WO2020107037A1

WO2020107037A1 - Compounds and methods for treating neurodegenerative diseases

Info

Publication number: WO2020107037A1
Application number: PCT/US2019/063113
Authority: WO
Inventors: Daniel A. MORDES; Kevin C. Eggan; Lee L. Rubin
Original assignee: President And Fellows Of Harvard College
Priority date: 2018-11-25
Filing date: 2019-11-25
Publication date: 2020-05-28
Also published as: WO2020107037A8

Abstract

Disclosed are compounds and methods for treating diseases and conditions related to toxic dipeptide repeats including Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD). Also disclosed are method of screening for agents that treat or prevent diseases and conditions related to toxic dipeptide repeats.

Description

COMPOUNDS AND METHODS FOR TREATING

NEURODEGENERATIVE DISEASES

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No.

62/771,149, filed on November 25, 2018, and U.S. Provisional Application No. 62/772,569, filed on November 28, 2019, the contents of which are hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Neurodegenerative diseases represent a major health, financial and emotional burden to society. One particularly devastating neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS) also known as Lou Gehrig’s disease. ALS is characterized by the progressive loss of motor neurons in the brain and spinal cord resulting in severe muscle atrophy and paralysis. ALS often affects patients in their 50s with survival typically limited to just 2-5 years from the time of disease onset. The only two FDA-approved therapies for ALS, riluzole and edavarone, demonstrate limited beneficial effects in select patient populations. ALS is on the same disease spectrum on a related neurodegenerative disease called frontotemporal dementia (FTD). FTD is characterized by devastating changes in behavior, personality, and language due to the progressive loss of cortical neurons in the brain. It is the second most common type of dementia for patients under 65 years old. There are no effective treatments to slow disease progression in FTD.

SUMMARY OF THE INVENTION

[0003] The most common genetic contributor to ALS is the presence of a hexanucleotide repeat expansion ( GGGGCC) mutation in the first intron of the gene

C90RF72. This variant is also commonly found in patients with FTD and with both ALS and FTD. The presence of this repeat expansion leads to the production of a variety of dipeptide repeat proteins (DPRs) which are detectable throughout the brain of affected individuals. Several DPRs have been shown to be toxic in model organisms, primary neurons, and human cell lines. We have established an in vitro model of C9<9///_’72-ALS/FTD disease using stem cell-derived neurons and synthetic toxic DPRs (Mordes et al, 2018). We have further developed this model to a high-throughput screening (384-well plate) format for chemical screening and drug testing. In this newer assay, we treat NGN2-cortical-like human neurons with toxic DPRs. We used this innovative translational approach to identify drug targets and potential small molecules for the treatment of C9( /i/_' 72-ALS/FTD.

[0004] Some aspects of the disclosure are directed to a method of treating or preventing a neurological disease, disorder or condition in a subject in need thereof, comprising administering to the subject an effective amount of an agent that reduces or eliminates dipeptide repeat (DPR) toxicity (e.g., GR-DPR and/or PR-DPR toxicity). In some embodiments, the subject has a mutation associated with production of DPR. In some embodiments, the mutation is a hexanucleotide repeat expansion (GGGGCC) mutation in the first intron of the gene C90RF72. In some embodiments, the DPR comprises poly-glycine- arginine (poly-GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats. In some embodiments, the neurological disease is one or both of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD). In some embodiments, the agent affects a pathway shown in FIG. 14. In some embodiments, the pathway is an adenosine A2A receptor, Aromatase, or PARP pathway. In some embodiments, the agent is a poly ADP ribose polymerase (PARP) inhibitor (e.g., a PARP inhibitor with CSF penetration). In some embodiments, the PARP inhibitor selectively inhibits one or more of PARP 1, PARP2, PARP3, PARP4, PARP9,

PARP 10, PARP 12, PAPR13, PAPR14, PARP 15, PARP 16, TNKS1, or TNKS2. In some embodiments, the PARP inhibitor selectively inhibits at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13 PARPs. In some embodiments, the PARP inhibitor selectively inhibits PARP1 and PARP2, PARP2 and PARP3, PARP1-PARP3, PARP1-PARP4, PARP1- PARP4 and PARP 15 -PARP 16, or PARP1-PARP4 and PARP 16.

