WO2023107893A2 - Traitement de maladies neurodégénératives par l'inhibition de l'ataxine-2 - Google Patents

Traitement de maladies neurodégénératives par l'inhibition de l'ataxine-2 Download PDF

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WO2023107893A2
WO2023107893A2 PCT/US2022/080927 US2022080927W WO2023107893A2 WO 2023107893 A2 WO2023107893 A2 WO 2023107893A2 US 2022080927 W US2022080927 W US 2022080927W WO 2023107893 A2 WO2023107893 A2 WO 2023107893A2
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atxn2
ataxin
agent
cells
nogo
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Aaron GITLER
Caitlin RODRIGUEZ
Garam Kim
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The Board Of Trustees Of The Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate

Definitions

  • TDP-43 A hyper-phosphorylated, ubiquitinated and cleaved form of TDP-43, known as pathologic TDP43, is a major disease-associated protein in nearly all cases of amyotrophic lateral sclerosis (ALS), and is also present in numerous other neurodegenerative disorders such as behavioral variant frontotemporal dementia (bvFTD), primary progressive aphasia (PPA), and limbic-predominant age-related TDP-43 encephalopathy (LATE).
  • ALS amyotrophic lateral sclerosis
  • PPA primary progressive aphasia
  • LATE limbic-predominant age-related TDP-43 encephalopathy
  • TDP-43 Despite the prominent presence of TDP-43 aggregates in multiple neurodegenerative diseases, targeting TDP-43 directly presents many challenges, in that it is tightly regulated and essential, and reducing its levels results in numerous deleterious effects, for example embryonic lethality during development, or motor phenotypes in adult mice. [0005] Other approaches have been used to target a modifier of TDP-43 aggregation and toxicity.
  • Ataxin-2 a polyglutamine (polyQ) protein for which long (>34) polyQ expansions cause spinocerebellar ataxia 2 (SCA2) and intermediate-length (22-34) repeats are a risk factor for ALS, is a potent modifier of TDP-43 aggregation and toxicity in multiple model systems including yeast, Drosophila, primary neurons, and mice.
  • the polyQ expansions lead to increased ataxin-2 protein levels, and antisense oligonucleotides (ASOs) targeting ataxin- 2 in vivo show marked protection against motor deficits and extend lifespan in TDP-43 overexpressing mice and in SCA2 mice.
  • ASOs antisense oligonucleotides
  • Ataxin-2 harbors a PAM2-domain, binds to poly(A)-binding protein, and contains an Lsm-domain commonly found in splicing factors. These domains likely promote ataxin-2 localization to mRNP granules and direct its role as a translational regulator.
  • Ataxin-2 functions in multiple types of mRNP granules such as P-bodies and stress granules, and its association with mRNP granules in Drosophila neurons is linked to translation-dependent long-term memory. Knockdown of ataxin-2 reduces recruitment of TDP-43 to stress granules. [0007] There is a critical need for alternative therapies to treat neurogenerative diseases associated with Ataxin-2, and comprising long and intermediate polyQ expansions. Described herein are methods and compositions for treating ATXN2 associated neurodegenerative diseases.
  • compositions and methods are provided for treating a mammalian subject for a neurodegeneration disease, including, without limitation, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia 2 (SCA2), etc., by inhibition of Ataxin-2 (ATXN-2).
  • An ATXN2 inhibitor is delivered to the subject is a dose effective to reduce ATXN2 protein levels or function, thereby reducing the symptoms of the neurodegenerative disease.
  • an ATXN2 inhibitor which may be referred to as an anti-ATXN2 agent, is a bisphosphonate.
  • an anti-ATXN2 bisphosphonate is an inhibitor of v-ATPase.
  • an anti-ATXN2 agent inhibits the NOGO receptor, NgR1 (also referred to as RTN4R).
  • NgR1 also referred to as RTN4R
  • An NgR1 inhibitor may be a genetic agent that inhibits NgR1 expression, or may interfere with NgR1 signaling, e.g. by competitive inhibition or blocking of the interaction between NgR1 and NOGO, neutralization of NOGO, NEP1-40 inhibitor, and the like.
  • a pharmaceutical formulation comprising an anti- ATXN2 agent as identified herein, and a pharmaceutically acceptable excipient.
  • the formulation may be provided in a unit dose.
  • the formulation may be suitable for parenteral or oral delivery.
  • the formulation may be suitable for direct delivery to the brain.
  • an effective dose of an anti-ATXN-2 agent is administered to an individual having, or at risk of having, a neurogenerative disease, in a dose effective to stabilize, reduce or prevent clinical symptoms of the disease.
  • a variety of neurogenerative diseases may be treated by practicing the methods, including ALS, SCA2, Parkinson’s disease, spinocerebellar ataxia type 1, Machado-Joseph Disease also known as spinocerebellar ataxia type 3, tauopathies, or other neurodegenerative diseases.
  • the neurodegenerative disease is ALS.
  • the neurodegenerative disease is SCA.
  • the anti-ATXN2 agent is a bisphosphonate, including without limitation etidronate and alendronate.
  • the anti-ATXN2 agent is an inhibitor of NgR1.
  • the individual may be human.
  • the individual may be monitored, e.g. before, during and/or after treatment, for inhibition of ATXN2.
  • the individual may be monitored for inhibition of NgR1.
  • the individual may be monitored for inhibition of vATPase.
  • the individual may be monitored for clinical indicia of disease.
  • a method is provided for treatment of a human subject for ALS, the method comprising administering an effective dose of etidronate or alendronate.
  • a method for treatment of a human subject for ALS the method comprising administering to the brain an effective dose of an NgR1 inhibitor.
  • a method for treatment of a human subject for SCA2 the method comprising administering to the brain an effective dose of an NgR1 inhibitor.
  • the effects of anti-ATXN2 agents on neurogenerative diseases may comprise a range of outcomes, which are optionally monitored following treatment. For instance, outcomes may include a reduction in symptoms associated with ALS, such as reduced muscle weakness, muscle atrophy, fasciculations, emotional lability, or respiratory muscle weakness relative to no treatment.
  • Methods of treatment disclosed herein may provide for a reduction in symptoms associated with ALS or SCA2 such as reduced ataxia, speech and swallowing difficulties, muscle wasting, or slow eye movement.
  • the methods may include administering to an individual suffering from a neurodegenerative disease such as ALS or SCA2 an effective dose of an anti-ATXN2 agent, where the treatment reduces or stabilizes clinical symptoms of the disease.
  • the individual is a human.
  • the anti-ATXN2 agent is combined with a secondary therapeutic agent, including without limitation riluzole, baclofen, quinine, phenytoin, glycopyrrolate, amitriptyline, benztropine, trihexyphenidyl, transdermal hyoscine, atropine, fluvoxamine, dextromethorphan, quinidine, gabapentin, etc.
  • a secondary therapeutic agent including without limitation riluzole, baclofen, quinine, phenytoin, glycopyrrolate, amitriptyline, benztropine, trihexyphenidyl, transdermal hyoscine, atropine, fluvoxamine, dextromethorphan, quinidine, gabapentin, etc.
  • more than one anti-ATXN2 agent may be administered to an individual.
  • the effective dose of each drug in a combination therapy may be lower than the effective dose of the same drug in a monotherapy
  • the two therapies are phased, for example where one compound is initially provided as a single agent, e.g. as maintenance, and where the second compound is administered during a relapse, for example at or following the initiation of a relapse, at the peak of relapse, etc.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0016] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings.
  • the patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale.
  • FIGs. 1A-1D Genome-wide CRISPR–Cas9 KO screens in human cells identify regulators of ataxin-2 protein levels.
  • A Pooled CRISPR–Cas9 screening paradigm. After transducing HeLa cells expressing Cas9 with a lentiviral sgRNA library, we fixed and co- immunostained the cells for ataxin-2 and a control protein ( ⁇ -actin or GAPDH). We then used FACS to sort top and bottom 20% ataxin-2 expressors relative to control protein levels (duplicate sorts per each control).
  • FIG. 2 Schematic of proteins encoded by selected hits (5% FDR), categorized by function and subcellular localization.
  • FIGs.3A-3F Inhibiting lysosomal v-ATPase leads to decreased ataxin-2 protein levels in vitro.
  • FIGs.4A-4K Small molecule drug Etidronate lowers ataxin-2 protein levels in human iPSC-derived neurons, mouse primary neurons, and in vivo in mice.
  • A Timeline of induced neuron differentiation in a human iPSC line with NGN2 stably integrated and drug treatment.
  • B Immunoblot on lysates from human iPSC-derived neurons treated with various doses of Etidronate.
  • FIG. 3B Quantification of Figure 3B, with ataxin-2 protein levels normalized to H2O- treated condition (mean ⁇ SD; analyzed using one-way ANOVA with post-hoc Dunnett’s multiple comparisons tests; *: p ⁇ 0.05).
  • D Timeline of primary neuron plating from embryonic mouse cortex and drug treatment.
  • E Immunoblot on lysates from mouse primary neurons treated with various doses of Etidronate.
  • F Quantification of the dose-dependent effect of Etidronate on ataxin-2 (normalized to control condition).
  • FIGs. 5A-5H Calibration steps prior to conducting genome-wide screens.
  • A Overview of screen optimization strategy.
  • HeLa cells expressing Cas9 were infected with a lentiviral sgRNA targeting ataxin-2, and puromycin was used to select for cells that received a guide. Cells were kept pooled to retain a mosaic population. These cells were then fixed in methanol, immunostained, and sorted using FACS for the top and bottom 25% of ataxin-2 expressors relative to a control protein (GAPDH or ⁇ -actin). The sorted and unsorted populations were Sanger sequenced and analyzed for insertions or deletions (indels) at the ATXN2 locus.
  • B Immunoblot of the WT and ATXN2 mosaic KO populations, as generated in panel a.
  • C Gating strategy for FACS.
  • FIGs.6A-6B Validation of screen results in neuroblastoma cell line SH-SY5Y, related to Figure 1.
  • A Validation of numerous top hit genes in SH-SY5Y cells using siRNA transfections and immunoblot analyses as in Fig.1 C and D.
  • FIGs. 7A-7D Other polyQ protein levels are unaltered and TDP-43 is slightly decreased in ATP6V1A KO cells.
