WO2024077262A2 - Procédés et compositions pour le silençage de l'expression d'elavl2 pour le traitement d'une maladie - Google Patents

Procédés et compositions pour le silençage de l'expression d'elavl2 pour le traitement d'une maladie Download PDF

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WO2024077262A2
WO2024077262A2 PCT/US2023/076274 US2023076274W WO2024077262A2 WO 2024077262 A2 WO2024077262 A2 WO 2024077262A2 US 2023076274 W US2023076274 W US 2023076274W WO 2024077262 A2 WO2024077262 A2 WO 2024077262A2
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nucleic acid
seq
acid inhibitor
nos
rna
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WO2024077262A3 (fr
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Fen-Biao GAO
Jonathan K. Watts
Gopinath Krishnan
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University Of Massachusetts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
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    • C12N2310/30—Chemical structure
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • the present disclosure relates to compositions and methods for reducing expression of ELAVL2.
  • a GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 is the most commonly known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • RNAs transcribed from these repeats in both sense and antisense directions can be translated into five dipeptide repeat (DPR) proteins — poly(GR), poly(GA), poly(GP), poly(PR), and poly(PA).
  • DPR dipeptide repeat
  • poly(GR) is a key neurotoxic species, as its expression strongly correlates with neurodegeneration in C9ORF72 patient brains.
  • poly(GR) is toxic in many cellular and animal models.
  • AS Os antisense oligonucleotides
  • compositions and methods that target genetic suppressors of poly(GR) toxicity without affecting the level of toxic poly(GR) protein.
  • Disclosed herein are gene silencers of these modifier genes and method of use thereof which offer clinical benefits by slowing down or blocking ongoing disease processes, even in the presence of disease-causing G4C2 repeats, poly(GR) and other dipeptide repeat (DPR) proteins.
  • nucleic acid inhibitor which is capable of reducing expression of ELAVL2 by 10% or more compared to a control where the nucleic acid inhibitor is not present, wherein said nucleic acid inhibitor hybridizes to at least five nucleotides within SEQ ID NO: 1.
  • the nucleic acid inhibitor hybridizes to at least five nucleotides within a region of SEQ ID NO: 2. In some embodiments, the nucleic acid inhibitor hybridizes to at least five nucleotides within a region of 1-450 or 2100-3983 of SEQ ID NO: 2.
  • the nucleic acid inhibitor is 15-30 nucleotides long. In some embodiments, the nucleic acid inhibitor is an antisense oligonucleotide or an RNAi molecule. In some embodiments, the RNAi molecule is siRNA.
  • the nucleic acid inhibitor comprises at least one modification.
  • the at least one modification comprises at least one modified sugar moiety; at least one modified inter- nucleotide linkage; and/or at least one modified nucleotide.
  • the at least one modification comprising an internucleotide linkage comprising: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, and/or carboxymethyl ester.
  • the at least one modification comprising a modified nucleotide comprises a peptide nucleic acid, a morpholino analogue, a locked nucleic acid (LNA) or other bridged or bicyclic nucleic acid analogue, and/or a combination thereof.
  • the at least one modification comprising a modified sugar moiety comprises: a 2 ’-fluoro sugar moiety, 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, and/or another 2'-O-alkyl or alkoxyalkyl modified sugar moiety.
  • the nucleic acid inhibitor further comprises a conjugate.
  • the conjugate comprises a therapeutic agent, a diagnostic agent, or an agent that increases cell uptake or penetration.
  • the conjugate comprises a peptide, a protein, an antibody or fragment thereof, a lipid, a neurotransmitter, a cationic polymer, a nanoparticle, a probe, or a carbohydrate.
  • the conjugate is a multivalent conjugate.
  • the multivalent conjugate comprises a multimeric antisense oligonucleotide or a multivalent siRNA.
  • a viral vector comprising the nucleic acid inhibitor of any preceding aspect, or which induces the intracellular expression of the nucleic acid inhibitor of any preceding aspect.
  • a pharmaceutical composition comprising the nucleic acid inhibitor of any preceding aspect.
  • the pharmaceutical composition further comprises a vehicle for delivery of the nucleic acid inhibitor.
  • the vehicle comprises a nanoparticle.
  • the vehicle comprises a viral vector.
  • nucleic acid comprising 90% or more identity to any one of SEQ ID NOS: 3-50, SEQ ID NOS: 54-64, or SEQ ID NOS: 82-150.
  • nucleic acid comprising 95% or more identity to any one of SEQ ID NOS: 3-50, SEQ ID NOS: 54-64, or SEQ ID NOS: 82-150.
  • nucleic acid comprising SEQ ID NOS: 17, 21, or 24.
  • nucleic acid comprising SEQ ID NOS: 41, 45, and 48.
  • nucleic acid comprising SEQ ID NOS: 57, 58, 59, 60, 61, or 62.
  • nucleic acid comprising SEQ ID NOS: 63 or 64.
  • the poly(GR) toxicity is caused by a GGGGCC (G4C2) repeat expansion in a first intron of C9ORF72.
  • the subject has, or is at risk of developing, amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the method comprises expression of ELAVL2 is reduced by 10% or more compared to a control wherein a modulator of ELAVL2 expression is not present. In some embodiments, one or more symptoms of a disease or disorder associated with poly(GR) toxicity is reduced by 10% or more compared to a control wherein a modulator of ELAVL2 expression is not present. In some embodiments, one or more symptoms of a disease or disorder associated with poly(GR) toxicity is prevented compared to a control wherein a modulator of ELAVL2 expression is not present.
