNASE4 fails to cleave both yeast tRNA (Fig. 3A) and HeLa rRNA (Fig. 3B), even with prolonged incubation, indicating that DEPC treatment has completely abolished the enzymatic activity of RNASE4. Fig. 3C shows that DEPC treatment also completely abolished the ability of RNASE4 to induce endothelial tubule formation. These results indicate that the
ribonucleolytic activity of RNASE4 is essential for angiogenesis.
The ability of WT and K40A RNASE4 to induce endothelial cell tube formation was analyzed. Each well of the 48-well plate was coated with 50 μΐ growth factor-reduced Matrigel. HUVEC Cells were seeded on the Matrigel at a density of 1.5 x 104 cells per well and cultured in the presence or absence wild-type RNASE4 protein, K40A RNASE4 protein or ANG for 4 h. Cells were fixed in 3.7% paraformaldehyde and photographed. The images were analyzed by ImageJ software to calculate the length or area of capillary-like structures as well as the numbers of circled tubular structure. Experiments were done in triplicate and were repeated at least three times.
As shown in Fig. 4A, both WT and K40A RNASE4 were able to induce tube formation of HUVEC cultures in Matrigel. ANG was included as a positive control.
RNASE4 had about 50% of the activity of ANG in this assay. For example, WT RNASE4 at 1 μg/ml and ANG at 0.5 μg/ml had an equivalent activity (Fig. 4B). The K40A variant of RNASE4 actually had a higher angiogenic activity than the WT RNASE4 and is only slightly lower than ANG. No endotoxin was detected in the preparation. Heat inactivation or proteolysis abolished all the activity, indicating that an intact peptide and structure is important. No angiogenic activity of RNase A/RNASE 1 , EDN/RNASE2, and
ECP/RNASE3 were detected in the same assay system (Fig. 5).
An ex vivo angiogenesis assay was performed using mouse aortic ring explant cultures. Thoracic aortic vessels from 2 month old C57BL/6J mice were dissected and transferred to a dish containing ice-cold MEM. The fibroadipose tissues were removed with microdissecting forceps and scissors. The aorta were cut into 0.5 mm pieces and washed extensively in cold PBS, and placed in Matrigel-coated 48-well plates. Each well was covered with another 50 μΐ Matrigel and incubated in human endothelial SMF basal growth medium in the presence or absence of testing materials for 15 days. New angiogenic sprouts were stained with 20μ1 MTT (5 mg/ml in PBS) for 2 h, photographed and analyzed by ImageJ.
Both WT RNASE4 and K40A variant were able to stimulate endothelial sprouts from the inner side of the aorta so that the sprouts grew outward in the inside -out flipped aortic rings (Fig. 6A) and inward in the unflipped ones (Fig. 6B). Quantitative analysis with the ImageJ program indicated that the area covered by endothelial branch in the control, ANG, K40A and WT RNASE4 were 0.05 ± 0.01, 0.67 ± 0.19, 0.61 ± 0.09, and 0.47 ± 0.07 mm2, respectively, in inside-out flipped culture (Fig. 6C); and were 0.04 ± 0.01, 0.27 ± 0.01, 0.32 ± 0.01, and 0.17 ± 0.02 mm2, respectively, in unflipped culture (Fig. 6D). Thus, RNASE4 elicits an angiogenic response in mouse aorta explant culture. Like the results with the in vitro endothelial cell tube formation assay (Fig. 4), the K40A variant has a higher activity than the WT RNASE4, indicating that a robust ribonucleolytic activity is not necessary for RNASE4 to induce angiogenesis. It is noteworthy that while WT RNASE4 has a slightly lower angiogenic activity as compared with ANG, the activity of K40A RNASE4 variant and ANG are comparable. To confirm the in vitro and ex vivo finding that R ASE4 is angiogenic, we examined the ability of R ASE4 to induce blood vessel formation in the Matrigel plug in vivo angiogenesis assay. A cold solution of 0.5 ml Matrigel with or without 0.5 μg/ml of the indicated protein was injected subcutaneously into C57BL/6 mice. The Matrigel plug was removed 4 days later and processed for immunohistochemical staining of blood vessels with anti-von Willebrand Factor (vWF) IgG.
As shown in Fig. 7A, a vigorous neovessel growth occurred in the Matrigel plugs in the presence of both WT R ASE4 and the K40A variant. ANG, used as a positive control, also elicited a strong angiogenic response. Quantitative analysis indicates that the vessel density per mm2 in control Matrigel and in those containing ANG, WT RNASE4 and K40A variant was 49 ± 7, 250 ± 10, 184 ± 39, and 189 ± 58, respectively (Fig. 7B). Thus, RNASE4 and its K40A variant are active in in vitro, ex vivo and in vivo angiogenesis assays. Taken together, these results demonstrate that human RNASE4 is angiogenic.
