Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense

Definitions

• the present disclosure relates generally to a method for selective reduction of tau in axons by preventing the transport of the Microtubule-associated protein tau (MAPT) mRNA encoding tau protein from the cell body into axons by blocking the interaction cT MAPT mRNA with hnRNP R, or by reducing hnRNP R levels.
• hnRNP R is an RNA-binding protein that interacts with the 3'UTR of MAPT mRNA. Molecules that inhibit such interaction between MAPT mRNA and hnRNP R, or that lower hnRNP R levels, reduce axonal tau protein.
• AD Alzheimer's disease
• AD is a neurodegenerative disorder and the most common form of late-onset dementia, affecting a substantial proportion of individuals aged 65 and over. It is characterized by progressive memory loss and is expected to increase dramatically over the coming decades as aging is the main risk factor. AD is caused by the accumulation of insoluble protein aggregates in the brain, including the formation of tau fibrils in neuronal axons, leading to neuron dysfunction and loss, which in turn results in progressive memory loss leading to a reduced ability to execute daily functions.
• Treating AD is challenging because of the disease’s complex etiology.
• two types of protein aggregates are present in the brain: extracellular accumulations of Amyloid-P (AP) protein (senile plaques, SPs) and intracellular fibrils of hyperphosphorylated tau protein (neurofibrillary tangles, NFTs).
• AP Amyloid-P
• SPs spikes
• NFTs hyperphosphorylated tau protein
• the temporal and spatial formation of NFTs correlates more closely with the cognitive impairments and the progression of the disease.
• most therapeutic approaches have focused on removing or delaying the formation of SPs, it is increasingly clear that preventing the formation of NFTs or halting their spread offers to be a promising therapeutic option.
• current therapeutic strategies aimed at preventing or slowing down the formation of plaques and tangles through antibody -based targeting of A or tau may induce unwanted side-effects.
• NFTs initially occur in the entorhinal cortex and the hippocampus, the site of memory formation affected first in AD, whereas SPs arise more diffusely throughout the brain. Furthermore, axon dysfunction is an early event in AD inducing neuronal degeneration through “dying back” mechanisms spreading from the damaged axons to neuronal cell bodies. Salvadores, N., et al., Axonal Degeneration in AD: The Contribution of Abeta and Tau. Front Aging Neurosci, 2020. 12: p. 581767.
• Tau is needed in the brain for axon maintenance by stabilizing the cytoskeleton through microtubule assembly. This function is impaired by hyperphosphorylation of tau leading to its fibrillization and toxic accumulation as NFTs in axons. This results in axonal tau aggregates, which develop early during AD and disrupt transport of RNAs and proteins required for axon and synapse maintenance. Robbins, M., et al., Synaptic tau: A pathological or physiological phenomenon? Acta Neuropathol Commun, 2021. 9(1): p. 149. This suggests that NFT formation is a critical step in the etiology underlying AD.
• tau is reduced globally — MAPT mRNA levels are reduced in a targeted manner through delivery of short double-stranded RNAs in the form of short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), or through delivery of antisense oligonucleotides (ASOs) that elicit RNase H-mediated mRNA degradation.
• siRNAs short interfering RNAs
• shRNAs short hairpin RNAs
• ASOs antisense oligonucleotides
• tau-specific antibodies are used that neutralize and/or remove pathological tau.
• Jadhav, S., et al. A walk through tau therapeutic strategies. Acta Neuropathol Commun, 2019. 7(1): p. 22.
• different antibodies have been developed that are specific for tau’s hyperphosphorylated form and that target various regions of tau.
• antibody-based strategies have shown some success by preventing tau seeding and the formation of NFTs, the strategies are limited by the occurrence of multiple tau isoforms and pathological fragments that might not be targeted simultaneously with individual antibodies.
• antibody delivery to the brain is inefficient and may require repeated administration. Additionally, immunization against targets in the brain, whether active or passive, might induce inflammatory cascades causing further complications and leading to acute disease states.