  • the method comprises modulation of expression of ELAVL2 is carried out by a nucleic acid inhibitor.
  • the nucleic acid inhibitor comprises an antisense oligonucleotide, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), small nucleolar RNA (sno-RNA), Piwi-interacting RNA (piRNA), or short hairpin RNA (shRNA) molecules.
  • the method comprises the nucleic acid inhibitor hybridizing to at least five nucleotides within SEQ ID NO: 1. In some embodiments, the method comprises the nucleic acid inhibitor hybridizing to at least five nucleotides within a region of SEQ ID NO: 2. In some embodiments, the method comprises the nucleic acid inhibitor hybridizing to at least five nucleotides within a region of 1-450 or 2100-3983 of SEQ ID NO: 2.
  • the method comprises the nucleic acid inhibitor comprising 90% or more identity to any one of SEQ ID NOS: 3-50, SEQ ID NOS: 54-64, or SEQ ID NOS: 82- 150. In some embodiments, the method comprises the nucleic acid inhibitor comprising 95% or more identity to any one of SEQ ID NOS: 3-50, SEQ ID NOS: 54-64, or SEQ ID NOS: 82- 150. In some embodiments, the method comprises the nucleic acid inhibitor comprising SEQ ID NOS: 17, 21, or 24. In some embodiments, the method comprises the nucleic acid inhibitor comprising SEQ ID NOS: 41, 45, or 48.
  • the method comprises the nucleic acid inhibitor comprises SEQ ID NOS: 57, 58, 59, 60, 61, or 62. In some embodiments, the method comprises the nucleic acid inhibitor comprises SEQ ID NOS : 63 or 64. Also disclosed herein is a method of screening for a nucleic acid inhibitor of ELAVL2, the method comprising contacting SEQ ID NO: 1 with complementary nucleic acids between 15-30 nucleotides long and determining which are effective at knocking down expression of ELAVL2.
  • the screening takes place inside of a cell.
  • the silencing RNA comprises an antisense oligonucleotide, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), small nucleolar RNA (sno-RNA), Piwi-interacting RNA (piRNA), or short hairpin RNA (shRNA) molecules.
  • siRNA short interfering RNA
  • dsRNA double stranded RNA
  • miRNA micro-RNA
  • small nucleolar RNA small nucleolar RNA
  • piRNA Piwi-interacting RNA
  • shRNA short hairpin RNA
  • FIGS. 1A, IB, and 1C show the Rbp9 (an ortholog of ELAVL2) knockdown (KD) in the eye also suppressed photoreceptor neuron degeneration and this suppressor effect is not due to a decrease in Poly(GR) levels in the fly eye. It was shown that Rbp9 knockdown by two independent RNAi suppressed poly(GR) toxicity in fly eye neurons (Panel A and B), confirming it is indeed a genetic suppressor. More importantly, rbp9 knockdown did not affect the expression level of poly(GR) (Panel C), showing rbp9 acts in a pathway downstream of poly(GR) expression.
  • Rbp9 an ortholog of ELAVL2 knockdown
  • FIG. 2 shows the screening of ASOs identified those that could effectively knockdown the expression level of ELAVL2 in iPSC-derived human C9ORF72- ALS/FTD patient motor neurons. Quantitative real-time PCR analysis confirming ASO-mediated reduction of ELAVL- 2 mRNA levels in human iPSC-derived motor neurons (3 weeks old). ASOs578-595 screened in C9 patient line40. ASOs 596-601 screened in C9patient line42. Expression of GAPDH mRNAs were used as endogenous controls. ELAVL-2 ASOs (5pM) treated for 72hr in two biologically independent experiment. Error bars represent mean ⁇ SD. FIGS.
  • FIG. 3A and 3B show confirmation of reduced ELAVL-2 mRNA and protein levels.
  • FIG. 3 A shows the quantitative real-time PCR analysis confirming ASO-mediated reduction of ELAVL-2 mRNA levels in human iPSC-derived motor neurons (3 weeks old).
  • ELAVL-2 AS Os (5pM) treated for one week in three biologically independent experiment. Error bars represent mean ⁇ SD.
  • FIG. 3B shows the western blot analysis confirming reduction in ELAVL2 protein levels in motor neurons treated with ELAVL2-ASO#599.
  • FIG. 4 shows ELAV2 is localized in both the nucleus and cytosol in iPSC-derived motor neurons.
  • FIG. 5 shows the generation of CRISPR deletion of ELAVL2 gene in two C9ORF72 iPSC lines.
  • FIGS. 6 A and 6B show the RNA-seq analysis of control (FIG. 6A) and C9ORF72 iPSC- derived (FIG. 6B) motor neurons with ELAVL2 knockdown.
  • FIGS. 7A and 7B show 24 specific antisense oligonucleotides (FIG. 7A) which were found to reduce expression of ELAVL2 (SEQ ID NOS: 3-24. Note that these correspond to the ASOs from FIG. 2); and the nucleic acid sequence encoding ELAVL2 (FIG. 7B), which shows where the specific ASOs from FIG. 7A bind.