  • FIG.8 MA plot 72 hours after treatment with NT vs. ATP6V1A siRNAs in HeLa cells. To determine whether there are broad transcriptional changes after knocking down a v- ATPase subunit, we performed RNA-seq after HeLa cells were treated with NT or ATP6V1A siRNAs. Few noteworthy transcriptional changes are seen (apart from ATP6V1A itself) upon knockdown of ATP6V1A (FDR ⁇ 0.01). Importantly, ATXN2 mRNA levels were not altered by ATP6V1A knockdown (yellow circle).
  • FIGs.9A-9D Treating human SH-SY5Y cells or mouse cortical neurons with another bisphosphonate leads to decreased ataxin-2 protein levels.
  • A Immunoblot on lysates from human SH-SY5Y cells with various doses of Alendronate.
  • B Quantification of the immunoblot in (B) reveals a dose-dependent effect of Alendronate on ataxin-2 protein levels (mean ⁇ SD, normalized to control condition).
  • C Immunoblot on lysates from mouse primary neurons treated with various doses of Alendronate.
  • D Quantification of the dose-dependent effect of Alendronate on ataxin-2 (mean ⁇ SD, normalized to control condition).
  • FIGs.11A-11D Treating mouse cortical neurons with Etidronate does not affect TDP- 43 localization.
  • FIGs.12A-12L Generation of the ataxin-2-HiBiT cell line and overview of the whole- genome siRNA screen.
  • A We engineered an endogenous C-terminal HiBiT fusion on ataxin- 2 using CRISPR-Cas9 genome editing in HEK293T cells. LgBiT compliments the HiBiT protein tag to form NanoBiT.
  • FFLuc firefly luciferase
  • B Antibody-based immunoblotting and HiBiT substrate-based detection on ataxin- 2-HiBiT cell lysates transfected with siRNA. Quantified in (C) and (D).
  • E HiBiT signal measured via luciferase assay on cells transfected with increasing doses of siRNA.
  • F Schematic of the whole- genome siRNA screen for regulators of ataxin-2 levels.
  • G Plot showing results of HiBiT replicates after filtering for changes in FFLuc. Datapoints in blue are primary screen hits that decrease ataxin-2. ATXN2 siRNA (red) was the strongest hit.
  • (H) GO- Slim Biological Process analysis of the hits from the primary screen.
  • FIGs. 13A-13I Targeting RTN4R lowers levels of ataxin-2.
  • A High confidence hits ranked by average HiBiT Z-score, then filtered for essentiality (gene effect, DepMap) and CNS expression (GTEx).
  • B HiBiT signal measured by luciferase assay in ataxin-2-HiBiT cells transfected with designated siRNA.
  • C Immunoblot for ataxin-2 and GAPDH on lysates derived from unedited HEK293T cells treated with designated siRNA. Ataxin-2 levels are quantified in (D). We performed RT-qPCR on RNA from cells treated as in (C). We probed for RTN4R transcript (E) or ATXN2 transcript (F) along with ACTB for normalization.
  • FIGs. 14A-14N RTN4R knockdown or inhibition of RTN4/NoGo-Receptor in mouse and human neurons and in mouse brain reduces ataxin-2 levels.
  • (B) Induced neuron differentiation in a human iPSC line with NGN2 stably integrated, as verified by Tuj1 (green) and NeuN (red) immunostaining, scale bar 20 ⁇ m. Treatment timeline for shRNA lentivirus or NEP1- 40 in human induced neurons (iNeurons). DPI days post induction.
  • C Immunoblot on lysates from mouse neurons treated with shRNA.
  • D Quantification of ataxin- 2 and RTN4/NoGo- Receptor levels.
  • E Immunoblot on lysates from iNeurons treated with shRNA.
  • (I) Immunocytochemistry and fluorescence microscopy on mouse neurons treated with 50 ⁇ M NEP1-40. MAP2 labels neurons, DAPI labels nuclei. Scale bar 20 ⁇ m.
  • FIGs. 15A-15E Reduction of ataxin-2 increases axonal regrowth after axotomy.
  • A Timeline for axotomy and regeneration experiment using mouse primary neurons grown in microfluidics chambers.
  • B Immunoblot on lysates from mouse neurons treated with Atxn2 shRNA.
  • FIGs.16A-16C Ataxin-2 screen results and filters.
  • FIGs. 17A-17B Shared protein domain between ataxin-2 and its regulators.
  • A InterPro domain enrichment of primary screen hits.
  • B Bottom: representation of ataxin-2 protein domains including the poly-glutamine stretch (polyQ), the LSm and LSm-associated (LSm AD) domains, and the PABP-interacting motif (PAM).
  • Top graph of the IUPred2 score, a prediction of protein disorder, for the amino acid sequence of ataxin-2.
  • FIGs. 18A-18B Further validation of screen results.
  • A Immunoblot of ataxin-2 and GAPDH levels after siRNA treatment in unedited HEK293T cell lysates.
  • B RT-qPCR on RNA from cells treated with non-targeting siRNA or siRNA targeting various splicing factors. We probed for ATXN2 transcript along with ACTB for normalization. Two-way ANOVA with multiple comparisons: WBP11 and AQR, p ⁇ 0.05. LSM5 and SNRPB, p ⁇ 0.01. ATXN2 and SNRPD2, p ⁇ 0.001. SFRS3, p ⁇ 0.0001. Error bars represent ⁇ SEM.
  • FIGs.19A-19D Further validation of RTN4R as a regulator of ataxin-2.
  • A Immunoblot on SH- SY5Y cell lysates after RTN4R siRNA treatment. Ataxin-2 levels are quantified in (B).
  • C Immunoblot on HEK293T cell lysates after ATXN2 siRNA treatment. RTN4/NoGo- Receptor levels are quantified in (D). Student’s t-test. *p ⁇ 0.05. Error bars represent ⁇ SEM.
  • FIGs. 20A-20B Knockdown of RTN4R does not alter the expression of other polyQ proteins or ATXN2L.
  • FIGs.21A-21B Knockdown of RTN4R reduces TDP-43 localization to stress granules.
  • A We treated HEK293T cells with siRNA, and subsequently treated for 30 minutes with 0.5mM sodium arsenite to induce stress granules.
  • FIGs.22A-22B Knockdown of RTN4R does not decrease ataxin-2 through autophagy pathways and does not increase general proteasome activity.
  • A We treated ataxin-2-HiBiT cells with siRNA, then treated for 24hr with autophagy inhibitor Bafilomycin A1 or DMSO. We performed a luciferase assay to measure HiBiT activity.
  • FIGs. 23A-23E Full immunoblot on neuron lysates treated with two different shRNA constructs targeting RTN4R and RT-qPCR on shRNA-treated primary neurons.
  • A Immunoblot of ataxin-2 and RTN4/NoGo-Receptor levels after 12 days of shRNA treatment in cortical neuron cultures.
  • FIGs.24A-24B RNA sequencing of RTN4R knockdown iNeurons.
  • FIGs. 25A-25C RTN4R knockdown effects in neurons.
  • A Immunoblot of the Sm- containing SNRPB and actin after designated shRNA treatment in iNeuron cultures. Quantification of SNRPB levels in (B).
  • C Mouse cortical neurons were treated for 12 days with non-targeting or RTN4R shRNA, and subsequently treated for 3 or 5 days with lentivirus expressing GFP or TDP-43.
  • Anti-ATXN2 agents include, for example, bisphosphonates, such as etidronate, alendronate, etc., other vATPase inhibitors, e.g. thonzonium, and inhibitors of Nogo-66 receptor 1 (NgR1), which are identified herein based on an ability to inhibit the function or activity of ATXN-2 associated gene products in a dose dependent manner.
  • anti-ATXN2 agents were found to achieve ATXN2 protein reduction through lysosomal v-ATPase inhibition, in the case of etidronate or thonzonium, or through competitive inhibition in the case of NEP1-40. [0043] Improvement in the use of disease-modifying therapies in neurological diseases is of great clinical interest.
  • the term "treating” is used to refer to both prevention of relapses, and treatment of pre- existing conditions.
  • the prevention of neurodegenerative disease may be accomplished by administration of the agent prior to development of a relapse, after an initial diagnosis.
  • "Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: inhibiting the disease symptom, i.e., arresting its development; or relieving the disease symptom, i.e., causing regression of the disease or symptom.
  • the treatment of ongoing disease where the treatment stabilizes or improves the clinical symptoms of the patient, is of particular interest.
  • onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. Reducing the severity of a relapse shall mean that the clinical indicia associated with a relapse are less severe in the presence of the therapy than in an untreated disease.
  • onset may refer to a relapse in a patient that has ongoing relapsing remitting disease.
  • the methods of the invention are specifically applied to patients that have been diagnosed with a neurodegenerative disease. Treatment is aimed at the treatment or reducing severity of relapses, which are an exacerbation of a pre-existing condition.
  • Diagnosis generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of disease states, stages of ALS or SCA, or responsiveness of ALS or SCA to therapy), and use of therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).
  • the term "biological sample” encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay.
  • the term encompasses blood, cerebral spinal fluid, and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components.
  • the term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
  • Ataxin-2 is a member of the Like-Sm (LSm) family of RNA-binding proteins and contains a polyglutamine (polyQ) repeat of ⁇ 22–23 amino acids in healthy individuals, whereas significant expansion of the polyQ repeat to over 34 amino acids is the genetic cause of SCA2.
  • Ataxin-2 intermediate-length polyQ repeat expansions (27–33 amino acids) are associated with increased risk of ALS, a neurodegeneration with abnormal cytoplasmic aggregation of TDP-43 called TDP-43 proteinopathy.
  • Ataxin-2 contains PAM2 in its C-terminal region along with the N-terminal LSm domain which directly binds to a 3′-UTR of specific mRNAs and promotes its stability and protein production.
  • ATXN2-associated genes may be any gene that has an influence on the ATXN2 gene or gene product.
  • ATXN2-associated genes may include ALG1, DPAGT1, RFT1, CFAP20, CLASRP, LUC7L3, PNISR, SNRPA, PAXBP, ATXN7L3, ENY2, USP22, CMTR2, CMTR2, ATP6V0C, ATP6V1A, ATP6V1B2, ATP6V1C1, ATP6V1D, LSM12, RP11-894J14.5, TSC1, TSC2, UBA3, RTN4R, EFHD2, TRAPPC1, MAP3K11, MFAP1, UNC84A, ZNF32, or ASB8.
  • the screenings disclosed herein have identified v-ATPases and RTN4R (NgR1) as suitable targets for inhibition to reduce ATXN2.
  • the V-ATPases are large multi-subunit complexes composed of two domains: a membrane integral V0 sector involved in proton translocation and a peripheral V1 domain catalyzing ATP hydrolysis.