[0005] In some embodiments, the PARP inhibitor is selected from Olaparib (AZD- 2281), AZD-2461, Niraparib (MK-4827), Rucaparib (AG-14699), AG-14361, Veliparib (ABT-888), Talazoparib (BMN673), CEP 9722, E7016, BGB-290, 3-aminobenzamide (INO- 1001), NMSpl 18, A-966492, PJ34 HC1, UPF 1069, ME0328, Pamiparib (BGB-290), NMS- P118, E7449, Picolinamide, Benzamide, NU1025, Iniparib (BSI-201), Cyproterone Acetate, Exemestane, Lafutidine, CGS-21680 HC1, Entocapone, PD184352, Ouabain, BGP-15 2HC1, IKK- 16, Istradefylline, KU0058948, DPQ, NU1025, EB-47, DiQ, DR2313, 4-ANI, ISQ, 2- hydroxybenzaminde, CNQ, 3-AB, INH2BP, PJ34, 6(5H)-phenanthridinone, 3-methyl-5-AIQ, TIQ-A, XAV939, or a pro-drug, analog or derivative thereof.

[0006] In some embodiments, the subject is also administered a second agent to treat or prevent a neurological disease, disorder or condition. In some embodiments, the second agent is antisense oligonucleotides (ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs. In some embodiments, the second agent is Edaravone or Riluzole.

[0007] In some embodiments, the subject does not have TDP-43 pathology in neuronal cells. In some embodiments, the subject does not have acute TDP-43 pathology. In some embodiments, the subject is at risk for developing a neurological disease, disorder or condition (e.g., has a mutation associated with, e.g., ALS and/or FTD). In some embodiments, the agent is orally administered. In some embodiments, the subject is a human.

[0008] Some aspects of the disclosure are directed to a composition comprising an agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell. In some

embodiments, the agent is a poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, the composition further comprises a second agent that is antisense

oligonucleotides (ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0009] Some aspects of the disclosure are directed to a method of screening for a candidate agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell, comprising providing a composition comprising neuronal cells contacted with a dipeptide repeat (DPR), contacting the composition with a test agent, comparing the viability of the neuronal cells contacted with the test agent to control neuronal cells not contacted with the test agent, and identifying a test agent as a candidate agent if the viability of the neuronal cells contacted with the test agent are increased as compared to the control neuronal cells not contacted with the test agent. In some embodiments, the DPR comprises poly-glycine-arginine (poly-GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats. In some embodiments, the neuronal cells are stem cell-derived. In some embodiments, the neuronal cell are cortical-like neurons, upper motor neurons, or lower motor neurons. In some embodiments, the screening protocol is the screening protocol shown in FIG. 10.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0011] FIGS. I A-1B show a screen of chemical modifiers of DPR toxicity. FIG. I A shows a screen of stem cell-derived NGN2-cortical-like human neurons contacted with poly- glycine-arginine (poly-GR) toxic dipeptide repeats (DPR) and a commercial library of compounds. An increased viability score indicates that the compound more effectively modified poly-GR toxicity. FIG. IB shows that screens of compounds against poly-GR toxicity and against poly-proline-arginine (poly-PR) toxicity using stem cell-derived NGN2- cortical-like human neurons identified 8 compounds effective against both poly-GR toxicity and poly-GR toxicity.

[0012] FIGS. 2A-2B show plots testing various PARP inhibitors at constant concentrations (and I 7-AAG, an Hsp90 inhibitor) in assays with stem ceil derived human neurons and toxic DPR. FIG. I A shows testing with NGN2 cortical-like neurons and FIG. IB show's testing with lower motor neurons (IMNs) derived from stem cells based on NGN2 expression and patterning factors. There are differences among their effects. ABT-888 (veliparib) was one of the compounds in the primary screen shown in FIG. I. BMN673 (talazoparib) may provide a greater effect.