  • FIG. 8 shows the quantitative real-time PCR analysis confirming siRNA-mediated reduction of ELAVL-2 mRNA levels in human iPSC-derived 2 weeks old motor neurons (isogenic control line). Expression of GAPDH mRNAs were used as endogenous controls. ELAVL-2 siRNAs (5pM ) treated for 72 h in one biologically independent experiment. Error bars represent mean + SD of two technical wells.
  • FIG. 9 shows the quantitative real-time PCR analysis confirming siRNA-mediated reduction of ELAVL-2 mRNA levels in human iPSC-derived 2 weeks old motor neurons.
  • ASOs2240-2249 screened in healthy control lines.
  • ASOs 2250-2259 screened in C9patient line.
  • Expression of GAPDH mRNAs were used as endogenous controls.
  • ELAVL-2 ASOs (5pM) treated for 72hr in two biologically independent experiment. Error bars represent mean ⁇ SD.
  • FIG. 10 shows the immunofluorescence analysis of ELAVL-2 protein cellular distribution in iPSC-derived motor neurons of 2 weeks old. Commercial ELAVL2 antibody used.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • an agent includes a plurality of agents, including mixtures thereof.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.
  • the statement that a formulation "may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • a “subject” means an individual.
  • the “subject” can include domesticated animals (e.g. , cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.
  • “Subject” can also include a mammal, such as a primate or a human.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • Detecting is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting a nucleic acid molecule in sample. Detection can include a physical readout, such as fluorescence output.
  • hybridization is defined as forming base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).
  • nucleic acid molecules which have been “isolated” include nucleic acids molecules purified by standard purification methods, as well as those chemically synthesized. Isolated does not require absolute purity, and can include nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.
  • nucleic acid is a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides.
  • a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer.
  • a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid.
  • modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA).
  • the major nucleotides of DNA are deoxyadenosine 5 '-triphosphate (dATP or A), deoxy guanosine 5 '-triphosphate (dGTP or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine 5 '-triphosphate (dTTP or T).
  • the major nucleotides of RNA are adenosine 5'- triphosphate (ATP or A), guanosine 5 '-triphosphate (GTP or G), cytidine 5 '-triphosphate (CTP or C) and uridine 5 '-triphosphate (UTP or U).
  • Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, as known in the art and discussed in more detail below.
  • modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 7-deaza-8-azaguanosine, 5-fluorouracil, 5 -bromouracil, 5 -chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine,
  • modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to: 2’ -substituted ribose analogues, arabinose, 2-fluoroarabinose, xylose, and hexose; morpholino sugar analogues, or bicyclic or tricyclic sugar moieties.
  • 2’ -substituted ribose groups may include 2’-fluoro, 2’-amino, 2’-O- methyl, 2’-(?-methoxyethyl, or other 2’-O-substitutions including alkyl, aryl, alkoxyalkyl, or aminoalkyl groups, among others.
  • modified components of the phosphate backbone include analogues such as a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or an analog thereof.
  • the phosphate linkage analogue may not necessarily include a phosphorus atom, as formacetal, amide, triazole or other linkage groups may replace the phosphate group in an oligonucleotide.
  • complementary binding occurs when the base of one nucleic acid molecule forms a hydrogen bond to the base of another nucleic acid molecule.
  • the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G).
  • the sequence 5'-ATCG-3' of one ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA.
  • the sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'.
  • Nucleic acid molecules can be complementary to each other even without complete hydrogen-bonding of all bases of each molecule. For example, hybridization with a complementary nucleic acid sequence can occur under conditions of differing stringency in which a complement will bind at some but not all nucleotide positions.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid molecule to proceed where there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid molecule (e.g., antisense oligonucleotide) to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or under conditions in which the assays are performed in the case of in vitro assays (e.g., hybridization assays).
  • the nucleic acid molecule e.g., antisense oligonucleotide
  • nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable or to specifically bind. That is, two or more nucleic acid molecules may be less than fully complementary and is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule.
  • a first nucleic acid molecule may have 10 nucleotides and a second nucleic acid molecule may have 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules, which may or may not form a contiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively.
  • complementary nucleic acid molecules may have wrongly paired bases - that is, bases that cannot form a traditional Watson- Crick base pair or other non- traditional types of pair (i.e., "mismatched" bases).
  • complementary nucleic acid molecules may be identified as having a certain number of "mismatches," such as zero or about 1, about 2, about 3, about 4 or about 5.
  • Perfectly or “fully” complementary nucleic acid molecules means those in which a certain number of nucleotides of a first nucleic acid molecule hydrogen bond (anneal) with the same number of residues in a second nucleic acid molecule to form a contiguous doublestranded region.
  • two or more fully complementary nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form one doublestranded region, with or without an overhang) or have a different number of nucleotides (e.g., one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand).
  • polymorphism is a variation in a gene sequence.
  • the polymorphisms can be those variations (DNA sequence differences, e.g., substitutions, deletions, or insertions) which are generally found between individuals or different ethnic groups and geographic locations which, while having a different sequence, produce functionally equivalent gene products.
  • the term can also refer to variants in the sequence which can lead to gene products that are not functionally equivalent.
  • Polymorphisms also encompass variations which can be classified as alleles and/or mutations which can produce gene products which may have an altered function.
  • Polymorphisms also encompass variations which can be classified as alleles and/or mutations which either produce no gene product or an inactive gene product or an active gene product produced at an abnormal rate or in an inappropriate tissue or in response to an inappropriate stimulus. Alleles are the alternate forms that occur at the polymorphism.
  • Polymorphisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation.