Example 3: RNASE4 stimulates neuronal differentiation and neurite outgrowth
The neurogenic activity of RNASE4 examined in P19 mouse embryonal carcinoma cells. PI 9 cells are pluripotent and have stem cell-like property. These cells are able to both self-renew and differentiate into various types of neural cells. P19 cell were seeded on PA6 monolayer and cultured in a-MEM plus 0.1% nonessential amino acids, 0.1% knockout serum replacement, 0.5 μΜ retinoic acid, and 200 ng/ml testing proteins for 24 h.
Embryonal bodies were counted under phase light microscope at 10 X magnification. The numbers of embryonal bodies from the entire well were counted. Experiments were done in triplicate and were repeated three times.
Both ANG and RNASE4 induced the formation of neurospheres or embroynal bodies (Fig. 8A). The number of these neurospheres in a 35 mm dish after 24h incubation was 218 ± 9 and 261 ± 64, respectively, in the presence of RNASE4 and ANG, representing a 7.3- and 8.7-fold increase over that formed in the presence of control protein BSA (30 ± 14) (Fig. 8B). As the formation of neurospheres is a strong indication of neuronal differentiation, these results indicate that both ANG and RNASE4 induce neurogenesis.
To examine the effect of RNASE4 on neurite outgrowth, cells were cultures as described above but for 216 h. Cells were fixed successively in 4% paraformaldehyde for 15 min at RT and cold methanol for 5 min at -20 °C, blocked with 0.1% gelatin, 0.5% BSA and 0.1% Tween 20 in TBS or 1 h at RT. Neurofilaments were stained by a rabbit anti- Neurofilament medium chain at 1 :500 dilution at 4°C overnight and Alexa 488-labeled goat anti-rabbit F(ab') at 1 :400 dilution for 1 h at RT.
ANG and RNASE4 were able to stimulate neurite outgrowth in a prolonged culture of P19 cell (Fig. 8C). After 9 days culture, the average length of neurofilaments in the presence of RNASE4 and ANG was 20.2 ± 0.8 and 20.7 ± 1.4 μιη, respectively, which is 3.0- and 3.1 -fold higher than that in the presence of BSA (6.7 ± 1.2 μιη) (Fig. 8D).
ANG and RNASE4 are also able to stimulate neurofilament growth of primary neurons. Brain cortical cells were isolated from C57/B6SJL embryos (E14) and seeded in 6 wells at a density of 1.1 x 106 per cm2 in neurobasal medium plus B27 and 0.5 mM L- glutamine for 36 h. The medium was changed and 10 μΜ Ara-c was added and cultured for another 6 days with medium change every two days. Ara-c was removed and the cells were cultured in neurobasal medium plus B27 in the presence or absence of testing material for additional 6 days. For hypothermia-induced degeneration, the cells were put at RT temperature for 40 min and returned to the cell culture incubator for additional 3 h.
Neurofilaments were stained as described above.
As shown in Fig. 9, mouse cortical neurons isolated from E14 embryos were able to survive for 12 days in neurobasal medium, but very little neurofilament growth was noted when only BSA was present (Fig. 9A). In the presence of B27, a serum- free supplement for growth and long-term viability of primary neurons, an extensive network of neurofilaments was formed. A similar network of neurofilaments was formed in the presence of RNASE4 and ANG. The length of neurofilaments formed in the presence of BSA, B27, RNASE4, and ANG were 295 ± 116, 903 ± 180, 804 ± 135, and 962 ± 122 μιη, respectively (Fig. 9B). Thus, the activity of ANG and RNASE4 in stimulating neurofilament growth from the primary culture of the mouse embryonic cortical neuron is comparable to or greater than that of the B27 positive control. These results clearly indicate that both RNASE4 and ANG have neurogenic activity.
The neural differentiation activity of RNASE4 was further examined by looking at e its ability to induce differentiation of mouse ES cells. Fig. 10 shows that both RNASE4 and ANG induced differentiation of mouse ES cells into GFAP -positive progenitor neurons. The length of neurofilaments in the presence of ANG and RNASE4 was 38.8 ± 2.3 and 36.8 ± 0.7 μιη, respectively, which is 3.1- and 3.0-fold higher than that in the negative control (BSA: 12.3 ± 0.7), and is 55% and 52% of that in the positive control (Retinoic acid: 70.5 ± 1.5). These results further demonstrate the neurogenesis activity of RNASE4 and angiogenin.