[0013] FIG. 3 shows a Drug Discovery and Development Timeline.

[0014] FIGS. 4A-4B show a small molecule screen in stem-cell derived neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Adapted from Yang et al, Cell Stem Cell , 12(6): 713-726 (2013).

[0015] FIG. 5 shows approved therapies for ALS.

[0016] FIG. 6 shows neurological manifestations of C9QRF72 hexanucleotide (GGGGCC)repeal expansion.

[0017] FIGS. 7A-7J show detection of dipeptide repeats in C90RF72-ALS/FTD patients. Adapted from Mon et al, Science (2013). [0018] FIG. 8 shows arginine-containing DPRs are toxic in Drosophila models. Adapted from Mizielinska et al. Science 2014.

[0019] FIG. 9 shows arginine-containing DPRs are toxic in Human Cortical-like Neurons.

[0020] FIG. 10 shows a screening protocol for finding agents that reduce or eliminate DPR toxicity in neurons.

[0021] FIG. 11 shows the results of a primary screen.

[0022] FIG. 12 is a micrograph showing control stem cell-derived neurons (left side) and stem cell-derived neurons contacted with DPR (left side), clearly showing that contact with DPR is toxic.

[0023] FIG. 13 shows a screen wherein stem cell-derived neurons and GR-DPR are contacted with test compounds and cell viability is measured.

[0024] FIG. 14 shows the pathways affected by the compounds found in the assay of FIG. 13 to reduce GR-DPR toxicity.

[0025] FIG. 15 shows that the adenosine A2A receptor, Aromatase, and PARP pathways each have more than one hit for compounds that reduce GR-DPR toxicity.

[0026] FIG. 16 shows a screen wherein stem cell-derived neurons and PR-DPR are contacted with test compounds and cell viability is measured.

[0027] FIG. 17 shows that the c-MET and PARP pathways each have more than one hit for compounds that reduce PR-DPR toxicity.

[0028] FIG. 18 shows PARP inhibitors.

[0029] FIG. 19 shows Velaparib is a known anti -cancer therapeutic.

[0030] FIG. 20 details the mechanism of action for PARP inhibitors.

[0031 ] FIG. 21 shows a secondary test of selected top compounds in NGN2 neurons at a single dose.

[0032] FIG. 22 shows the relative selectivity of PARP inhibitors. Adapted from Wahlberg et ak, Nature Biotechnology (2012).

[0033] FIG. 23 shows the expression of selected PARPs in NGN2 neurons. Adapted fromNehme et al, Cell Reports (2018).

[0034] FIG. 24 shows micrographs detailing increased expression of PARP 1 in ALS patient brains. Adapted from Appel et al, Neurology (2004). [0035] FIG. 25 provides proposed mechanisms for C90RF72-ALS/FTD. See Taylor JP, Nature (2016) 539(7628): 197-206.

[0036] FIGS. 26A-26E demonstrate identification of C90RF72-associated transcriptional changes. FIG. 26A provides a diagram of RNA-seq datasets obtained from the frontal cortex and cerebellum. FIG. 26B shows a comparison of the differentially expressed transcripts in C90RF72-ALS and sporadic ALS and a comparison of the differentially expressed transcripts by brain region. FIG. 26C provides a correlation of the fold change of changed transcripts in C90RF72-ALS that were common to the cortex and the cerebellum. FIG. 26D shows a protein chaperone network.

[0037] FIG. 27 demonstrates marked upregulation of HSFl-target genes in

C90RF72-ALS and FTD. See Mordes et al. ACTA Neuropathologica Comm. (2018) 6:55.

[0038] FIG. 28 demonstrates selective C90RF72 DPR toxicity in stem cell-derived motor neurons.

[0039] FIGS. 29A-29C demonstrate DPR toxicity in NGN2 cortical-like neurons. FIG. 29A provides a schematic for differentiating neurons. FIG. 29B shows gene expression of MAPT and BRN2 at DO, D4, D14, and D21 of the differentiation protocol, and provides a representative image of MAP2. FIG. 29C shows decrease in viability when neurons are treated with DPR.