  • the integral V0 domain is a 260 kDa complex containing six different subunits (a, c, c’, c”, d, and e)
  • the V1 domain is a 640 kDa complex including eight different subunits (A, B, C, D, E, F, G, and H).
  • H + - ATPase subunits have multiple isoforms encoded by separate genes that are located throughout the genome with differing tissue expression patterns. For example, there are two isoforms for the B, E, d, and e subunits; three for the C and G subunits; and four for the A subunit. Some of the isoforms have different expression patterns in various tissues. For example, the d1 subunit is ubiquitously expressed while the d2 homolog is expressed only in the kidney, osteoclast and lung. Similarly, the G1 isoform is expressed ubiquitously while G2 and G3 isoforms are found mainly in neuronal tissue and kidney.
  • V-ATPase is equipped with the accessory subunits ATP6AP1 and ATP6AP2.
  • ATP6AP1 also known as Ac45
  • ATP6AP1 functions to guide the V- ATPase to certain subcellular compartments such as neuroendocrine regulated secretory vesicles and regulates their activity, the intragranular pH and Ca 2+ -dependent exocytotic membrane fusion.
  • ATP6AP2 was first identified as the C-terminal fragment of the (pro) renin receptor (PRR) for renin and prorenin. Ablation of PRR in cardiomyocytes reveals that PRR is an integral component for the stability and assembly of V0 subunits.
  • V-ATPase is a key accessory protein for V-ATPase functions in the CNS and essential for stem cell self-renewal and neuronal survival.
  • V-ATPase is responsible for generating the H + - electrochemical gradient in synaptic vesicles, which drives the refilling of newly formed synaptic vesicles with neurotransmitter.
  • Synaptic vesicle V-ATPase also participates in the step of fusion.
  • V-ATPase Relying on its H + -pumping ability, V-ATPase modulates multiple cellular activities including endosome maturation and trafficking, protein processing and degradation via different autophagic pathways in multiple vesicle organelles such as lysosome and endosome.
  • the acidic environment of the lysosomes is critical for not only the function of lysosomes but also many cellular processes related to lysosomes.
  • V-ATPase is also involved in pH sensing, nutrient signalling, and scaffold for protein-protein interactions.
  • Nogo-66 receptor 1 (NgR1, alternatively referred to as RTN4R) is a glycosylphosphatidyl inositol-linked protein that belong to the leucine-rich repeat superfamily. Through binding to myelin-associated inhibitors, NgR1 contributes to the inhibition of axonal regeneration after spinal cord injury. NgR1 has been shown to limit axonal sprouting, plasticity, and regeneration in the adult CNS after binding to myelin-associated inhibitors, such as Nogo, MAG, and OMgp.
  • myelin-associated inhibitors such as Nogo, MAG, and OMgp.
  • NgR1 binds with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in chondroitin sulfate proteoglycan-mediated inhibition of axon growth from cultured neurons.
  • NgRs lack a transmembrane domain, and require the formation of complexes with coreceptors to trigger downstream signaling pathways.
  • a large number of cis-interacting proteins have been reported to bind to NgR1: adaptors, such as LINGO-1 or AMIGO3; and signaling coreceptors, such as TROY and/or P75NTR.
  • NgR Nogo receptor
  • NgR1 Nogo receptor 1
  • Knockout of NgR1 extends the critical period for experience-dependent plasticity in the ocular dominance paradigm in visual cortex, as well as extending critical period plasticity for acoustic preference.
  • NgR1 neuronal activity induction
  • various forms of neuronal activity induction have been demonstrated to rapidly downregulate NgR1 expression in mice, as seen with wheel running, kainic acid, electroconvulsive seizures and amphetamine.
  • Regulation of the Nogo receptor homologs NgR2 and NgR3 by neuronal activity have also been reported.
  • NgR1 has been demonstrated to regulate synaptic plasticity. Ablation or blocking of NgR1 enhances hippocampal long-term potentiation (LTP) in slice preparations, and appears to be mediated by the binding of NgR1 to selected ligands such as Nogo-A, and fibroblast growth factor-2 (FGF2).
  • LTP hippocampal long-term potentiation
  • NgR1 ⁇ / ⁇ mice The extension of the critical period plasticity into adulthood is also reflected in an increase in spine turnover in the cortex of adult NgR1 ⁇ / ⁇ mice, and a shift toward immature spine subtypes in the hippocampus of NgR1 ⁇ / ⁇ mice.
  • modulation of NgR1 has resulted in mild memory phenotypes.
  • Forebrain NgR1 overexpression impairs long-term memory, without affecting short-term memory, whilst constitutive knockout of NgR1 impairs working memory without affecting spatial memory.
  • NgR1 ⁇ / ⁇ mice also exhibit improved extinction learning in cued conditioned fear.
  • an "antagonist,” or “inhibitor” agent refers to a molecule which, when interacting with (e.g., binding to) a target protein, decreases the amount or the duration of the effect of the biological activity of the target protein (e.g., interaction between leukocyte and endothelial cell in recruitment and trafficking).
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules that decrease the effect of a protein. Unless otherwise specified, the term “antagonist” can be used interchangeably with “inhibitor” or “blocker”.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” can be used interchangeably.
  • analog is used herein to refer to a molecule that structurally resembles a molecule of interest, but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the starting molecule, an analog may exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher potency at a specific receptor type, or higher selectivity at a targeted receptor type and lower activity levels at other receptor types) is an approach that is well known in pharmaceutical chemistry. [0068] Anti-ATXN2 agent.
  • an anti-ATXN2 agent blocks the activity or function of an ATXN2 or an ATXN2-associated gene or gene product, such that ATXN2 protein levels are reduced, or ATXN2 protein function and/or activity is reduced, particularly with respect to human AXTN2.
  • an ATXN2 inhibitor which may be referred to as an anti-ATXN2 agent, is a bisphosphonate.
  • an anti-ATXN2 bisphosphonate is an inhibitor of v-ATPase.
  • Other inhibitors of v-ATPase are also useful as anti-ALXN2 agents, e.g. thonzonium, alexidine, and the like.
  • an anti-ATXN2 agent inhibits the NOGO receptor, NgR1 (also referred to as RTN4R).
  • NgR1 also referred to as RTN4R.
  • An NgR1 inhibitor may be a genetic agent that inhibits NgR1 expression, or may interfere with NgR1 signaling, e.g. by competitive inhibition or blocking of the interaction between NgR1 and NOGO, neutralization of NOGO, NEP1-40 inhibitor, and the like.
  • Bisphosphonates is a bisphosphonate. Without being limited by the theory, bisphosphonates may inhibit ataxin-2 by inhibition of v- ATPase through incorporation into molecules of newly formed adenosine triphosphate (ATP).
  • the bisphosphonate is etodronate. In some embodiments the bisphosphonate is alendronate. In some embodiments the bisphosphonate is tiludronate. In some embodiments, suitability of a bisphosphonate may be determined by assessing its effect on the inhibition of vATPase.
  • Bisphosphonates are chemically stable derivatives of inorganic pyrophosphate (PPi), a naturally occurring compound in which 2 phosphate groups are linked by esterification. Like their natural analogue PPi, bisphosphonates have a very high affinity for bone mineral because they bind to hydroxyapatite crystals.
  • bisphosphonates In addition to their ability to inhibit calcification, bisphosphonates inhibit hydroxyapatite breakdown, thereby effectively suppressing bone resorption.
  • the core structure of bisphosphonates differs from PPi in that bisphosphonates contain a central nonhydrolyzable carbon; the phosphate groups flanking this central carbon are maintained. Nearly all bisphosphonates in current clinical use also have a hydroxyl group attached to the central carbon (termed the R 1 position).
  • the flanking phosphate groups provide bisphosphonates with a strong affinity for hydroxyapatite crystals in bone, whereas the hydroxyl motif further increases a bisphosphonate’s ability to bind calcium.
  • Non–nitrogen-containing bisphosphonates e.g. etidronate, clodronate, and tiludronate
  • Non–nitrogen-containing bisphosphonates may be categorized as first-generation bisphosphonates. Because of their close structural similarity to PPi, non–nitrogen-containing bisphosphonates become incorporated into molecules of newly formed adenosine triphosphate (ATP) by the class II aminoacyl– transfer RNA synthetases after osteoclast-mediated uptake from the bone mineral surface.
  • ATP adenosine triphosphate
  • Second- and third-generation bisphosphonates include, for example, alendronate, risedronate, ibandronate, pamidronate, and zoledronic acid, and have nitrogen-containing R 2 side chains. It is believed that nitrogen-containing bisphosphonates bind to and inhibit the activity of farnesyl pyrophosphate synthase, a key regulatory enzyme in the mevalonic acid pathway critical to the production of cholesterol, other sterols, and isoprenoid lipids.
  • bisphosphonates include once-weekly, e.g. alendronate or risedronate; and monthly, e.g. ibandronate or risedronate, oral formulations.
  • IV formulation are also available, e.g. for pamidronate, ibandronate, and zoledronic acid, which for most clinical conditions require even less frequent dosing, and have eliminated the gastrointestinal adverse effects incurred by some patients managed with oral bisphosphonates.
  • Such formulations are believed to have pharmacodynamic equivalence to daily dosing of each drug.
  • Daily dose formulations are also available.
  • the dose of bisphosphonate will depend on the periodicity of administration, as well as the specific drug that is chosen.
  • the dose may be, for example, equivalent to that conventionally used for treating bone diseases.
  • the conventional dose may be from about 5, about 10 mg, about 20 mg up to about 40 mg daily for an adult, or an equivalent dosage administered weekly, e.g.70 milligrams (mg) once a week.
  • the conventional dose may be from about 5 mg/kg, up to about 20 mg/kg.
  • tiludronate for example, 200-400 mg tiludronic acid is administered every 3 months.
  • Such dosages may be adjusted based on response of the neurologic disease, e.g. about 1.5X the conventional dosing for bone disease, about 2X, about 2.5X or more, up to the maximum tolerated dose.
  • Additional inhibitors of a lysosomal v-ATPase include, without limitation, thonzonium, alexidine, YM-175, bafilomycin, concanamycin, archazolid, lobatamide, apicularen, oximidine, cruentaren etc.
  • the dose will depend on the periodicity of administration, as well as the specific drug that is chosen.
  • the dosage of, for example, bafilomycin A1 may be administered to achieve less than the maximum inhibition level of 0.5 ⁇ M.