• A0 and tau immunotherapies An additional challenge for A0 and tau immunotherapies is to identify the isoform and aggregate species that needs to be targeted in order to achieve a therapeutic outcome.
• Both A0 and tau exist as fragments of different length or splice isoforms, respectively, and their aggregation progresses from an oligomeric state towards fibrillary deposits.
• tau concentration is selectively decreased from the axons of neurons.
• One such method can include an inhibition, reduction and/or depletion of the RNA-binding protein hnRNP R, which may lead to a reduction in plaques and tangles.
• the present invention discloses a method for preventing the transport of the Microtubule-associated protein tau (MAPT mRNA encoding tau protein from the cell body into axons.
• MAPT mRNA Microtubule-associated protein tau
• reduced mRNA transport local tau protein synthesis in axons is decreased and lower tau protein levels selectively in axons are achieved, retaining the tau levels in the somatodendritic compartment.
• the reduced availability of newly synthesized axonal tau protein limits the levels of tau protein that can be hyperphosphorylated and transsynaptically transmitted, thereby reducing the formation of NFTs and the spreading of tau pathology. This should interfere with the progression of AD.
• the method allows tau to continue to function in neuronal cell bodies, while axonal NFT formation is blocked. Preventing the local accumulation of tau in axons achieves a more specific removal of tau aggregates, with fewer side-effects than the prior art methods.
• Fig. 1A shows results of individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP).
• Fig. IB shows RNA co-immunoprecipitation results.
• Fig. 1C shows compartmentalized cultures of mouse motoneurons.
• Fig. ID shows quantitative PCR (qPCR) results.
• Fig. 2A shows fluorescent in situ hybridization (FISH).
• Fig. 2B shows quantification of the FISH signal.
• Fig. 3A shows immunostaining of motoneurons.
• Fig. 3B shows quantification of tau immunosignals.
• Fig. 4 depicts the creation and accumulation of tau fibrils in AD and compares it against the proposed mechanism/method of the present disclosure to reduce tau pathology in AD.
• Fig. 5 shows the design of antisense oligonucleotides (ASOs).
• FIGs. 6A-6B demonstrate that hnRNP R binds to Mapt mRNA.
• FIG. 6A includes UCSC genome browser views showing hnRNP R binding sites along the Mapt pre-mRNA revealed by individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP).
• FIGs. 7A-7D demonstrate that reduction in hnRNP R function reduces Mapt mRNA levels in axons of motoneurons.
• FIG. 7A is a number of images of fluorescent in situ hybridization (FISH) of Mapt mRNA in motoneurons cultured from Hnrnpr+!+ and -/- mice at DIV 5. Scale bars: 10 pm and 5 pm (inset).
• FIG. 7C is a schematic of a microfluidic chamber for compartmentalized neuron cultures.
• FIG. 7D is a graphical illustration of the results of quantitative PCR of Mapt mRNA from somatodendritic and axonal RNA of Hnrnpr+!+ and - I- mouse motoneurons at DIV 7.
• FIGs. 8A-8C demonstrate that reduction in hnRNP R function reduces tau protein levels in axons of motoneurons.
• FIG. 8A-8C demonstrate that reduction in hnRNP R function reduces tau protein levels in axons of motoneurons.
• FIG. 8A is several images of tau immunostaining of motoneurons cultured from Hnrnpr+I+ and -I- mice at DIV 5, with proximal and distal regions of the axon marked. GFP expression was used for visualization of neuronal morphology and for normalization of tau levels. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 8B and 8C are graphical illustrations of the quantification of the tau (FIG. 8B) and Tubulin (FIG.
• FIG. 9 is a representation of the design of two antisense oligonucleotides (MAPT- ASO1 and MAPT-ASO2) for blocking the interaction between hnRNP R and Mapt mRNA.
• FIGs. 10A-10B demonstrate oligonucleotide uptake in motoneurons and hippocampal neurons.