[0040] FIGS. 30A-30C summarize a chemical screen for modifiers of DPR toxicity. FIG. 30A shows a screen of stem cell-derived NGN2-cortical-like human neurons contacted with poly-GR DPR and a commercial library of compounds. An increased viability score indicates that the compound more effectively modified poly-GR toxicity. FIG. 30B shows the pathways affected by the compounds found in the assay of FIG. 30A to reduce GR-DPR toxicity. FIG. 30C shows that screens of compounds against poly-GR toxicity and against poly-PR toxicity using stem cell-derived NGN2-cortical-like human neurons identified 8 compounds effective against both poly-GR toxicity and poly-GR toxicity.

[0041] FIG. 31 provides an initial characterization of C90RF72-BAD mice.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Some aspects of the disclosure are directed to a method of treating or preventing a neurological disease, disorder or condition in a subject in need thereof, comprising administering to the subject an effective amount of an agent that reduces or eliminates dipeptide repeat (DPR) toxicity.

[0043] Non-limiting examples of neurological disease, disorder and conditions of the present disclosure include poly glutamine expansion disorders (e.g., HD,

dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado-Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e.g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Guillain-Barre syndrome, ischemia stroke, Krabbe disease, kuru, Lewy body dementia, multiple sclerosis, multiple system atrophy, non-Huntingtonian type of Chorea, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), SteeleRichardson-Olszewski disease, schizophrenia, late onset psychosis, autism spectrum disorder, a movement disorder, and Tabes dorsalis.

[0044] In some embodiments, the neurological disease, disorder or condition is one associated with a mutation associated with production of DPR. In some embodiments, the mutation is a hexanucleotide repeat expansion (GGGGCC) mutation in the first intron of the gene C90RF72. In some embodiments, the DPR comprises poly-glycine-arginine (poly-GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats. In some embodiments, the neurological disease is Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), or both ALS and FTD. In some embodiments, the neurological disease is Spinocerebellar ataxia type 36.

[0045] The term“agent” as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In some embodiments, the agent is selected from the group consisting of a nucleic acid, a small molecule, a polypeptide, and a peptide. In certain embodiments, agents are small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

[0046] “Small molecule” is defined as a molecule with a molecular weight that is less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic, a small molecule may be more permeable to cells, less susceptible to degradation, and less apt to elicit an immune response than large molecules.

[0047] As used herein, the term“polypeptide” or“protein” is used to designate a series of amino acid residues connected to the other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The term“polypeptide” refers to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. The term “peptide” is often used in reference to small polypeptides, but usage of this term in the art overlaps with "protein" or "polypeptide." Exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, as well as both naturally and non-naturally occurring variants, fragments, and analogs of the foregoing.

[0048] The term“nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The terms“nucleic acid” and“polynucleotide” are used interchangeably herein and should be understood to include double-stranded

polynucleotides, single-stranded (such as sense or antisense) polynucleotides, and partially double-stranded polynucleotides. A nucleic acid often comprises standard nucleotides typically found in naturally occurring DNA or RNA (which can include modifications such as methylated nucleobases), joined by phosphodi ester bonds. In some embodiments a nucleic acid may comprise one or more non-standard nucleotides, which may be naturally occurring or non-naturally occurring (i.e., artificial; not found in nature) in various embodiments and/or may contain a modified sugar or modified backbone linkage. Nucleic acid modifications (e.g., base, sugar, and/or backbone modifications), non-standard nucleotides or nucleosides, etc., such as those known in the art as being useful in the context of RNA interference (RNAi), aptamer, CRISPR technology, polypeptide production, reprogramming, or antisense-based molecules for research or therapeutic purposes may be incorporated in various embodiments. Such modifications may, for example, increase stability (e.g., by reducing sensitivity to cleavage by nucleases), decrease clearance in vivo, increase cell uptake, or confer other properties that improve the translation, potency, efficacy, specificity, or otherwise render the nucleic acid more suitable for an intended use. Various non-limiting examples of nucleic acid modifications are described in, e.g., Deleavey GF, et al, Chemical modification of siRNA. Curr. Protoc. Nucleic Acid Chem. 2009; 39: 16.3.1-16.3.22; Crooke, ST (ed.) Antisense drug technology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J. (ed.) Therapeutic oligonucleotides, RSC biomolecular sciences. Cambridge: Royal Society of Chemistry, 2008; U. S. Patent Nos. 4,469,863; 5,536,821 ; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929, 226; 5,977,296; 6,140,482; 6,455,308 and/or in PCT application publications WO 00/56746 and WO