  • an anti-ATXN2 agent is antibody or a fragment thereof that specifically binds to a lysosomal v-ATPase subunit, such as a ATP6V0C, ATP6V1A, ATP6V1B2, ATP6V1C1, or ATP6V1D gene product.
  • a lysosomal v-ATPase subunit such as a ATP6V0C, ATP6V1A, ATP6V1B2, ATP6V1C1, or ATP6V1D gene product.
  • any NgR1 inhibitor may be used. These include, without limitation, NgR1 antagonist peptide (NAP2) or variants thereof, etc.
  • NEP1-40 and NAP2 are described in GrandPre et al (Nature.2002 May 30;417(6888):547-51.) and Sun et al.
  • the polypeptide sequence of human NEP1-40 is RIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNS.
  • Alternative inhibitors include RTN4/NoGo-Receptor decoy, for example AXER-204 is a human fusion protein that acts as a soluble decoy/trap for Nogo-A (see, for example, clinical NCT03989440, herein specifically incorporated by reference).
  • Brain-specific angiogenesis inhibitors are adhesion-GPCRs whose extracellular sequences are composed of an N-terminal domain, 4–5 thrombospondin type-1 repeats (TSRs), a hormone-binding domain (HBD), and a GAIN domain, which have been shown to be inhibitors of NgR1 (see, for example, Wang et al. (2021) Cell Nov 24;184(24):5869-5885.e25, herein specifically incorporated by reference).
  • TSRs thrombospondin type-1 repeats
  • HBD hormone-binding domain
  • GAIN domain which have been shown to be inhibitors of NgR1 (see, for example, Wang et al. (2021) Cell Nov 24;184(24):5869-5885.e25, herein specifically incorporated by reference).
  • TSRs thrombospondin type-1 repeats
  • HBD hormone-binding domain
  • GAIN domain GAIN domain
  • Antagonists of interest include antibodies or fragments thereof, soluble receptors, receptor or ligand fragments, conjugates of receptors and Fc regions, and the like.
  • antibody or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure that has a specific shape which fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins (IgG, IgM, IgA, IgE, IgD, etc.), from all sources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, turkey, emu, other avians, etc.) are considered to be “antibodies.”
  • Antibodies utilized in the present invention may be polyclonal antibodies, although monoclonal antibodies are preferred because they may be reproduced by cell culture or recombinantly, and may be modified to reduce their antigenicity.
  • Antibody fusion proteins may include one or more constant region domains, e.g.
  • a soluble receptor-immunoglobulin chimera refers to a chimeric molecule that combines a portion of the soluble adhesion molecule counterreceptor with an immunoglobulin sequence.
  • the immunoglobulin sequence preferably, but not necessarily, is an immunoglobulin constant domain.
  • the immunoglobulin moiety may be obtained from IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG1 or IgG3.
  • a straightforward immunoadhesin combines the binding region(s) of the "adhesin" protein with the hinge and Fc regions of an immunoglobulin heavy chain.
  • nucleic acid encoding the soluble adhesion molecule will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N- terminal fusions are also possible.
  • the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CH1 of the heavy chain or the corresponding region of the light chain.
  • the precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics.
  • Antibodies that have a reduced propensity to induce a violent or detrimental immune response in humans such as anaphylactic shock
  • which also exhibit a reduced propensity for priming an immune response which would prevent repeated dosage with the antibody therapeutic are preferred for use in the invention.
  • These antibodies are preferred for all administrative routes, including intrathecal administration.
  • humanized, chimeric, or xenogenic human antibodies, which produce less of an immune response when administered to humans, are preferred for use in the present invention.
  • Chimeric antibodies may be made by recombinant means by combining the murine variable light and heavy chain regions (VK and VH), obtained from a murine (or other animal- derived) hybridoma clone, with the human constant light and heavy chain regions, in order to produce an antibody with predominantly human domains.
  • VK and VH murine variable light and heavy chain regions
  • the production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated fully herein by reference).
  • Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody.
  • polyclonal or monoclonal antibodies may be produced from animals which have been genetically altered to produce human immunoglobulins, such as the Abgenix XenoMouse or the Medarex HuMAb ⁇ technology.
  • single chain antibodies Fv, as described below can be produced from phage libraries containing human variable regions.
  • immunoglobulin fragments comprising the epitope binding site (e.g., Fab’, F(ab’)2, or other fragments) are useful as antibody moieties in the present invention.
  • Such antibody fragments may be generated from whole immunoglobulins by ficin, pepsin, papain, or other protease cleavage.
  • “Fragment” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
  • “Fv” immunoglobulins for use in the present invention may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., poly-glycine or another sequence which does not form an alpha helix or beta sheet motif).
  • a peptide linker e.g., poly-glycine or another sequence which does not form an alpha helix or beta sheet motif.
  • Small molecule agents that inhibit, for example, vATPase or NgR1 encompass numerous chemical classes, though typically they are organic molecules, e.g. small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • Candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries, for example.
  • Libraries of candidate compounds can also be prepared by rational design. (See generally, Cho et al., Pac. Symp. Biocompat.305-16, 1998); Sun et al., J. Comput. Aided Mol. Des.12:597-604, 1998); each incorporated herein by reference in their entirety).
  • libraries of GABA A inhibitors can be prepared by syntheses of combinatorial chemical libraries (see generally DeWitt et al., Proc. Nat. Acad. Sci. USA 90:6909-13, 1993; International Patent Publication WO 94/08051; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al., Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem. Soc.117:5588-89, 1995; Nestler et al., J. Org. Chem.59:4723-24, 1994; Borehardt et al., J. Am. Chem.
  • Candidate antagonists can be tested for activity by any suitable standard means.
  • the antibodies may be tested for binding against the adhesion molecule of interest.
  • antibody candidates may be tested for binding to an appropriate cell line, e.g. leukocytes or endothelial cells, or to primary tumor tissue samples.
  • the candidate antibody may be labeled for detection (e.g., with fluorescein or another fluorescent moiety, or with an enzyme such as horseradish peroxidase).
  • Suitable conditions shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions” as used herein means physiological conditions.
  • a “subject” or “patient” in the context of the present teachings is generally a mammal, for example a human. Mammals other than humans can be advantageously used as subjects that represent animal models of inflammation. A subject can be male or female.
  • To “analyze” includes determining a set of values associated with a sample by measurement of a marker (such as, e.g., presence or absence of a marker or constituent expression levels) in the sample and comparing the measurement against measurement in a sample or set of samples from the same subject or other control subject(s).
  • a marker such as, e.g., presence or absence of a marker or constituent expression levels
  • the markers of the present teachings can be analyzed by any of various conventional methods known in the art.
  • To “analyze” can include performing a statistical analysis to, e.g., determine whether a subject is a responder or a non-responder to a therapy (e.g., an anti- ATXN2 treatment as described herein).
  • a “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • a pharmaceutically acceptable excipient, diluent, carrier and adjuvant includes both one and more than one such excipient, diluent, carrier, and adjuvant.
  • a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
  • it may be desirable to administer an anti ATXN2 agent into the brain region e.g. ventricles, CSF, etc.
  • a continuous delivery device includes, for example, an implanted device that releases a metered amount of an anti- ATXN2 agent continuously over a period of time.
  • Convection-enhanced delivery is a technique that generates a pressure gradient at the tip of an infusion catheter to deliver therapeutics directly through the interstitial spaces of the central nervous system.
  • Dosage unit refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.
  • “Pharmaceutically acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like.
  • Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid).
  • Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., C1-6 alkyl esters.
  • a pharmaceutically acceptable salt or ester can be a mono-acid-mono- salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified.
  • Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • a "therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
  • Neurodegenerative diseases refers to diseases and conditions that are characterized by progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.
  • neurodegenerative disease may include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease, Canavan disease, Cerebral palsy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Diabetic neuropathy, Frontotemporal lobar degeneration, Glaucoma, Guillain- Barre syndrome, Hereditary spastic paraplegia, Huntington's disease, HIV associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Motor neuron disease, Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Neuroborreliosis, Niemann Pick disease, Parkinson's disease, Pelizaeus- Merzbacher Disease, Peripheral neuropathy, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff's disease, Schilder's
  • Amyotrophic lateral sclerosis is a group of rare neurological diseases that mainly involve the nerve cells (neurons) responsible for controlling voluntary muscle movement. It is characterized by steady, relentless, progressive degeneration of corticospinal tracts, anterior horn cells, bulbar motor nuclei, or a combination. Symptoms vary in severity and may include muscle weakness and atrophy, fasciculations, emotional lability, and respiratory muscle weakness. Diagnosis involves nerve conduction studies, electromyography, and exclusion of other disorders via MRI and laboratory tests. Current treatment is supportive. The majority of ALS cases (90 percent or more) are considered sporadic.
  • ALS asymmetric symptoms
  • Weakness progresses to the forearms, shoulders, and lower limbs. Fasciculations, spasticity, hyperactive deep tendon reflexes, extensor plantar reflexes, clumsiness, stiffness of movement, weight loss, fatigue, and difficulty controlling facial expression and tongue movements soon follow.
  • Other symptoms include hoarseness, dysphagia, and slurred speech; because swallowing is difficult, salivation appears to increase, and patients tend to choke on liquids.
  • a pseudobulbar affect occurs, with inappropriate, involuntary, and uncontrollable excesses of laughter or crying.
  • Sensory systems, consciousness, cognition, voluntary eye movements, sexual function, and urinary and anal sphincters are usually spared. Death is usually caused by failure of the respiratory muscles; 50% of patients die within 3 yr of onset, 20% live 5 yr, and 10% live 10 yr. Survival for > 30 yr is rare.
  • the drugs riluzole (Rilutek) and edaravone (Radicava) have been approved to treat certain forms of ALS. Riluzole is believed to reduce damage to motor neurons by decreasing levels of glutamate, which transports messages between nerve cells and motor neurons.
  • Animal models for ALS include mutations in the SOD1 gene. Missense mutations in the SOD1 gene on chromosome 21 were the first identified causes of autosomal dominant FALS. SOD1 is a ubiquitous cytoplasmic and mitochondrial enzyme which functions in a dimeric state to catalyse the breakdown of harmful reactive oxygen species (ROS), thereby preventing oxidative stress.
  • ROS reactive oxygen species
  • Sod1 ⁇ / ⁇ mice do not have any motor neuron loss, but they have a significant distal motor axonopathy, demonstrating the important role of SOD1 in normal neuronal function.