  • sequence identity is expressed in terms of the identity between the sequences.
  • Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
  • NCBI National Center for Biotechnology
  • NCBI National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894
  • BLASTN is used to compare nucleic acid sequences
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75. 15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.
  • sample such as a biological sample
  • biological samples include all clinical samples useful for detection of a methylated nucleotide, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; urine; sputum; or CVS samples.
  • a sample includes blood obtained from a human subject, such as whole blood or serum.
  • test nucleic acid molecule refers to a nucleic acid molecule whose detection, quantitation, qualitative detection, characterization, or a combination thereof, is intended.
  • the test nucleic acid molecule can be a defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of DNA or RNA containing a gene or portion thereof of interest).
  • the nucleic acid molecule need not be in a purified form.
  • Various other nucleic acid molecules can also be present with the test nucleic acid molecule.
  • test nucleic acid molecule can be a specific nucleic acid molecule (which can include RNA or DNA), for which the detection of a particular polymorphism is intended.
  • a test nucleic acid includes a viral nucleic acid molecule, or a bacterial nucleic acid molecule. Purification or isolation of the test nucleic acid molecule, if needed, can be conducted by methods known to those in the art, such as by using a commercially available purification kit or the like.
  • contacting means placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fully- and partially- solvated molecules.
  • RNA ribonucleic acid
  • RNA includes double-stranded (ds) RNA, singlestranded (ss) RNA, isolated RNA (such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), altered RNA (which differs from naturally occurring RNA by the addition, deletion, substitution or alteration of one or more nucleotides), or any combination thereof.
  • altered RNA can include addition of nonnucleotide material, such as at one or both ends of an RNA molecule, internally at one or more nucleotides of the RNA, or any combination thereof.
  • Nucleotides in RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as naturally occurring nucleotides, non-naturally occurring nucleotides, chemically-modified nucleotides, deoxynucleotides, or any combination thereof. These altered RNAs may be referred to as analogs or analogs of RNA containing standard nucleotides (i.e., standard nucleotides, as used herein, are considered to be adenine, cytidine, guanidine, thymidine, and uridine).
  • Antisense activity means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
  • Antisense compound means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • Antisense inhibition means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
  • dsRNA refers to any nucleic acid molecule comprising at least one ribonucleotide molecule and capable of inhibiting or down regulating gene expression, for example, by promoting RNA interference ("RNAi") or gene silencing in a sequence-specific manner.
  • RNAi RNA interference
  • the dsRNAs of the instant disclosure may be suitable substrates for Dicer or for association with RISC to mediate gene silencing by RNAi.
  • One or both strands of the dsRNA can further comprise a terminal phosphate group, such as a 5 '-phosphate or 5', 3 '- diphosphate.
  • dsRNA molecules in addition to at least one ribonucleotide, can further include substitutions, chemically-modified nucleotides, and non-nucleotides.
  • dsRNA molecules comprise ribonucleotides up to about 100% of the nucleotide positions.
  • dsRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example, meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siNA), siRNA, micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide, chemically-modified dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), or the like.
  • a double- stranded structure may be formed by a self-complementary nucleic acid molecule or by annealing of two or more distinct complementary nucleic acid molecule strands.
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • dsRNA molecules of this disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level or any combination thereof.
  • target nucleic acid refers to any nucleic acid sequence whose expression or activity is to be altered (e.g., ELAVL2).
  • the target nucleic acid can be DNA, RNA, or analogs thereof, and includes single, double, and multi-stranded forms.
  • target site or “target sequence” is meant a sequence within a target nucleic acid (e.g., mRNA) that, when present in an RNA molecule, is “targeted” for cleavage by RNAi and mediated by a dsRNA construct of this disclosure containing a sequence within the antisense strand that is complementary to the target site or sequence.
  • off-target effect or “off-target profile” refers to the observed altered expression pattern of one or more genes in a cell or other biological sample not targeted, directly or indirectly, for gene silencing or antisense therapy.
  • an off-target effect can be quantified by using a DNA microarray to determine how many non-target genes have an expression level altered by about two-fold or more in the presence of a candidate antisense oligonucleotide or siRNA, or analog thereof specific for a target sequence, such as an ELAVL2 mRNA.
  • gene as used herein, especially in the context of "target gene” or “gene target” for gene silencing means a nucleic acid molecule that encodes an RNA or a transcription product of such gene, including a messenger RNA (mRNA, also referred to as structural genes that encode for a polypeptide), an mRNA splice variant of such gene, a functional RNA (fRNA), or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), microRNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
  • mRNA messenger RNA
  • fRNA functional RNA
  • ncRNA non-coding RNA
  • stRNA small temporal RNA
  • miRNA microRNA
  • snRNA small nuclear RNA
  • siRNA small nucleolar RNA
  • rRNA
  • gene silencing refers to a partial or complete loss-of-function through targeted inhibition of gene expression in a cell, which may also be referred to as RNAi "knockdown,” “inhibition,” “down-regulation,” or “reduction” of expression of a target gene, such as a human FGF2 gene.
  • RNAi knockdown
  • inhibition inhibition
  • down-regulation or “reduction” of expression of a target gene
  • the extent of silencing may be determined by methods described herein and known in the art (see, e.g., PCT Publication No. WO 99/32619; Elbashir et ah, EMBO J. 20:6S77, 2001).