01/14398. Different modifications may be used in the two strands of a double-stranded nucleic acid. A nucleic acid may be modified uniformly or on only a portion thereof and/or may contain multiple different modifications. Where the length of a nucleic acid or nucleic acid region is given in terms of a number of nucleotides (nt) it should be understood that the number refers to the number of nucleotides in a single-stranded nucleic acid or in each strand of a double-stranded nucleic acid unless otherwise indicated. An“oligonucleotide” is a relatively short nucleic acid, typically between about 5 and about 100 nt long.

[0049] In some embodiments, the agent is a poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor selectively inhibits PARPl, PARP2, or PARP3. In some embodiments, the agent selectively inhibits PARPl and PARP2, PARPl and PARP3, or PARP2 and PARP2. In some embodiments, the agent inhibits PARPl, PARP2 and PARP3. . In some embodiments, the PARP inhibitor selectively inhibits one or more of PARPl, PARP2, PARP3, PARP4, PARP9, PARP10, PARP 12, PAPR13, PAPR14, PARP 15, PARP16, TNKS1, or TNKS2. In some embodiments, the PARP inhibitor selectively inhibits at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13 PARPs. In some embodiments, the PARP inhibitor selectively inhibits PARPl and PARP2, PARP2 and PARP3, PARPl -PARP 3, PARP1-PARP4, PARP1-PARP4 and PARP 15 -PARP 16, or PARP1- PARP4 and PARP 16. As used herein,“selectively inhibits” means that the agent

preferentially inhibits at least one PARP by at least 1.1-fold, 1.2-fold, 1.3-fold, 1-4-fold, 1-5- fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, or more than it inhibits another PARP.

[0050] In some embodiments, the PARP inhibitor is Olaparib (AZD-2281), AZD- 2461, Niraparib (MK-4827), Rucaparib (AG-14699), AG-14361, Veliparib (ABT-888), Talazoparib (BMN673), CEP 9722, E7016, BGB-290, 3-aminobenzamide (INO-lOOl), NMSpl l8, A-966492, PJ34 HC1, UPF 1069, ME0328, Pamiparib (BGB-290), NMS-P118, E7449, Picolinamide, Benzamide, NU1025, Iniparib (BSI-201), Cyproterone Acetate, Exemestane, Lafutidine, CGS-21680 HC1, Entocapone, PD184352, Ouabain, BGP-15 2HC1, IKK- 16, Istradefylline, KU0058948, DPQ, NU1025, EB-47, DiQ, DR2313, 4-ANI, ISQ, 2- hydroxybenzaminde, CNQ, 3-AB, INH2BP, PJ34, 6(5H)-phenanthridinone, 3-methyl-5-AIQ, TIQ-A, XAV939, or a pro-drug, analog or derivative thereof.

[0051] In some embodiments, the subject is also administered a second agent to treat or prevent a neurological disease, disorder or condition. In some embodiments, the first and second agent are co-formulated. In some embodiments, the first and second agent are administered simultaneously. In some embodiments, the first and second agent are administered within a time of each other to produce overlapping therapeutic effects in the patient. When the first and second agent are administered simultaneously or within a time of each other to produce overlapping therapeutic effects, the agents may be administered by the same or a different route of administration (e.g., oral versus infusion).

[0052] In some embodiments, the second agent is antisense oligonucleotides (ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs.