  • the significant loss of motor neurons in transgenic mice expressing mutant SOD1 is likely to result from a toxic gain-of-function.
  • SCA2 The spinocerebellar ataxia type 2 (SCA2) is one of the most common polyglutamine (polyQ) disorders. Caused by a dominant expansion of a CAG repeat tract (CAGexp) at ATXN2, SCA2 is related to a polyQ with more than 32–33 glutamines in ataxin-2. Disease usually starts in adulthood and clinical picture is not homogeneous.
  • Main symptoms are related to cerebellar dysfunction, and include ataxic gait, cerebellar dysarthria as well as dysmetria. Other symptoms include uncoordinated movement (ataxia), speech and swallowing difficulties, muscle wasting, slow eye movement, and sometimes dementia . Severe saccade slowing and peripheral neuropathy are very frequent and affect more than 50% of case series . Besides, several other manifestations might appear, such as pyramidal findings, extrapyramidal syndromes (including dystonic movements and parkinsonism), lower motor neuron findings, cognitive deterioration, and others. ATXN2 expansion explains most but not all variability in age at onset (AO) of symptoms, and it was related to presence of some neurological findings such as dystonic movements and parkinsonism.
  • AO age at onset
  • SCA2 Mean (SD) age at onset was around 30 to 33 (14) years and median survival was 68 [95% CI: 65–70] years, usually after a wheelchair period.
  • SCA2 mouse model was a transgenic line SCA2-58Q.
  • a full-length human ATXN2 gene with 58 CAG repeats was expressed under Purkinje cell protein 2 (Pcp2) promoter specifically in the cerebellar PCs of the mice.
  • Pcp2 Purkinje cell protein 2
  • the present disclosure provides methods for treating neurodegenerative diseases, including ALS and SCA2.
  • the methods comprise administering to the subject an effective amount of an agent that is an anti-ATXN2 agent as a single agent or combined with an additional one or more agent(s).
  • Methods of treatment may include determining the effectiveness of therapy by monitoring clinical indicia for stabilization or reduction of adverse disease symptoms.
  • administration may be oral.
  • polypeptides such as NEP1-40, antibodies, etc.
  • CED Convection-enhanced drug delivery
  • Nanoparticles as drug or gene carriers may be used, e.g. in combination with CED. Transport of particles through the extracellular space of tissues is hindered by the large size of nanoparticles (10-100 nm), which are much larger than small molecule drugs or therapeutic proteins that more easily penetrate the brain extracellular matrix (ECM). However, nanoparticles may be able to penetrate brain tissue provided that particles are less than 100 nm in diameter, are neutral or negatively charged, and are not subject to rapid elimination mechanisms.
  • the agent is delivered as continuous intraventricular CNS administration.
  • intraventricular administration is combined with systemic administration, for example utilizing an implantable device to deliver the agent.
  • the implantable device is an osmotic pump.
  • the device may be implanted intraventricularly, for example, with a conventional stereotaxic apparatus.
  • a continuous delivery device includes, for example, an implanted device that releases a metered amount of an agent continuously over a period of time.
  • the device may be implanted so as to release the anti-ATXN2 agent into the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • An example of such devices is an osmotic pump, which operates because of an osmotic pressure difference between a compartment within the pump, called the salt sleeve, and the tissue environment in which the pump is implanted.
  • the high osmolality of the salt sleeve causes water to flux into the pump through a semipermeable membrane which forms the outer surface of the pump. As the water enters the salt sleeve, it compresses the flexible reservoir, displacing the test solution from the pump at a controlled, predetermined rate. The rate of delivery is controlled by the water permeability of the pump’s outer membrane. Thus, the delivery profile of the pump is independent of the drug formulation dispensed. Drugs of various molecular configurations, including ionized drugs and macromolecules, can be dispensed continuously in a variety of compatible vehicles at controlled rates.
  • the anti-ATXN2 agent is combined with a therapeutic dose of a drug used to treat ALS or SCA2 such as riluzole, baclofen, quinine, phenytoin, glycopyrrolate, amitriptyline, benztropine, trihexyphenidyl, transdermal hyoscine, atropine, fluvoxamine, dextromethorphan, quinidine, or gabapentin.
  • a drug used to treat ALS or SCA2 such as riluzole, baclofen, quinine, phenytoin, glycopyrrolate, amitriptyline, benztropine, trihexyphenidyl, transdermal hyoscine, atropine, fluvoxamine, dextromethorphan, quinidine, or gabapentin.
  • the active agents may be administered in separate formulations, or may be combined, e.g. in a unit dose.
  • the formulation may
  • the combined therapies are administered concurrently, where the administered dose of any one of the compounds may be a conventional dose, or less than a conventional dose.
  • the two therapies are phased, for example where one compound is initially provided as a single agent, e.g. as maintenance, and where the second compound is administered during a relapse, for example at or following the initiation of a relapse, at the peak of relapse, etc.
  • administering the therapeutic compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • the administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously.
  • the delivery systems employ a number of routinely used pharmaceutical carriers.
  • an effective dose of an anti-ATXN2 agent of the invention is administered alone, or combined with additional active agents for the treatment of a condition as listed above.
  • the effective dose may be from about 1 ng/kg weight, 10 ng/kg weight, 100 ng/kg weight, 1 ⁇ g/kg weight, 10 ⁇ g/kg weight, 25 ⁇ g/kg weight, 50 ⁇ g/kg weight, 100 ⁇ g/kg weight, 250 ⁇ g/kg weight, 500 ⁇ g/kg weight, 750 ⁇ g/kg weight, 1 mg/kg weight, 5 mg/kg weight, 10 mg/kg weight, 25 mg/kg weight, 50 mg/kg weight, 75 mg/kg weight, 100 mg/kg weight, 250 mg/kg weight, 500 mg/kg weight, 750 mg/kg weight, for example up to about 500 mg/kg weight, and the like.
  • the dosage may be administered multiple times as needed, e.g.
  • the dosage may be administered orally.
  • Examples of doses for polypeptide drugs e.g. peptides, antibodies, etc.
  • 0.05 mg/kg to about 10 mg/kg may include, but are not necessarily limited to a range from about 0.05 mg/kg to about 10 mg/kg (e.g., from about 0.1 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 7.5 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 4 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 7.5 mg/kg, from about 0.5 mg/kg to about 5 mg/kg, from about 0.5 mg/kg to about 4 mg/kg, from about 0.5 mg/kg to about 3 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 7.5 mg/kg,from about 1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 4 mg/kg, from about 1 mg/kg to about 3 mg/kg, about 1 mg/
  • Examples of doses for small molecules, e.g. bisphosphonates can vary widely, but may be 0.05 ⁇ g/kg to about 20 mg/kg (e.g., from about 0.1 ⁇ g/kg to about 10 mg/kg, from about 0.1 ⁇ g/kg to about 7.5 mg/kg, from about 0.1 ⁇ g/kg to about 5 mg/kg, from about 0.1 ⁇ g/kg to about 4 mg/kg, from about 0.1 ⁇ g/kg to about 3 mg/kg, from about 0.5 ⁇ g/kg to about 10 mg/kg, from about 0.5 ⁇ g/kg to about 7.5 ⁇ g/kg, from about 0.5 ⁇ g/kg to about 5 mg/kg, from about 0.5 ⁇ g/kg to about 4 mg/kg, from about 0.5 ⁇ g/kg to about 3 mg/kg, from about 1 ⁇ g/kg to about 10 mg/kg, from about 1 ⁇ g/kg to about 7.5 mg/kg, from about 1 ⁇ g/kg to about 5 mg/kg, from
  • compositions can be administered in a single dose, or in multiple doses, usually multiple doses over a period of time, e.g. daily, every-other day, weekly, semi-weekly, monthly etc. for a period of time sufficient to reduce severity of the inflammatory disease, which can comprise 1, 2, 3, 4, 6, 10, or more doses.
  • Determining a therapeutically or prophylactically effective amount of an agent according to the present methods can be done based on animal data using routine computational methods. The effective dose will depend at least in part on the route of administration.
  • Pharmaceutical Compositions [00122] The above-discussed compounds can be formulated using any convenient excipients, reagents and methods. Compositions are provided in formulation with a pharmaceutically acceptable excipient(s).
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • the subject compound is formulated in an aqueous buffer.
  • Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5mM to 100mM.
  • the aqueous buffer includes reagents that provide for an isotonic solution.
  • Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like.
  • the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80.
  • the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4oC.
  • Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.
  • the subject compound is formulated for sustained release.
  • the anti-ATXN2 agent is formulated with a second agent in a pharmaceutically acceptable excipient(s).
  • the subject formulations can be administered orally, subcutaneously, intramuscularly, parenterally, or other route, including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical or injection into an affected organ.
  • Each of the active agents can be provided in a unit dose of from about 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 5 ⁇ g, 10 ⁇ g, 50 ⁇ g, 100 ⁇ g, 500 ⁇ g, 1 mg, 5 mg, 10 mg, 50, mg, 100 mg, 250 mg, 500 mg, 750 mg or more.
  • the anti-ATXN2 agent may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients.
  • a pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used.
  • suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders.
  • suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Preferred carriers are edible oils, for example, corn or canola oils.
  • Polyethylene glycols, e.g. PEG are also good carriers.
  • Example 1 Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2
  • Antisense oligonucleotide therapy targeting ATXN2 a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis— has entered clinical trials in humans. Additional methods to lower ataxin-2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in defining how ataxin-2 is normally regulated.
  • FACS fluorescence activated cell sorting
  • v-ATPases lysosomal vacuolar ATPases
  • Figure 3A Lysosomal v-ATPases help to maintain an acidic pH ( ⁇ 4.5) in the lysosome, by pumping protons from the cytosol to the lumen via consumption of ATP (Maxson and Grinstein, 2014).
  • siRNAs and immunoblotting to confirm that knocking down numerous v-ATPase subunits leads to decreased ataxin-2 protein levels (Figure 3B and 3C).
  • Etidronate ( ⁇ 206 Daltons), which was FDA-approved in 1977 as a drug to treat Paget disease of bone (Altman et al., 1973). Interestingly, Paget disease of bone has been connected to TDP-43 proteinopathy (Gitcho et al., 2009; Neumann et al., 2007; Watts et al., 2004). Etidronate is a bisphosphonate whose chemical structure and high affinity for bone minerals very selectively induces apoptosis in osteoclasts, popularizing their use in skeletal disorders like osteoporosis over multiple decades (Drake et al., 2008).