  • quantification of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of this disclosure, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA level or protein level or activity, for example, by equal to or greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
  • the term "therapeutically effective amount” means an amount of antisense oligonucleotide(s) or RNAi that is sufficient to result in a decrease in severity of disease symptoms, an increase in frequency or duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease, in the subject (e.g., human) to which it is administered.
  • One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on such factors as the subject's size, the severity of symptoms, and the particular composition or route of administration selected.
  • the nucleic acid molecules of the instant disclosure individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease, disorder, or condition, the nucleic acid molecules can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment.
  • ALS Amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • ALS occurs in both familial (ELAF) and sporadic (ELAE) forms.
  • ELAF familial
  • ELAE sporadic
  • a significant number of ELAF cases are associated with expansions of a non-coding G4C2 hexanucleotide repeats in the C9ORF72 gene.
  • FTD familial frontotemporal dementia
  • FTD familial frontotemporal dementia
  • ⁇ 5% of ELAE ⁇ 5% of ELAE.
  • the closest mammalian homolog of the fly gene rbp9 is ELAVL2.
  • Multiple AS Os targeting human ELAVL2 were designed and screened to determine their knockdown efficiency in human iPSC-derived motor neurons. Those ASOs which were found to be effective are found in Table 1 that are SEQ ID NOS: 3-26 and 27-50, and Table 5 that are SEQ ID NOS: 63-64 and 133-150.
  • Multiple siRNA’s targeting ELAVL2 were also designed and screened to determine their knockdown efficiency. The siRNAs which were found to be effective are found in Table 3 that are SEQ ID NOS: 57-62 and 99-132.
  • nucleic acid inhibitors and methods of using them, to target these modifier genes. This can offer clinical benefits because they may slow down or block ongoing disease processes, even in the presence of disease-causing G4C2 repeats RNA and DPR proteins. Also disclosed are methods of screening for other inhibitors of ELAVL2 which can be used to treat diseases and disorders related to G4C2 expansion.
  • nucleic acid inhibitor which is capable of reducing expression of ELAVL2.
  • This nucleic acid molecule can reduce expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any amount in between or below these numbers. This reduction in expression is compared to a control where the nucleic acid is not present.
  • One of skill in the art will understand how to compare the results of inhibition by the nucleic acid inhibitor compared to a control where a negative control sequence or no nucleic acid inhibitor is present.
  • SEQ ID NO: 1 represents the genomic locus of chromosome 9 which encodes ELAVL2 and includes introns as well as exons (from human hg38 assembly, chr9:23689906- 23826667, reverse complement).
  • SEQ ID NO: 2 represents the nucleic acid which specifically encodes ELAVL2.
  • Contemplated herein is a nucleic acid inhibitor which hybridizes to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides within SEQ ID NO: 1 or SEQ ID NO: 2.
  • Contemplated herein is a nucleic acid inhibitor which hybridizes to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • nucleic acid inhibitor which hybridizes to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • nucleic acid inhibitor does not need to hybridize completely within SEQ ID NO: 1 or SEQ ID NO: 2, but should hybridize substantially enough within this region as to cause an inhibition in expression of ELAVL2. This is referred to herein as “partial hybridization” within the specified region.
  • partial hybridization within the specified region.
  • nucleic acid inhibitor which hybridizes completely or partially within certain areas of SEQ ID NO: 1 or 2. For example, there are certain regions of SEQ ID NO: 2 which can be considered “hot spots’’ so that nucleic acid inhibitors designed against those regions show very high probability of abrogating ELAVL2 expression.
  • nucleic acid inhibitors that hybridize to at least 5 contiguous nucleotides within the region of nucleotide number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • nucleic acid inhibitors that hybridize to at least 5 contiguous nucleotides within the region of nucleotide number 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108 , 2109, 2110, 2111, 2112 , 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123 , 2124, 2125, 2126, 2127 , 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138 , 2139, 2140, 2141, 2142 , 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153 , 2154, 2155, 2156, 2157 , 2158, 21
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOS: 3-50, 63-64, and 133-150.
  • SEQ ID NOS: 3-50 can be found in Table 1, and it is noted that SEQ ID NOS: 3-26 correspond to unmodified nucleic acid inhibitors, while SEQ ID NOS: 27-50 are examples of modified nucleic acid inhibitors.
  • SEQ ID NOS: 63-64 and 133-150 which are also examples of modified nucleic acid inhibitors, can be found in Table 5.
  • nucleotides prefaced with “e” are 2’-0-M0E nucleotides
  • nucleotides prefaced with “d” are deoxy nucleotides
  • # indicates phosphorothioate (PS) linkage.
  • PS phosphorothioate
  • an inhibitor for use in the central nervous system, can be synthesized with phosphorothioate or phosphoramidate linkages at the terminal positions, phosphodiester linkages between sugar-modified nucleotides, phosphorothioate linkages throughout the stretch of deoxynucleotides.
  • a subset of positions within the deoxynucleotide stretch can be modified with a phosphonate or phosphoramidate linkage (for example 1 , 2, or 3 linkages within the stretch of deoxynucleotides might typically be modified with a phosphonate or phosphoramidate linkage).
  • Other patterns of backbone modifications are known to one of skill in the art.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOS: 54-62 and 82-132.