[0053] As used herein,“treat,”“treatment,” or“treating” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a condition is reduced or halted. That is,“treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment.

[0054] The methods described herein may lead to a reduction in the severity or the alleviation of one or more symptoms of the disorder (e.g., ALS and/or FTD). As used herein, “prevent” when used in reference to a disease, disorder or medical condition, refers to reducing or eliminating the likelihood of development of the disease, disorder or medical condition.

[0055] As used herein, the term“administering,” refers to the placement of the agent as disclosed herein into a subject by a method or route which results in delivery to a site of action. The agent can be administered by any appropriate route which results in an effective treatment in the subject. Thus administration via oral ingestion is specifically contemplated. However, with appropriate formulation, other routes are contemplated, including, for example, intranasally, intraarterially; intra-coronary arterially; orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, or by other means known by those skilled in the art. The agents are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. In some embodiments, the agent is orally administered.

[0056] A“therapeutically effective amount” is an amount of an agent that is sufficient to produce a statistically significant, measurable change in, for example, blood glucose clearance. Such effective amounts can be gauged in clinical trials as well as animal studies. A treatment is considered“effective treatment,” as the term is used herein, if any one or all of the signs or symptoms are improved or ameliorated, e.g., by at least 10% following treatment with an agent as described herein. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill.

[0057] In some embodiments, the subject is a mouse, rat, rabbit, dog, cat, non-human primate, or human. In some embodiments, the subject is a human. [0058] Some aspects of the disclosure are directed to a composition comprising an agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell. The agent may be any agent disclosed herein and is not limited. In some embodiments, the agent is a poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, the composition comprises a second agent. The second agent may be any agent described herein and is not limited. In some embodiments, the second agent is antisense oligonucleotides (ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs. In some embodiments, the composition further comprises pharmaceutically acceptable carrier, diluent, or excipient.

[0059] Some aspects of the disclosure are directed to a method of screening for a candidate agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell, comprising providing a composition comprising neuronal cells contacted with a dipeptide repeat (DPR), contacting the composition with a test agent, comparing the viability of the neuronal cells contacted with the test agent to control neuronal cells not contacted with the test agent, and identifying a test agent as a candidate agent if the viability of the neuronal cells contacted with the test agent are increased as compared to the control neuronal cells not contacted with the test agent.

[0060] In some embodiments, the DPR comprises poly-glycine-arginine (poly-GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats. In some embodiments, the neuronal cells are stem cell-derived. In some embodiments, the stem cells are induced pluripotent stem cells (iPS). In some embodiments, the iPS cells are derived from a subject having a neurological disease, disorder or condition (e.g., ALS and/or FTD). In some embodiments, the neuronal cells are derived from stem cells via an NGN2 induction protocol (see, e.g., Nehme et al., Cell Reports, Vol. 23, pp. 2509-2523 (2018)). In some embodiments, the neuronal cell are cortical-like neurons, upper motor neurons, and/or lower motor neurons.

[0061] Methods of determining viability of a cell are not limited and may be any method known in the art. In some embodiments, the method for measuring viability is tryphan blue dye exclusion.

[0062] In some embodiments, the method of screening is a high-throughput screening method.

[0063] The terms“decrease,”“reduce,”“reduced,”“reduction,”“decrease,” and “'inhibit” are all used herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt,“reduce,”“reduction” or“decrease” or“inhibit” typically means a decrease by at least 10% as compared to a reference level and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , up to and including, for example, the complete absence of the given entity or parameter as compared to the reference level, or any decrease between 10-99% as compared to the absence of a given treatment.

[0064] The terms“increased,”“increase” or“enhance” or“activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms“increased”,“increase” or“enhance” or“activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or more as compared to a reference level.