  • Alendronate ( ⁇ 249 Daltons) is a second-generation bisphosphonate (also known as Fosamax®), whereas Thonzonium ( ⁇ 511 Daltons) inhibits the v-ATPase through a different mechanism (uncoupling the proton transport and ATPase activity of the v-ATPase proton pumps).
  • Thonzonium and Alendronate decreased ataxin-2 protein levels across a wide range of doses ( Figure S5C, S5D, S6C and S6D), they became toxic to mouse neurons at concentrations greater than 10 ⁇ M. This toxic dose has been previously reported for Thonzonium (Chan et al., 2012). Etidronate, however, was not toxic even at doses up to 100 ⁇ M (highest concentration tested) ( Figure 4E and 4F). The differences in toxic doses align with their known differences in IC 50 (the concentration of drug t hat is needed to inhibit a biological process by half) (David et al., 1996).
  • Etidronate s well-known safety, small size, and predicted ability to cross the BBB, we chose to focus on Etidronate for the following experiments. Still, it is noteworthy that three different v-ATPase inhibitors—working through distinct mechanisms—potently decrease ataxin-2 protein levels in neurons without causing the type of toxicity seen in osteoclasts. [00139]
  • Etidronate s effects on TDP-43 levels and/or localization, since TDP-43 nuclear depletion and cytoplasmic aggregation are pathologic hallmarks of ALS (Neumann et al., 2006).
  • Alendronate a structural analog of Etidronate—is similarly effective in decreasing ataxin-2 levels in neurons in vitro, indicating the potential to further develop and/or test structural analogs of bisphosphonates that can most effectively cross the BBB and decrease ataxin-2 levels in the brain.
  • Etidronate Given Etidronate’s known safety profile and our demonstration of its ability to lower ataxin-2 levels in the brain in vivo, we postulate that Etidronate could serve as a starting compound for future optimization to decrease ataxin-2 levels in the brain.
  • Example 2 we present a complementary screen for regulators of ataxin-2 using a distinct approach: a whole-genome arrayed siRNA-based screen in a HEK293T cell line containing an 11-amino acid HiBit tag in frame with ataxin-2. Perhaps due to one screen being conducted in HeLa cells versus the other in HEK293T cells—or one being a CRISPR-Cas9- based knockout screen versus the other being a siRNA HiBit screen—the screens led to very distinct hits (only one gene in common – LSM12).
  • both sets of screens did reveal several converging themes of ataxin-2 regulation, such as the presence of many spliceosome components and LSm domain-containing proteins.
  • the lack of hit overlap is largely due to differences in the cell types (i.e., different genes expressed) and screening systems (CRISPR knockout vs. HiBit knockdown).
  • Another contributor to differences between the two screens could stem from one screen being pooled (all cells mixed together and competing against one another) while the other is arrayed (analyzed one by one).
  • Low-passage HeLa cells were transfected at 40-50% confluency in a 100 mm dish with lentiviral concentrations such that the infection rate was ⁇ 20%, to reduce the chance that a single cell will incorporate multiple lentiviral particles.
  • the culturing media was changed to blasticidin (10 ⁇ g/mL)-containing DMEM, high glucose, GlutaMAXTM, HEPES media media (Gibco) to select for cells that incorporated Cas9-Blast.
  • the blasticidin- containing DMEM media was replaced every 24 hours until a control plate in parallel of the same quantity of non-Cas9-infected HeLa cells exhibited complete cell death.
  • Cas9-BFP cells were clonally isolated using FACS.
  • Genome-wide deletion library production and titering All gRNA oligonucleotides were constructed on a microfluidic array, then lentivirus was generated using standard protocols. Briefly, all guides were pooled together at roughly the same concentration (10 sgRNAs per gene targeting ⁇ 21,000 human genes and ⁇ 10,000 safe-targeting sgRNAs), which were cloned into a lentiviral backbone. This pool was used to transfect low-passage HEK293T cells at 70-80% confluency in 150 mm dishes, from which the resulting supernatant contained all 25,000 sgRNAs, with each sgRNA represented ⁇ 1000 times.
  • HeLa-Cas9 cells were cultured in DMEM, high glucose, GlutaMAXTM, HEPES media (Gibco) containing 10% FBS (Omega) and 1% penicillin-streptomycin (P/S) (Gibco) in 150 mm plates.
  • the virus titering was performed such that 5-10% of cells were mCherry-positive, to reduce the chance that a single cell will incorporate multiple gRNAs.24 hours after infection, media was changed to DMEM media containing puromycin (1 ⁇ g/mL) to select for infected cells. The puromycin-containing media was replaced every 24 hours until >90% of cells were mCherry- positive, and an uninfected control plate containing HeLa-Cas9 cells exhibited complete cell death when subjected to puromycin selection. The cells were grown for an additional five days to give Cas9 ample time to cut. [00146] Fixation and IF.
  • the cells were then rinsed in PBS, resuspended in PBS containing 2 mM EDTA, and taken directly to the FACS facility for sorting.
  • Fluorescence activated cell sorting To identify genetic modifiers of ataxin-2 protein levels, the cell suspension was sorted using a BD FACSAria II cell sorter with a 70 ⁇ m nozzle (BD Biosciences). Cell populations containing the lowest and highest 20% of ataxin-2 levels— relative to ⁇ -actin or GAPDH— were sorted, respectively. Each sorted population, as well as the unsorted (starting) population, were spun down at 600g for 20 minutes at room temperature before extracting genomic DNA. [00148] Genomic DNA extraction, PCR amplification, and next-generation sequencing.
  • Genomic DNA was extracted immediately after pelleting using the Blood and Tissue DNeasy Maxi Kit (QIAGEN, 51194). The DNA was isolated according to the manufacturer’s instructions, with the exception of eluting with buffer EB, rather than buffer AE. After PCR amplification using Agilent Herculase II Fusion DNA Polymerase Kit, deep sequencing was performed on an Illumina NextSeq 550 platform to determine library composition. Guide composition between the sorted top 20% and the unsorted (starting) populations were compared using Cas9 high Throughput maximum Likelihood Estimator (casTLE) (Morgens et al., 2016) to determine genes that, when knocked out, increased or decreased ataxin-2 protein levels.
  • casTLE Cas9 high Throughput maximum Likelihood Estimator
  • HeLa cells (ATCC ® ) were cultured in DMEM, high glucose, GlutaMAXTM, HEPES media (Gibco) containing 10% f etal-bovine serum (FBS) (Omega) and 1% penicillin-streptomycin (P/S) (Gibco) in a controlled humidified incubator at 37°C with 5% CO 2 .
  • DMEM high glucose
  • GlutaMAXTM HEPES media
  • FBS f etal-bovine serum
  • P/S penicillin-streptomycin
  • s iRNA reverse transfection experiments were conducted similarly as for HeLa, except for performing knockdown for 96 hours in two doses (a second dose given after 24 hours with a full media change) and complexing with Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Gibco) for 20 minutes prior to addition of cells. Cells were cultured in 24-well plates. [00151] Generating ATXN2 mosaic knockout cell line.
  • a sgRNA oligonucleotide targeting the first shared exon in ATXN2 was cloned into a lentiviral backbone that contains mCherry and a puromycin resistance cassette. This construct was used to transfect low-passage HEK293T cells at 70-80% confluency in 100 mm plates.
  • HeLa-Cas9 cells (cultured in DMEM, high glucose, GlutaMAXTM, HEPES media containing 10% FBS and 1% penicillin- streptomycin in 100 mm plates) were then infected with the lentiviral media generated above, such that ⁇ 50% of cells were mCherry-positive. 24 hours after infection, the media was changed to puromycin-containing media (1 ⁇ g/mL) to select for cells that received a sgRNA. The puromycin- containing media was replaced every 24 hours until >90% of cells were mCherry-positive, and an uninfected control plate containing HeLa-Cas9 cells exhibited complete cell death upon subjection to puromycin selection.
  • Protein concentrations were determined using bicinchoninic acid (Invitrogen 23225) assays. Samples were denatured at 70°C in LDS sample buffer (Invitrogen NP0008) containing 2.5% 2- mercaptoethanol (Sigma-Aldrich) for 10 minutes. Samples were run on 4–12% Bis–Tris gels (Thermo Fisher) using gel electrophoresis, then wet-transferred (Bio-Rad Mini Trans- Blot Electrophoretic Cell 170-3930) onto 0.45 ⁇ m nitrocellulose membranes (Bio-Rad 162- 0115) at 100V for 90 minutes.
  • LDS sample buffer Invitrogen NP0008
  • 2- mercaptoethanol Sigma-Aldrich
  • Imaging Blocking Buffer (LiCOr 927-40010) was applied to membranes for one hour then replaced with Odyssey Blocking Buffer containing antibodies against ataxin-2 (1:1000, ProteinTech 21776-1-AP) and ⁇ -actin (1:2000, Thermo Fisher Scientific MA1-744) and placed on a shaker overnight at room temperature. After rinsing three times in PBS-Tween (0.1%) for 10 minutes each, membranes were incubated in Odyssey Blocking Buffer containing HRP-conjugated anti-rabbit IgG (H+L) (1:2000, Life Technologies 31462) or anti-mouse IgG (H+L) (1:2000, Fisher 62-6520) secondary antibodies for one hour.
  • RNA Quantification After reverse transfection with siRNAs in 12-well plates as described in the ‘Cell culture and siRNA transfection’ section, the PureLink® RNA Mini Kit was used to isolate RNA with DNase digestion (Thermo Fisher Scientific 12183025). To convert RNA to cDNA, we used the Applied Biosystems High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific 4368813).
  • RNA-sequencing was performed using TaqManTM Universal Master Mix II (Thermo Fisher Scientific4440040), using 1 ⁇ L of 20X TaqMan gene- specific expression assay to the reaction and our probes of interest (Thermo Fisher Scientific; human ATXN2: Hs00268077_m1, human ACTB: Hs01060665_g1).
  • the Delta-Delta Ct method was run on the thermocycler and visualized on Thermo Fisher ConnectTM, from which relative expression values were averaged across all biological/technical replicates per condition.
  • RNA-seq After knocking down a v- ATPase subunit, we performed RNA-seq after HeLa cells were treated with NT or ATP6V1A siRNAs for 72 hours, as described in the 'Cell culture and siRNA transfection' section. Briefly, we isolated RNA using the PureLink® RNA Mini Kit with DNase digestion (Thermo Fisher Scientific 12183025), then determined RNA quantity and purity by optical density measurements of OD260/280 and OD230/260 using a NanoDrop spectrophotometer.