  • SEQ ID NOS: 54-62 and 82-132 can be found in Table 3, and it is noted that SEQ ID NO: 54-56 and 82-98 are examples of unmodified nucleic acid inhibitors, SEQ ID NO: 57-59 and 99-115 are examples of modified nucleic acid inhibitors targeting an antisense (as) strand, and SEQ ID NO: 60-62 and 116-132 are examples of modified nucleic acid inhibitors targeting a sense strand.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 24.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 45.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 63. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 64.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 54. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 55.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 56.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 57. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 58.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 59.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 60. Further, disclosed are nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 61.
  • nucleic acid inhibitors comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62.
  • the nucleic acid inhibitor comprises SEQ ID NO: 17. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 21. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 24. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 41. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 45. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 48.
  • the nucleic acid inhibitor comprises SEQ ID NO: 63. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 64.
  • the nucleic acid inhibitor comprises SEQ ID NO: 54. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 55. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 56.
  • the nucleic acid inhibitor comprises SEQ ID NO: 57. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 58. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 59.
  • the nucleic acid inhibitor comprises SEQ ID NO: 60. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 61. In some embodiments, the nucleic acid inhibitor comprises SEQ ID NO: 62.
  • nucleotides prefaced with “m” are 2’-O-methyl
  • nucleotides prefaced with “f” are 2’-fluoro
  • the strands prefaced with “P” are 5 ’-phosphate
  • # indicates phosphorothioate (PS) linkage.
  • 5 ’-phosphate analogues for example vinylphosphate or substituted phosphates, can be used.
  • nucleic acid inhibitor described herein can be any length.
  • a typical antisense oligonucleotide is typically between 15 and 30 nucleotides long.
  • contemplated herein are nucleic acids which are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long, or longer.
  • the nucleic acid inhibitor described herein can be an antisense oligonucleotide (ASO) or an RNAi molecule, such as siRNA.
  • ASO antisense oligonucleotide
  • siRNA RNAi molecule
  • Types of antisense and RNAi molecules are disclosed in Watts et al. (Watts JK, Corey DR. Silencing disease genes in the laboratory and the clinic. J Pathol. 2012 Jan;226(2):365-79. doi: 10.1002/path.2993).
  • Also contemplated herein is short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), small nucleolar RNA (sno-RNA), Piwi-interacting RNA (piRNA), or short hairpin RNA (shRNA) molecules.
  • the nucleic acid inhibitor is an antisense oligonucleotide or an RNAi molecule
  • it can be modified.
  • modifications include, but are not limited to, at least one modified sugar moiety; at least one modified inter-nucleotide linkage; and/or at least one modified nucleotide.
  • modifications can include, for example, those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins.
  • the internucleoside phosphate linkages such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins.
  • modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3', 5' -substituted oligonucleotide having a sugar which, at both its 3' and 5’ positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position).
  • Other modified oligonucleotides are capped with a nuclease resistance- conferring bulky substituent at their 3' and/or 5' end(s), or have a substitution in one nonbridging oxygen per nucleotide.
  • Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.
  • modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.
  • Oligonucleotides which are self-stabilized are also considered to be modified oligonucleotides useful in the methods of the invention (Tang et al . (1993) Nucleic Acids Res. 20:2729-2735).
  • oligonucleotides comprise two regions: a target hybridizing region; and a self-complementary region having an oligonucleotide sequence complementary to a nucleic acid sequence that is within the self-stabilized oligonucleotide.
  • Such modifications can comprise an intemucleotide linkage comprising: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, and/or carboxymethyl ester.
  • the at least one modification can comprise a modified nucleotide comprises a peptide nucleic acid, a morpholino analogue, a locked nucleic acid (LNA) or other bridged or bicyclic nucleic acid analogue, and/or a combination thereof.
  • the at least one modification can comprise a modified sugar moiety comprises: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, and/or a 2’-O-alkyl modified sugar moiety.
  • the nucleic acid inhibitor described herein can include a conjugate.
  • conjugates are known to those of skill in the art. Examples of modifications can be found in Winkler (Oligonucleotide conjugates for therapeutic applications. Ther Deliv. 2013 Jul;4(7) :791 -809), herein incorporated by reference in its entirety for its teaching concerning conjugates.
  • Nucleic acid inhibitors described herein can be conjugated to a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life.
  • the conjugate can comprise a therapeutic agent, a diagnostic agent, or an agent that increases cell uptake or penetration. More specifically, the conjugate can comprise a peptide, a protein, an antibody or fragment thereof, a lipid, a neurotransmitter, a cationic polymer, a nanoparticle, a probe, or a carbohydrate.
  • the conjugate can be a multivalent conjugate, such as a multimeric antisense oligonucleotide or multivalent siRNA, for example.
  • multivalent siRNA it can suppress one gene at several sites, or suppress multiple genes all at once.
  • ASO monomers to one or more targets can be co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers, for example, that are stable outside of cells, but cleaved inside cells, releasing the active ASO monomers.
  • vectors such as viral vectors, which can be used to deliver the nucleic acid inhibitors.
  • viral vectors which can be used to deliver the nucleic acid inhibitors.
  • ASOs nucleic acids of interest
  • the viral vector can also be used to induce intracellular expression of the nucleic acid inhibitors.
  • Antisense sequences of the invention may be delivered alone in vivo or in association with a vector.
  • a “vector” is any vehicle capable of facilitating the transfer of the antisense sequence to the cells and preferably cells expressing ELAVL2.