[0065] As used herein the term“comprising” or“comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0066] The term“consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0067] As used herein the term“consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0068] The term“statistically significant” or“significantly” refers to statistical significance and generally means a“p” value greater than 0.05 (calculated by the relevant statistical test). Those skilled in the art will readily appreciate that the relevant statistical test for any particular experiment depends on the type of data being analyzed. Additional definitions are provided in the text of individual sections below. [0069] Definitions of common terms in cell biology and molecular biology can be found in“The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R. (2000); Immunology by Wemer Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al.

(eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Cun-ent Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al, eds.

[0070] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al, Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001) and Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995) which are both incorporated by reference herein in their entireties.

[0071] As used herein, the terms“proteins” and“polypeptides” are used

interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms“protein”, and“polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.“Protein” and“polypeptide” are often used in reference to relatively large polypeptides, whereas the term“peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms“protein” and“polypeptide” are used interchangeably herein when refining to a gene product and fragments thereof.

[0072] Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0073] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0074] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these

embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0075] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0076] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are

representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. [0077] The articles“a” and“an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0078] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0079] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by“about” or“approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by“about” or“approximately”, the invention includes an embodiment in which the value is prefaced by“about” or“approximately”.

[0080] “Approximately” or“about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered“isolated”.

EXAMPLES:

[0081] Example 1 :

[0082] We screened a commercial library of over 450 annotated compounds most of which have known targets or mechanism of actions, including some that are already-approved for clinical use and some that are in clinical development. We used kenpaullone, which was previously identified as having a neuroprotective effect in mouse stem cell-derived neurons, as a potential positive control.

[0083] We performed a primary screen for modifiers of poly-GR toxicity (Figure 1 A) and then another screen for modifiers of poly -PR toxicity. Interestingly, there was a significant degree of overlap between the top hits for these two screens, suggesting that these DPRs may exert their toxic effects in a similar manner (Figure IB). Of the top hits from the GR screen were two PARP inhibitors and of the top hits from the PR screen were two PARP inhibitors with ABT-888 (veliparib) being common to both. We have validated that at least two PARP inhibitors improve neuronal viability in response to DPR-mediated neurotoxicity. The compounds that we have identified as being neuroprotective may be applicable as potential treatments for other forms of ALS and FTD.

[0084] Other labs and companies are developing antisense oligonucleotides (ASOs) to reduce the expression of GGGGCC repeats and toxic DPRs in C90RF72-ALS/FTD. Any compounds that we identified as being neuroprotective would be complimentary to ASO- based therapy.

[0085] Example 2:

[0086] The most common genetic contributor to ALS is a hexanucleotide (GGGGCC) repeat expansion within the first intron of C90RF72. Carriers of the C90RF72 expansion can also present with frontotemporal dementia (FTD) and atypical Parkinsonian syndromes. These initially diverse diagnoses can progress towards the inclusion of neurological features from each condition. Three distinct mechanisms have been proposed for how the C90RF72 expansion contributes to the development of ALS. First, C90RF72-ALS brains display reduced abundance of C90RF72 transcripts, suggesting that a loss-of-function mechanism may contribute to disease. Although complete loss of C90RF72 expression in mice leads to fatal autoimmunity and changes in microglia, no obvious signs of neurodegeneration or neural dysfunction have yet been reported in these animals. Second, mutant transcripts containing the GGGGCC repeats form intranuclear RNA foci that may sequester RNA binding proteins and lead to nucleolar stress. Finally, dipeptide repeat proteins (DPRs) were found to be translated from both sense and antisense transcripts containing these repeats. These aggregation-prone DPRs, including poly -glycine-arginine, are toxic when expressed in several model systems and have been shown to affect several cellular processes, including RNA processing, nucleocytoplasmic import and export, and endoplasmic reticulum stress.

[0087] Distinct transcriptional changes have been identified in C90RF72-ALS/FTD patient brains, including activation of a heat shock factor-1 (HSFl)-associated response. The expression of poly-GR in Drosophila neurons and the treatment of human stem cell-derived neurons with synthetic poly-GR increased levels of HSF1 and HSF1 -associated transcripts. This suggested that DPRs are sufficient to drive disease-associated transcriptional changes and that the effects of DPRs can be modelled in vitro. Consistent with results in other cell types, poly-GR and poly-PR are observed to be toxic to human stem cell-derived NGN2 cortical-like neurons. A high-through put screening platform has been established to identify cellular pathways that modify the toxic effects of DPRs and may provide a venue for therapeutic development in C90RF72-ALS/FTD.