  • RNA libraries were sequenced on an Illumina Nova-Seq 6000 machine.
  • the cortices were dissected out and dissociated into single-cell suspensions with a papain dissociation system (Worthington Biochemical Corporation) and plated onto poly-L-lysine (Sigma Aldrich)-coated plates (0.1% (wt/vol)) at a density of 350,000 cells per well in 24-well plates.
  • the neurons were grown in Neurobasal medium (Gibco) supplemented with P/S (Gibco), GlutaMAX (Invitrogen), and B-27 serum-free supplements (Gibco) at 37°C with 5% CO 2 .
  • iPSCs-derived neurons were induced utilizing a Tet-On induction system to express the transcription factor Ngn2. Briefly, iPSCs were maintained in mTeSR1 medium (Stemcell Technologies) on Matrigel-coated plates (Fisher Scientific CB-40230).
  • doxycycline (2 ⁇ g/mL) was added to the media to induce Ngn2 expression, followed by puromycin (2 ⁇ g/mL) treatment to rapidly and efficiently induce iNeurons.
  • the differentiating iNeurons were dissociated using Accutase (STEMCELL Technologies) and resuspended in a culture medium consisting of Neurobasal media (Thermo Fisher), N2 (Thermo Fisher), B-27 (Thermo Fisher), BDNF/GDNF (R&D Systems) on Matrigel-coated assay plates. This resuspension mixture was then plated onto Matrigel-coated 24-well plates.
  • Half-media changes were performed every 2-3 days.6-7 days after Ngn2 induction, the cells were treated with various doses of Etidronate (dissolved in media) or water (control, all to equal volumes) and lysed 24 hours later for protein collection.
  • Etidronate dissolved in media
  • water control, all to equal volumes
  • SH-SY5Y cells they were seeded at a density of 5x10 5 cells per well in 24-well plates. One day after plating, the cells were treated with various doses of Alendronate or Thonzonium (dissolved in media, all to equal volumes) and lysed 24 hours later for protein collection.
  • Immunocytochemistry, microscopy, and image quantification were performed every 2-3 days.6-7 days after Ngn2 induction, the cells were treated with various doses of Etidronate (dissolved in media) or water (control, all to equal volumes) and lysed 24 hours later for protein collection.
  • WT or ATP6V1A KO HeLa cells were grown on poly-L-lysine-coated glass coverslips [0.1% (wt/vol)] in 24-well plates (four wells per condition), then fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were rinsed 3 times with PBS then blocked with 1% BSA containing 0.4% Triton X- 100 for one hour. After overnight primary antibody incubation (1:1000 ataxin-2, ProteinTech 21776-1-AP; 1:1000 ⁇ -actin, Thermo Fisher Scientific MA1-744), cells were rinsed 3 times with PBS prior to incubating with fluorescent secondary antibodies (1:500) for one hour.
  • coverslips were mounted using Prolong Diamond Antifade mount containing DAPI (Thermo Fisher Scientific). All steps were carried out at room temperature. Images were acquired using a Zeiss LSM 710 confocal microscope (three fields-of-view per coverslip, four coverslips per condition). Images were processed and analyzed using ImageJ (version 2.1.0). Quantification of fluorescence intensities was conducted on >270 cells per condition. [00159] To determine whether Etidronate treatment led to changes in TDP-43 protein levels and localization via immunocytochemistry, we followed the same protocol as above, except that we plated primary neurons from mouse embryos (E16.5) in 24-well plates on glass coverslips at 350,000 cells/well density.
  • RTN4R knockdown or treatment with a peptide inhibitor, was sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro and Rtn4r knockout mice have reduced ataxin-2 levels in vivo.
  • ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy.
  • siRNA targets that decrease ataxin-2 were highly enriched for components of the spliceosome. Targeting one of the strongest hits, RTN4R, or peptide inhibition of RTN4R (also known as RTN4/NoGo- receptor), a regulator of neurite development, reduced ataxin-2 levels in vitro and in vivo. Moreover, knockdown of ataxin-2 promoted axonal regeneration in primary cortical neurons following injury. These results leverage the robust effect of ataxin-2 as a disease modifier to define novel therapeutic targets.
  • HiBiT is an 11-amino acid peptide subunit of the NanoBiT luciferase enzyme that is inert on its own but binds with high affinity to LgBiT, reconstituting NanoBiT. This allows for easy and specific detection of ataxin-2 using an antibody-free blotting method ( Figure 12B-D).
  • siRNAs that significantly impacted expression levels of the FFluc control in the same direction as ataxin-2 levels—resulting in 348 primary screen hits (Data Table 2).
  • DepMap is a resource of essential genes across a range of cell types (Meyers et al., 2017, McFarland et al., 2018), usually used to identify cancer vulnerabilities that can be exploited. However, for neurodegeneration targets, we sought to avoid essential genes, thus filtered these out of our hit list. We also filtered hits for central nervous system expression (GTEx database, cortical pTPM>15) (2013) ( Figure 13A).
  • RTN4R the gene that encodes RTN4/NoGo- Receptor.
  • RTN4/NoGo-Receptor has been implicated in axon regeneration, sprouting, and plasticity (Fournier et al., 2001, McGee et al., 2005, Wang et al., 2020, Wang et al., 2011, Akbik et al., 2013, Bhagat et al., 2016, Kim et al., 2004, Fink et al., 2015), and targeting its ligand NoGo- A modulates mutant SOD1 mouse models of ALS (Jokic et al., 2006, Bros-Facer et al., 2014, Fournier et al., 2001, Yang et al., 2009).
  • RTN4/NoGo-Receptor has several glia- and neuron- derived ligands including the three gene products of the RTN4 gene—NoGo-A, B, and C—as well as oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG) (Schwab, 2010, Domeniconi et al., 2002, Wang et al., 2002).
  • OMgp oligodendrocyte myelin glycoprotein
  • MAG myelin-associated glycoprotein
  • the HiBiT system allows for monitoring protein degradation (Riching et al., 2018).
  • siRNA knockdown of RTN4R treatment of cells with a proteasome inhibitor (Figure 13G), but not an autophagy inhibitor ( Figure 22A), resulted in an increase in ataxin-2 to levels comparable to the non-targeting control, indicating that ataxin- 2 is degraded by the proteasome.
  • RTN4R knockdown did not increase 20S proteasome activity ( Figure S7B), suggesting a specific effect on ataxin-2 proteasomal degradation caused by RTN4R knockdown.
  • NEP1-40 is a peptide that acts as a competitive RTN4/NoGo-Receptor antagonist. It is a fragment of the luminal region of NoGo-A, B and C that binds to RTN4/NoGo-Receptor to prevent ligand signaling ( Figure 13H) (GrandPré et al., 2002). NEP1-40 treatment decreased ataxin-2 levels in the ataxin-2-HiBiT cells ( Figure 13I). Thus, targeting RTN4R by either genetic knockdown or with a peptide inhibitor leads to decreased levels of ataxin-2. [00171] Knockdown or inhibition of the RTN4/NoGo-Receptor lowers ataxin-2 levels in mouse and human neurons.
  • mice cortical neurons and human iPSC-derived neurons (iNeurons) ( Figure 14A and 14B) (Bieri et al., 2019).
  • Treatment of mouse cortical neurons and human iNeurons with lentiviruses expressing shRNA targeting mouse or human RTN4R respectively resulted in about a 50% reduction of ataxin-2 (Figure 14C-F), without affecting levels of ATXN2 mRNA ( Figure 23).
  • Application of the NEP1-40 inhibitor peptide caused a dose-dependent reduction of ataxin-2 in both mouse cortical neurons and human iNeurons ( Figure 14G-L).
  • Ataxin-2 has been implicated in two neurodegenerative diseases - ALS and SCA2 - efforts are underway to target it therapeutically including the use of ASOs targeting ATXN2 (Becker et al., 2017, Scoles et al., 2017).
  • Example 1 we identified v- ATPase as a target to lower ataxin-2 levels and Etidronate and other bisphosphonates as potent small molecule inhibitors of ataxin-2 levels.
  • RTN4/NoGo-Receptor is an optimal target for therapeutic reduction of ataxin-2 because in addition to gene-based strategies there are several ways in which it may be targeted including receptor inhibition, demonstrated here, as well as neutralization of its ligands (Schwab, 2010).
  • a RTN4/NoGo-Receptor Decoy (Wang et al., 2011, Wang et al., 2020) is being investigated in a clinical trial for chronic spinal cord injury (ClinicalTrials.gov identifier: NCT03989440). With additional targets and strategies to lower ataxin-2 levels in hand, combination therapies can be used to have maximum therapeutic benefit and to mitigate potential negative effects of relying on a single strategy.
  • Methods [00177] Cell culture and transfection.
  • HEK293T cells were maintained in a 37°C incubator with 5% CO 2 in DMEM with Glutamax and HEPES (Thermo Fisher Scientific, cat# 10564-029), 10% fetal bovine serum (vol/vol; Invitrogen, cat# 16000-044), and 1% Pen/Strep (vol/vol; Invitrogen, cat# 15140-122).
  • 25 ⁇ L of lysate was placed in two wells of an opaque white, flat bottom 96-well assay plate (Sigma Aldrich, cat# CLS3990).
  • HiBiT detection 25 ⁇ L of HiBiT lytic reagent (1:25 substrate, 1:50 LgBiT) was added to the lysate (Promega, cat# N3050).
  • FFluc detection 25 ⁇ L of ONE-Glo Assay Buffer (Promega, cat# E6120) was added to the lysate. Plates were incubated in the dark with gentle rotation for 10 minutes, then luminescence was measured on a Tecan Spark plate reader (Tecan).
  • HiBiT signal was normalized to the FFluc signal for each individual well of the original transfected plate then normalized to the non-targeting/untreated control, this value is represented in bar graphs.
  • Genome Editing HEK293T cells (ATCC) were transfected using LipofectamineTM CRISPRMAXTM Cas9 Transfection Reagent (Thermo Fisher Scientific, cat# cmax00003), TrueCut Cas9 Protein V2 (Thermo Fisher Technology, cat# A36497), purified sgRNA and ssDNA (IDT).72 hr later, cells were lifted and re-plated at a density of ⁇ 1 cell/well in a 96-well plate.
  • Protein was transferred onto a nitrocellulose membrane (Bio-Rad, cat# 162-0115) at 4°C for 1hr and 45 minutes in 1X NuPAGE® Transfer Buffer (Life Technologies, cat# NP0006-1). Blocking and antibody incubation was performed in 2% BSA (Sigma Aldrich, cat# A7906) in PBS + 0.1% tween-20 (PBST). Washes were performed in PSBT.