  • the vector transports the antisense sequence to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the ASO sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: lend virus such as HIV-1, retrovirus, such as moloney murine leukemia virus, adenovirus, adeno-associated virus (AAV); SV40-type viruses; Herpes viruses such as HSV-1 and vaccinia virus.
  • lend virus such as HIV-1
  • retrovirus such as moloney murine leukemia virus, adenovirus, adeno-associated virus (AAV)
  • SV40-type viruses such as Herpes viruses such as HSV-1 and vaccinia virus.
  • Herpes viruses such as HSV-1 and vaccinia virus.
  • Retrovirus-based and lentivirus-based vectors that are replication-deficient (i.e., capable of directing synthesis of the desired AON, but incapable of producing an infectious particle) have been approved for human gene therapy trials. They have the property to integrate into the target cell genome, thus allowing for a persistent transgene expression in the target cells and their progeny.
  • the ASO can be delivered using an AAV vector.
  • the human parvovirus Adeno-Associated Virus is a dependovirus that is naturally defective for replication which can be repurposed into a vector able to elicit prolonged expression of therapeutic cargo within infected cells.
  • the present invention relates to an AAV vector comprising or expressing a nucleic acid inhibitor (antisense sequence or interfering RNA), targeting ELAVL2.
  • the present invention relates to an AAV vector comprising an ASO as described above, targeting ELAVL2.
  • the nucleic acid inhibitors of the present invention can be presented in the form of a pharmaceutical composition.
  • the pharmaceutical composition can comprise, for example, a vehicle for delivery of the nucleic acid inhibitor.
  • a pharmaceutical composition of the present invention may also include a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc.
  • the composition will generally be in the form of a liquid, although this needs not always to be the case.
  • Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, mineral oil, etc.
  • the formulation can also include lubricating agents, wetting agents, emulsifying agents, preservatives, buffering agents, etc.
  • the present invention involves the administration of a nucleic acid inhibitor and is thus somewhat akin to gene therapy.
  • nucleic acids are often delivered in conjunction with lipids (e.g. cationic lipids or neutral lipids, or mixtures of these), frequently in the form of liposomes or other suitable micro- or nanostructured material (e.g. micelles, lipocomplexes, dendrimers, emulsions, cubic phases, etc.).
  • lipids e.g. cationic lipids or neutral lipids, or mixtures of these
  • suitable micro- or nanostructured material e.g. micelles, lipocomplexes, dendrimers, emulsions, cubic phases, etc.
  • compositions of the invention are generally administered via enteral or parenteral routes, e.g. intravenously (i.v.), intra-arterially, subcutaneously, intramuscularly (i.m.), intracerebrally, intracerebroventricularly (i.c.v.), intrathecally (Lt.), intraperitoneally (i.p.), although other types of administration are not precluded, e.g. via inhalation, intranasally, topical, rectally, intraosseous, eye drops, ear drops administration, etc.
  • enteral or parenteral routes e.g. intravenously (i.v.), intra-arterially, subcutaneously, intramuscularly (i.m.), intracerebrally, intracerebroventricularly (i.c.v.), intrathecally (Lt.), intraperitoneally (i.p.), although other types of administration are not precluded, e.g. via inhalation, intranasally, topical, rectally,
  • sterile injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispensing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • delivery may be either local (i.e. in situ, directly into tissue such as muscle tissue) or systemic, usually delivery will be local to affected muscle tissue, e.g. to skeletal muscle, smooth muscle, heart muscle, etc.
  • techniques such as electroporation, sonoporation, a “gene gun” (delivering nucleic acid-coated gold particles), etc. may be employed.
  • nucleic acid inhibitors to treat disorders and diseases associated with poly(GR) toxicity in neuronal cells in a subject in need thereof, the method comprising modulating expression of ELAVL2.
  • the poly(GR) toxicity can be caused by a GGGGCC (G4C2) repeat expansion in a first intron of C9ORF72.
  • G4C2 repeat expansion is associated with ALS and FTD. Therefore, disclosed herein is a method of treating or preventing ALS or FTD by using nucleic acid inhibitors to modulate expression of ELAVL2. Further disclosed herein is a method of treating disorders and diseases other than those that are C9ORF72 -related.
  • treat is meant to reduce the symptoms or clinical manifestations by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control where a nucleic acid inhibitor of ELAVL2 is not given.
  • One measurement to determine the effectiveness of treatment could be the reduction in the death of motor neurons in a subject being given the nucleic acid inhibitors of ELAVL2.
  • ALS ALS
  • the symptoms of ALS which can be reduced include, but are not limited to, muscle twitches in the arm, leg, shoulder, or tongue; muscle cramps; tight and stiff muscles (spasticity); muscle weakness affecting an arm, a leg, the neck, or diaphragm; slurred and nasal speech; or difficulty chewing or swallowing.
  • the reduction in clinical manifestation can be measured by measuring the frontal and/or temporal lobes, as FTD is known to cause them to shrink. Therefore, the nucleic acid inhibitors disclosed herein can reduce the shrinkage of these lobes.
  • Symptoms of FTD which can be ameliorated by the nucleic acid inhibitors disclosed herein include, but are not limited to, changes in their personalities and become socially inappropriate, impulsive or emotionally indifferent, or loss of language. These can be measured by a clinician, and effectiveness of the treatments disclosed herein can be determined by monitoring symptoms or by monitoring clinical manifestations.