Claims

CLAIMS What is claimed is:

1. A method of treating or preventing a neurological disease, disorder or condition in a subject in need thereof, comprising administering to the subject an effective amount of an agent that reduces or eliminates dipeptide repeat (DPR) toxicity.

2. The method of claim 1, wherein the subject has a mutation associated with production of DPR.

3. The method of claim 2, wherein the mutation is a hexanucleotide repeat expansion (_' GGGGCC) mutation in the first intron of the gene C90RF72.

4. The method of claims 1-3, wherein the DPR comprises poly -glycine-arginine (poly- GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats.

5. The method of claims 1-4, wherein the neurological disease is one or both of

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).

6. The method of claims 1-5, wherein the agent is a poly ADP ribose polymerase

(PARP) inhibitor.

7. The method of claim 6, wherein the PARP inhibitor selectively inhibits one or more of PARP 1, PARP2, PARP3, and PARP4.

8. The method of claim 6, wherein the PARP inhibitor inhibits PARPl.

9. The method of claims 6-8, wherein the PARP inhibitor is selected from Olaparib (AZD-2281), AZD-2461, Niraparib (MK-4827), Rucaparib (AG-14699), AG-14361, Veliparib (ABT-888), Talazoparib (BMN673), CEP 9722, E7016, BGB-290, 3- aminobenzamide (INO-1001), NMSpl l8, A-966492, PJ34 HC1, UPF 1069, ME0328, Pamiparib (BGB-290), NMS-P118, E7449, Picolinamide, Benzamide, NU1025, Iniparib (BSI-201), Cyproterone Acetate, Exemestane, Lafutidine, CGS-21680 HC1, Entocapone, PD184352, Ouabain, BGP-15 2HC1, IKK-16, Istradefylline,

KU0058948, DPQ, NU1025, EB-47, DiQ, DR2313, 4-ANI, ISQ, 2- hydroxybenzaminde, CNQ, 3-AB, INH2BP, PJ34, 6(5H)-phenanthridinone, 3-methyl- 5-AIQ, TIQ-A, XAV939, or a pro-drug, analog or derivative thereof.

10. The method of claims 1-10, wherein the subject is also administered a second agent to treat or prevent a neurological disease, disorder or condition.

11. The method of claim 10, wherein the second agent is antisense oligonucleotides

(ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs.

12. The method of claims 1-11, wherein the agent is orally administered.

13. The method of claims 1-12, wherein the subject is a human.

14. A composition comprising an agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell.

15. The composition of claim 14, wherein the agent is a poly ADP ribose polymerase (PARP) inhibitor.

16. The composition of claims 14-15, further comprising a second agent that is antisense oligonucleotides (ASOs) that reduce the expression of GGGGCC repeats and toxic DPRs.

17. The composition of claims 14-16, further comprising a pharmaceutically acceptable excipient.

18. A method of screening for a candidate agent that reduces or eliminates dipeptide repeat (DPR) toxicity in a cell, comprising:

a. providing a composition comprising neuronal cells contacted with a dipeptide repeat (DPR),

b. contacting the composition with a test agent,

c. comparing the viability of the neuronal cells contacted with the test agent to

control neuronal cells not contacted with the test agent, and

d. identifying a test agent as a candidate agent if the viability of the neuronal cells contacted with the test agent are increased as compared to the control neuronal cells not contacted with the test agent.

19. The method of claim 18, wherein the DPR comprises poly -glycine-arginine (poly- GR) and/or poly-proline-arginine (poly-PR) dipeptide repeats.

20. The method of claims 18-19, wherein the neuronal cells are stem cell-derived.

21. The method of claims 18-20, wherein the neuronal cell are cortical -like neurons or lower motor neurons.