  • siRNA pools were tested in duplicate for both FFluc and HiBiT on 4 separate plates. Each plate had three controls: siTOX control siRNA, nontargeting control siRNA, and ATXN2 siRNA (Horizon Discovery, cat# L-011772-00) as a toxicity, negative, and positive control, respectively. All siRNAs were reverse transfected using 10 ⁇ L of Opti-MEM, 0.075 ⁇ L/well of Dharmafect 1, and 10 ⁇ L siRNA (final concentration of 50nM). Cells were seeded at 1000 cells/well in 30 ⁇ L of media (as above without Pen/Strep) in solid white 384-well plates, then placed in a 37°C incubator with 5% CO 2 . After 3 days, plates were removed from the incubator.
  • HiBiT lytic reagent was made by diluting Nano-Glo® HiBiT Lytic Substrate (1:50) and LgBiT Protein (1:100) in Nano-Glo® HiBiT Lytic Buffer.
  • 10 ⁇ L of HiBiT lytic reagent was dispensed into wells with a Multidrop Reagent Dispenser (Thermo Scientific), and rapidly shook for 15 seconds.
  • FFluc detection 10 ⁇ L of ONE-Glo Assay Buffer was dispensed into wells and rapidly shook for 15 seconds.
  • Luminescence was read on a Tecan Infinite M1000 Pro (Tecan).
  • Z-scores were calculated for each siRNA replicate using the in-plate standard deviation, then averaged for both HiBiT and FFluc conditions respectively. The average Z- scores were used to filter for primary screen hits. siRNAs were only considered if they had an average FFluc Z-score greater than -1, this cutoff removed any siRNA treatment that resulted in FFluc levels decreased more than one standard deviation from the mean. Since directionality of effect matters specifically when searching for a treatment that decreases ataxin-2, we implemented this cutoff for FFluc to ensure that our negative control was not similarly affected indicating a global and non-specific effect on the treated cells.
  • siRNAs were categorized as negative regulators of ataxin-2 expression if they had an average HiBiT Z-score less than - 1.65.102 of these were retested in a secondary screen by reselecting siRNAs from the original library stock. The same experimental parameters applied, except for the performance of a single replicate for FFluc detection. High confidence hits were chosen if they had a greater than 30% decrease in average HiBiT levels relative to the non- targeting siRNA control run in parallel on the same plate. High confidence hits were ranked by average HiBiT Z-score, then were filtered further to determine the most optimal therapeutic target.
  • HEK293T cells were grown on 10cm culture dishes to 80-90% confluency. Cells were transfected using Lipofectamine 3000. Briefly, 10 ⁇ g of shRNA vector (Mission® shRNA, Sigma Aldrich), 2.9 ⁇ g pRSV-REV, 5.8 ⁇ g pMDLg/pRRE, 3.5 ⁇ g pMD2.G, and 40 ⁇ L of P3000 was added to 1mL of Opti-MEM. This was combined with another 1mL of Opti-MEM with 40 ⁇ L of Lipofectamine 3000.
  • Neurons were grown in Neurobasal media supplemented with B-27 at 1:50 (Life Technologies, cat# 17504-044), Glutamax at 1:100 (Invitrogen, cat# 35050-061) and Pen/Strep. Neurons were maintained in a 37°C incubator with 5% CO2 with half media changes every 4 to 5 days. [00188] At DIV 5, neurons were transduced with virus as above (Mission® shRNA, mouse RTN4R: TRCN0000436683).24hr later, a half media change was performed. Neurons were maintained for 12 days after transduction prior to harvesting for experimentation.
  • Vacuum- assisted axotomy was achieved by complete aspiration of media from the axonal compartment/inner chamber, allowing for an air bubble to dislodge and shear axons(Tong et al., 2015). Media was replaced and completely aspirated a second time. Media was replaced carefully to prevent the inclusion of any air bubbles in the inner chamber. A half media change was performed the next day. Neurons were allowed to regrow axons for a total of 48hr prior to fixation. [00190] iNeuron culture.
  • iPSC-derived neurons iNeurons
  • iPS cells were maintained with mTeSR1 medium (Stemcell Technologies, cat# 85850) on Matrigel (Fisher Scientific, cat# CB-40230).
  • NGN2 was expressed by adding doxycycline (2 ⁇ g/ml) and selection with puromycin (2 ⁇ g/ml) for rapid and highly efficient iNeuron induction.
  • iNeurons were dissociated and grown in Neurobasal medium containing N-2 supplement (Gibco, cat# 17502048), B-27 supplement, BDNF (R&D Systems) and GDNF (R&D Systems) on Matrigel-coated plates.
  • N-2 supplement Gibco, cat# 17502048
  • B-27 supplement B-27 supplement
  • BDNF R&D Systems
  • GDNF R&D Systems
  • iNeurons were transduced with virus as above (Mission® shRNA, human RTN4R: TRCN0000061558).24hr later, a half media change was performed.
  • iNeurons were maintained for 3 days before being re-transduced with a half dose of virus, then maintained for another 5 days prior to harvesting for experimentation.
  • NEP1- 40 treatment 1mg of NEP1- 40 was diluted as above.
  • the peptide was added at 7 days post induction to the final concentration specified along with vehicle (0 ⁇ M). Cells were maintained for 48hr prior to lysis.
  • Cells were blocked in 2% BSA for an hour, then incubated in primary antibody for at least 2 hours at room temperature [ataxin-2 (Novus), MAP2 (Synaptic Systems, cat# 188004), Tuj1 (BioLegend, cat# 802001), NeuN (EMD Millipore, cat# MAB377)].
  • Cells were washed 3 times with PBS-MC, followed by incubation in species-specific Alexa Fluor®- labeled secondary antibody (Thermo Fisher Scientific) for 1 hour at 1:1000. Cells were subsequently washed and placed in ProLong Gold Antifade with DAPI (Thermo Fisher Scientific, cat# P36931), and imaged using a Leica DMI6000B fluorescent microscope.
  • RNA Quantification and Sequencing Cells were reverse transfected with siRNA in 12- well plates as described above. RNA was isolated using the PureLink® RNA Mini Kit according to the kit protocol with DNase digestion (Thermo Fisher Scientific, cat# 12183025).500ng of RNA was used to make cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, cat# 4368813).
  • RNA sequencing polyA- selected libraries were prepared in biological duplicate for each shRNA condition using the TruSeq Stranded mRNA kit from Illumina and sequenced on a HiSeq 4000 (Illumina) with paired end, 75 nucleotide-long reads. GEO accession number GSE200530. qPCR was performed in a 20 ⁇ L reaction using TaqManTM Universal Master Mix II (Thermo Fisher Scientific, cat# 4440040) and 25ng of RNA.
  • ANDREEV D. E., O'CONNOR, P. B., FAHEY, C., KENNY, E. M., TERENIN, I. M., DMITRIEV, S. E., CORMICAN, P., MORRIS, D. W., SHATSKY, I. N. & BARANOV, P. V.2015. Translation of 5' leaders is pervasive in genes resistant to eIF2 repression. Elife, 4, e03971. [00199] ANDRUSIAK, M. G., SHARIFNIA, P., LYU, X., WANG, Z., DICKEY, A. M., WU, Z., CHISHOLM, A. D.
  • LRRK2 modifies ⁇ -syn pathology and spread in mouse models and human neurons. Acta Neuropathol, 137, 961-980. [00204] BOEYNAEMS, S. & GITLER, A. D.2019. Axons Gonna Ride 'til They Can't No More. Neuron, 104, 179-181. [00205] BROS-FACER, V., KRULL, D., TAYLOR, A., DICK, J. R., BATES, S. A., CLEVELAND, M. S., PRINJHA, R. K. & GREENSMITH, L.2014.
  • ALS Genetics Gains, Losses, and Implications for Future Therapies. Neuron, 108, 822-842.
  • KIM G., NAKAYAMA, L., BLUM, J. A., AKIYAMA, T., BOEYNAEMS, S., CHAKRABORTY, M., COUTHOUIS, J., TASSONI-TSUCHIDA, E., RODRIGUEZ, C. M., BASSIK, M. C. & GITLER, A. D.2021.
  • Small molecule v-ATPase inhibitor Etidronate lowers levels of ALS protein ataxin-2. bioRxiv, 2021.12.20.473567.
  • Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron, 44, 439-51.
  • LAGIER-TOURENNE C., POLYMENIDOU, M. & CLEVELAND, D. W.2010. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet, 19, R46-64.
  • LASTRES-BECKER I., NONIS, D., EICH, F., KLINKENBERG, M., GOROSPE, M., KOTTER, P., KLEIN, F. A., KEDERSHA, N. & AUBURGER, G. 2016. Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation.
  • Pbp1p a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation. Mol Cell Biol, 18, 7383-96.
  • p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR). Cell, 184, 689-708.e20.
  • SCHWAB M. E. & STRITTMATTER, S. M. 2014. Nogo limits neural plasticity and recovery from injury. Curr Opin Neurobiol, 27, 53-60.
  • SCOLES D. R., MEERA, P., SCHNEIDER, M. D., PAUL, S., DANSITHONG, W., FIGUEROA, K. P., HUNG, G., RIGO, F., BENNETT, C. F., OTIS, T. S. & PULST, S. M.2017.
  • LGI1 is a Nogo receptor 1 ligand that antagonizes myelin-based growth inhibition. J Neurosci, 30, 6607-12. [00246] TONG, Z., SEGURA-FELIU, M., SEIRA, O., HOMS CORBERA, A., ANTONIO, J., R ⁇ O, J., ADE, J. & SAMITIER, J. 2015. A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies. RSC Advances, 5, 0-1.
  • P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature, 420, 74-8.

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Abstract

La présente invention concerne des compositions et des procédés pour traiter un sujet mammifère atteint d'une maladie neurodégénérative, telle que la sclérose latérale amyotrophique (ALS) et l'ataxie spinocérébelleuse 2 (SCA2). Des aspects de la composition comprennent des formulations comprenant un agent anti-ATXN2 et un excipient pharmaceutiquement acceptable, l'administration de l'agent anti-ATXN2 conduisant à une réduction des taux ou de la fonction de la protéine ATXN2.
PCT/US2022/080927 2021-12-06 2022-12-05 Traitement de maladies neurodégénératives par l'inhibition de l'ataxine-2 WO2023107893A2 (fr)

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