  • the amount of a nucleic acid inhibitor to be administered will be an amount that is sufficient to induce amelioration of unwanted ALS or FTD symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition of the patient, etc. and may be determined on a case by case basis. The amount may also vary according to other components of a treatment protocol (e.g. administration of other medicaments, etc.).
  • a suitable dose is generally in the range of from about 0.5 mg to about 500 mg, and more usually from about 5 mg to about 150 mg per dose. In a specific example, the dose can be between 8 mg to 100 mg. Such doses are typically repeated after several weeks to several months. In a specific example, the treatment is given every three to four months.
  • nucleic acid inhibitors ASOs or siRNAs, for example
  • ASOs or siRNAs may be delivered using conjugation or formulation approaches that allow delivery to the central nervous system through the bloodstream, but these approaches would typically require higher doses.
  • a viral -based delivery of the nucleic acid inhibitor will depend on different factors such as the virus that is employed, the route of delivery (intramuscular, intravenous, intraarterial or other), but may typically range from 10 9 to 10 15 viral particles/kg. Those of skill in the art will recognize that such parameters are normally worked out during clinical trials. Further, those of skill in the art will recognize that, while disease symptoms may be completely alleviated by the treatments described herein, this need not be the case. Even a partial or intermittent relief of symptoms may be of great benefit to the recipient.
  • treatment of the patient may be a single event, or the patient is administered with the ASOs on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart.
  • This screen can, in particular, be directed to the regions of the ELAVL2 locus described above, such as between nucleotides 1-450 and 2100-3983 of SEQ ID NO: 2.
  • tools can be used to screen for potential nucleic acid inhibitors. For example, Gagnon et al. (Gagnon KT, Corey DR. Guidelines for Experiments Using Antisense Oligonucleotides and Double-Stranded RNAs.
  • Nucleic Acid Ther. 2019 Jun;29(3): 116- 122) provides detailed examples of how to screen for ASOs, and this reference is incorporated in its entirety for its teaching concerning screening for ASOs. Also disclosed herein are nucleic acid inhibitors identified by these methods which are capable of targeting ELAVL2 nucleic acid.
  • Example 1 Antisense Oligonucleotides (ASOs) effective against EL VL2 (set 1).
  • SEQ ID NOS: 3-26 correspond to unmodified nucleic acid inhibitors, while SEQ ID NOS: 27-50 are examples of modified nucleic acid inhibitors. Referring to SEQ ID NOS: 27- 50, the nucleotides prefaced with “e” are 2’-0-M0E nucleotides, and the nucleotides prefaced with “d” are deoxy nucleotides. # indicates phosphorothioate (PS) linkage.
  • Table 2 Top 3 ELAVL2-ASQ’s (set 1) from Table 1.
  • Example 2 Tables of nucleotide sequences of ELAVL2-siRNAs.
  • SEQ ID NOS: 51-56 and 65-98 correspond to unmodified mRNA and siRNA inhibitors, while SEQ ID NOS: 57-62 and 99-132 are examples of modified anti-sense (as) and sense strand siRNAs.
  • SEQ ID NOS: 57-62 and 99- 132 the nucleotides prefaced with “m” are 2’-O-methyl, the nucleotides prefaced with “f” are 2 ’-fluoro, and as strands prefaced with “P” are 5 ’-phosphate. # indicates phosphorothioate (PS) linkage.
  • TegChoI a 3’ TEG-linked cholesterol conjugate used to improve delivery to cells in culture. In vivo these can be delivered, for example, as divalent asymmetric siRNAs or C16-conjugated siRNAs. Sequences disclosed herein can be used with or without TegChoI.
  • Table 4 Top 3 ELAVL2-siRNAs from Table 3.
  • Example 3 Tables of nucleotide sequences of ELAVL2-ASO’s (set 2)
  • SEQ ID NOS: 133-150 are examples of modified nucleic acid inhibitors. Referring to SEQ ID NOS: 133-150, the nucleotides prefaced with “e” are 2’-0-M0E nucleotides, and the nucleotides prefaced with “d” are deoxy nucleotides. # indicates phosphorothioate (PS) linkage.
  • Table 6 Top 2 ELAVL2-ASO’s (set 2) from Table 5.
  • Example 4 Poly(GR) as a neurotoxic species.
  • DPR dipeptide repeat
  • poly(GR) is a key neurotoxic species causing neurodegeneration in cellular and animal models.
  • DPR dipeptide repeat
  • poly(GR) expression in flies or human motor neurons are highly toxic.
  • an inducible mouse model was established in which (GR)80 is expressed in the brain at -5-15% of the level in C9ORF72 patient brains and demonstrated that low-level poly(GR) expression alone can elicit several FTD/ALS-like behavioral and cellular phenotypes.

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Abstract

Sont divulguées des compositions et des procédés pour réduire l'expression d'ELAVL2. Il a été déterminé que l'expansion de répétition GGGGCC (G4C2) dans le premier intron de C9ORF72 est la cause génétique la plus communément connue de la sclérose latérale amyotrophique (SLA) et de la démence frontotemporale (FTD). La réduction de l'expression de l'ELAVL2 réduit l'effet aval de l'expansion de répétition G4C2, ce qui permet de fournir des méthodes de traitement pour des maladies et des troubles associés à G4C2.
PCT/US2023/076274 2022-10-07 2023-10-06 Procédés et compositions pour le silençage de l'expression d'elavl2 pour le traitement d'une maladie WO2024077262A2 (fr)

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