The subcellular localization of RNASE4 was examined using an affinity purified polyclonal antibody. Fig. 11 shows that endogenous RNASE4 is detectable on cell surface (indicated with arrows) and cytoplasm (indicated with triangles) of both HUVEC (Fig. 11A) and P19 cells (Fig. 1 IB). When exogenous RNASE4 was added to the cells, it was predominately accumulated in the nuclei (indicated with stars) of both cell lines. These results indicate that RNASE4 undergoes nuclear translocation similarly to ANG.
Example 4: RNASE4 protects stress-induced neuronal degeneration
To determine whether RNASE4 possesses neuroprotective activity, effect in preventing hypothermia-induced fragmentation of neurofilament derived from mouse embryonic cortical neuron culture was examined. As shown in Figure 9, a robust network of neurofilaments formed when mouse embryonic cortical neurons were cultured in the presence of B27. These neurofilaments fragmented into small pieces after being subjected to 25 °C for 40 min in the presence of control protein BSA (Fig. 12A). By contrast, both RNASE4 or ANG prevented hypothermia-induced neurofilament fragmentation, indicating that both are neuroprotective. The average length of the neurofilaments after being subjected to hypothermia was 386 ± 88, 881 ± 160, and 971 ± 186 μιη in the cultures containing BSA, RNASE4, and ANG, respectively (Fig. 12B). Thus, RNASE4 and ANG have comparable neuroprotective activity. This is also the first report that ANG prevents stress-induced degeneration of primary neurons in culture.
To confirm the neuroprotective activity, the effects of RNASE4 and ANG on serum withdrawal-induced neuronal degeneration of P19-derived neurofilaments was examined. As shown in Fig. 12C, retinoic acid-induced P19 neurofilaments underwent fragmentation when subjected to serum starvation in the presence of BSA. However, no significant fragmentation was observed with the same treatment but in the presence of RNASE4 or ANG. ImageJ analyses indicated that the length of P19 cell-derived neurofilaments after 1 h serum starvation in the presence of BSA, RNASE4, and ANG was 4.2 ± 0.4, 9.7 ± 0.7, and 12.6 ± 1.3 μιη, respectively. RNASE4 had no effect on PA6 cell proliferation (Fig. 13). Example 5: RNASE4 slows weight loss and enhances neuromuscular function in a mouse model for ALS
The beneficial effect of exogenous RNASE4 on S0D1G93A mice was examined. First, in situ hybridization (ISH) was used to examine the mRNA level of RNASE4 in ALS and control spinal cords. Human RNASE4 and mouse Rnase4 DNA were amplified by PCR from HeLa cell DNA and C57B6/SJL mouse tail genomic DNA as templates. The primers were as follows: Human RNASE4, forward, 5'-
GGGTAATACGACTCACTATAGGGCGAaccttatttctcctgccccttg-3' (up case: T7 sequence); reverse, 5'-aagcccagcctcattcattacag-3'. Mouse Rnase4, forward, 5'-
GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3 ' ; reverse, 5 ' -gtg ctggttcttgccctgtatcta-3 ' . cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer's protocol . Control cRNA probes were generated from the control DNA template included in the kit. For in situ hybridization, the slides were deparaffmized with Xylene (2 x 10 min), washed in sequence with 100, 75, 50, and 25% ethanol and PBS each for 5min. The slides were then treated with 2 μg/ml of Proteinase K for 10 min, washed with 2 mg/ml of Lysine and PBS each for 5min, and incubated in acetylation buffer (0.25% acetic acid anhydride in 0.1 M Triethanolamine, pH 8.0) for 20 min. After two washes with 4 x SSC, the slides were prehybridized in
Hybridization buffer (5 x SSC, 0.5 mg/ml heparin, 0.8 mg/ml salmon sperm DNA) at 45 °C for 60 min. cRNA probes were added onto the slides and incubated at 45 °C overnight. Hybridization signals were detected and visualized with the digoxigenin Nucleic Acid Detection Kit from Roche following manufacturer's protocol.
Strong staining of RNASE4 mRNA in motor neurons was observed in non-ALS spinal cord, but was significantly decreased in ALS spinal cords (Fig. 14A). Quantitative analysis of the ISH images indicated that the total photon counts of RNASE4 mRNA staining per motor neurons of ALS and non-ALS spinal cord were 2.98 ± 0.71 x 104 and 9.67 ± 1.21 x 104, respectively, indicating that RNASE4 expression decreased by 69%> in ALS. Similarly, the mRNA level of mouse Rnase4 is also decreased in the spinal cord motor neurons of S0D1G93A mice as compared to that of WT mice (Fig. 14B). The photon counts per spinal cord motor neuron in WT and S0D1G93A mice were 6.26 ± 0.62 x 103 and
4.27 ± 0.34 x 103, respectively, representing a 32% decrease in Rnase4 expression in the spinal code motor neurons of S0D1G93A mice.
The therapeutic activity of RNASE4 protein in ALS treatment was examined.
SOD1 G93A mice^ at 1 1 weeks of age, were treated with weekly i.p. injection of PBS or RNASE4 protein at 10 μg per mouse. The neuromuscular function of the mice was examined by their performance in a rota-rod assay. As shown in Fig. 15 A, weekly treatment of RNASE4 increased the time the mice could stay on a rotarod (20 rpm) from week 14 to week 18 to various degrees. The greatest difference was recorded at week 15, when the R ASE4-treated mice were able to stay on the rotarod for 333 ± 106 sec, whereas PBS- treated animals were able to stay on it for only 181 ± 68 sec. R ASE4 treatment also decreased the rate of body weight decrease (Fig. 15B). In the early phase of the treatment (week 12 to 14), a slight increase in body weight in both control and treatment groups was observed. The average body weight of the PBS and R ASE4 groups at week 12 was essentially the same at 23.45 ± 0.70 and 23.53 ± 0.59 g, respectively. At week 15, when no weight loss was observed, the difference in body weight was already obvious at 23.87 ± 0.73 g for the PBS group and 24.24 ± 0.63 g for R ASE4 group, representing a 1.5% difference. This difference increased as the disease progressed and the animals started to lose weight. At week 17, the body weight of PBS and R ASE4 groups was 21.87 ± 0.76 and 22.93 ± 0.69 g, respectively, representing a difference of 4.6%. At weeks 20, and 21, the difference in body weight in the control and treatment group was 14.3 and 16.8%, with the p values of 0.007 and 0,007, respectively. These findings demonstrate that R ASE4 treatment slow loss of body weight in an ALS disease model.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:
1. A method of treating or preventing a neurodegenerative disease in a subject comprising administering to the subject an RNASE4 protein or a biologically active fragment thereof.
2. The method of claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) or progressive bulbar palsy (PBP).
3. The method of claim 2, wherein the neurodegenerative disease is ALS.
4. The method of claim 1, wherein the RNAS4 protein or biologically active fragment thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, [WT RNASE4 protein sequence], SEQ ID NO: 2 [RlOW variant], SEQ ID NO: 3 [E48D variant], SEQ ID NO: 4 [V75I variant], SEQ ID NO: 5 [A98V variant] or SEQ ID NO: 6 [K40A variant] .
5. The method of claim 4, wherein the RNASE4 protein or biologically active fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6 [K40A variant], wherein the amino acid sequence does not have a lysine at position 40.
6. The method of claim 4, wherein the RNASE4 protein or biologically active fragment thereof comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.
7. The method of claim 6, wherein the RNASE4 protein or biologically active fragment thereof comprises an amino acid sequence of SEQ ID NO: 6.
8. The method of claim 1, wherein the RNASE4 protein or biologically active fragment thereof has less than 10% of the ribonucleo lytic activity of a wild-type RNASE4 protein having a sequence of SEQ ID NO: 1.
9. The method of claim 1, further comprising administering to the subject an ANG protein or biologically active fragment thereof.
10. The method of claim 1, wherein the genome of the subject comprises an T allele at single nucleotide polymorphism rs37484338.
11. The method of claim 1 , wherein the subject has reduced expression or activity of RNASE4.
12. A method of treating or preventing a neurodegenerative disease in a subject comprising administering to the subject an agent that enhances the activity or expression or RNASE4.
13. The method of claim 12, wherein the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal SMA, PLS, PMA or PBP.
14. The method of claim 12, wherein the agent is a small molecule, a nucleic acid or a polypeptide.
15. The method of claim 12, wherein the agent enhances expression of RNASE4 by contacting the ANG/RNASE4 promoter.
16. The method of claim 15, wherein the agent also enhances expression of ANG by contacting the ANG/RNASE4 promoter.
17. The method of claim 12, further comprising administering to the subject ANG protein.
18. The method of claim 12, further comprising administering to the subject an agent that enhances ANG activity or expression.
19. The method of claim 12, wherein the genome of the subject comprises an A allele at single nucleotide polymorphism rs37484338.
20. The method of claim 12, wherein the subject has reduced expression or activity of RNASE4
21. A method of reducing neurodegeneration in a subject comprising administering to the subject an RNASE4 protein or a biologically active fragment thereof.
22. The method of claim 21, wherein the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal SMA, PLS, PMA or PBP.
23. The method of claim 22, wherein the neurodegenerative disease is ALS.
24. The method of claim 21, wherein the RNAS4 protein or biologically active fragment thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, [WT RNASE4 protein sequence], SEQ ID NO: 2 [RlOW variant], SEQ ID NO: 3 [E48D variant], SEQ ID NO: 4 [V75I variant], SEQ ID NO: 5 [A98V variant] or SEQ ID NO: 6 [K40A variant] .
25. The method of claim 24, wherein the R ASE4 protein or biologically active fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6 [K40A variant], wherein the amino acid sequence does not have a lysine at position 40.
26. The method of claim 24, wherein the RNASE4 protein or biologically active fragment thereof comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.
27. The method of claim 26, wherein the RNASE4 protein or biologically active fragment thereof comprises an amino acid sequence of SEQ ID NO: 6.
28. The method of claim 21 , wherein the RNASE4 protein or biologically active fragment thereof has less than 10% of the ribonucleo lytic activity of a wild-type RNASE4 protein having a sequence of SEQ ID NO: 1.
29. The method of claim 21, further comprising administering to the subject an ANG protein or biologically active fragment thereof.
30. The method of claim 21, wherein the genome of the subject comprises an A allele at single nucleotide polymorphism rs37484338.
31. The method of claim 21 , wherein the subject has reduced expression or activity of RNASE4.
32. A method of reducing neurodegeneration in a subject comprising administering to the subject an agent that enhances the activity or expression or RNASE4.
33. The method of claim 32, wherein the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal SMA, PLS, PMA or PBP.
34. The method of claim 32, wherein the agent is a small molecule, a nucleic acid or a polypeptide.
35. The method of claim 32, wherein the agent enhances expression of RNASE4 by contacting the ANG/RNASE4 promoter.
36. The method of claim 35, wherein the agent also enhances expression of ANG by contacting the ANG/RNASE4 promoter.
37. The method of claim 32, further comprising administering to the subject ANG protein.
38. The method of claim 32, further comprising administering to the subject an agent that enhances ANG activity or expression.
39. The method of claim 32, wherein the genome of the subject comprises an T allele at single nucleotide polymorphism rs37484338.
40. The method of claim 32, wherein the subject has reduced expression or activity of R ASE4
41. A method of identifying a subject as having increased risk of neurodegenerative disease comprising the step of detecting in a biological sample from the subject a mutation to a R ASE4 coding region, wherein the presence of a mutation to the R ASE4 coding region identifies the subject as having an increased risk of neurodegenerative disease.
42. The method of claim 41 , wherein the neurodegenerative disease is ALS, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal SMA, PLS, PMA or PBP
43. The method of claim 42, wherein the neurodegenerative disease is ALS.
44. The method of claim 41, wherein the mutation to the R ASE4 coding causes reduced R ASE4 expression or activity in the subject.
45. The method of claim 41, wherein the mutated R ASE4 coding region encodes an R ASE4 signal peptide that comprises a serine at signal peptide position -13.
46. The method of claim 45, wherein the mutated R ASE4 coding region comprises a thymidine at position -39.
47. An R ASE4 protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40.
48. The RNASE4 protein of claim 47, wherein the amino acid sequence has an alanine at position 40.
49. The RNASE4 protein of claim 47, wherein the amino acid sequence is identical to SEQ ID NO: l, except that the amino acid sequence does not have a lysine at position 40.
50. The RNASE4 protein of claim 47, wherein the amino acid sequence is identical to SEQ ID NO:6.
51. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence at least 90% identical to SEQ ID NO: 6, wherein the amino acid sequence does not have a lysine at position 40.
52. The isolated nucleic acid molecule of claim 51 , wherein the encoded amino acid sequence has an alanine at position 40.
53. The isolated nucleic acid molecule of claim 51 , wherein the encoded amino acid sequence is identical to SEQ ID NO: l, except that the amino acid sequence does not have a lysine at position 40.
54. The isolated nucleic acid molecule of claim 51 , wherein the encoded amino acid sequence is identical to SEQ ID NO:6.
55. The isolated nucleic acid molecule of claim 41 comprising a nucleic acid sequence of SEQ ID NO: 12.
56. A method of determining whether a test agent is a candidate therapeutic agent for treating or preventing a neurodegenerative disease comprising: a) contacting a cell with the test agent; and b) detecting the expression of RNASE4 by the cell; wherein a test agent that increases the expression of RNASE4 is a candidate therapeutic agent for treating or preventing a neurodegenerative disease.
PCT/US2013/021177 2012-01-13 2013-01-11 Methods and compositions for the treatment of neurodegenerative disease WO2013106672A1 (en)

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