• FIG. 10A are immunofluorescence images of mouse motoneurons (MN) treated with different concentrations of a Cy3-labeled sense oligonucleotide at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIG. 10B are immunofluorescence images of untreated (Ctrl) mouse hippocampal neurons (HN), and hippocampal neurons treated with 10 pM of a Cy3 -labeled sense oligonucleotide at DIV 25. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 11A-F demonstrate that treatment with MAPT-ASO1 and -ASO2 reduces axonal Mapt mRNA levels.
• FIG. 11A is images of Mapt FISH in untreated (Ctrl) mouse motoneurons and motoneurons treated with MAPT-ASO1 or -ASO2 at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 1 IB-11C are graphical representations quantifying the Mapt FISH signal in the somata (FIG. 1 IB) and axons (FIG. 11C) of motoneurons. Statistical analysis was performed using a Kruskal-Wallis test with Dunn’s multiple comparisons test.
• FIG. 1 ID is images of Mapt FISH in untreated (Ctrl) mouse hippocampal neurons and hippocampal neurons treated with MAPT-ASO1 or -ASO2 at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 11E-11F are graphical representations, quantifying the Mapt FISH signal in the somata (FIG. 1 IE) and axons (FIG. 1 IF) of hippocampal neurons.
• Statistical analysis was performed using a Kruskal-Wallis test with Dunn's multiple comparisons test.
• FIGs. 12A-12D demonstrate that treatment with MAPT-ASO1 and -ASO2 reduces axonal tau protein levels.
• FIG. 12A is images of tau immunostaining of untreated (Ctrl) mouse motoneurons and motoneurons treated with MAPT-ASO1 or -ASO2 at DIV 11, with proximal and distal regions of the axon marked. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 12B-12D are graphical illustrations quantifying the tau immunosignal in the somata (FIG. 12B) and proximal (FIG. 12C) and distal (FIG. 12D) axonal regions of untreated and treated motoneurons.
• FIGs. 13A-13B demonstrate MAPT-ASO2 uptake in motoneurons and hippocampal neurons.
• FIG. 13 A is immunofluorescence images of untreated (Ctrl) mouse motoneurons, and motoneurons treated with 10 pM of a Cy3 -labeled scramble oligonucleotide as control or MAPT-ASO2 at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIG. 13B is immunofluorescence images of untreated (Ctrl) mouse hippocampal neurons, and hippocampal neurons treated with 10 pM of a Cy3-labeled scramble oligonucleotide or MAPT-ASO2 at DIV 25. Scale bars: 10 pm and 5 pm (inset).
• FIGs 14A-14F demonstrate that treatment with MAPT-ASO2 reduces axonal Mapt mRNA levels relative to treatment with a scramble oligonucleotide.
• FIG. 14A is images of Mapt FISH in untreated (Ctrl) mouse motoneurons and motoneurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 14B- 14C are graphical illustrations quantifying the Mapt FISH signal in the somata (FIG. 14B) and axons (FIG. 14C) of motoneurons.
• FIG. 14D is images of Mapt FISH in untreated (Ctrl) mouse hippocampal neurons and hippocampal neurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 14E-14F are graphical illustrations quantifying the Mapt FISH signal in the somata (FIG. 14E) and axons (FIG. 14F) of hippocampal neurons.
• FIGs. 15A-15C demonstrate a reduced tau synthesis in axons of neurons treated with MAPT-ASO2.
• FIG. 15A is images of newly synthesized tau protein in untreated (Ctrl) mouse motoneurons and motoneurons treated with scramble oligonucleotide or MAPT-ASO2 using a puromycin labeling with proximity ligation assay (Puro-PLA) at DIV 6. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 15B and 15C are graphical illustrations quantifying the tau Puro-PLA signal in the somata (FIG. 15B) and axons (FIG. 15C) of motoneurons.
• FIGs. 16A-16G demonstrate that treatment with MAPT-ASO2 reduces axonal tau protein levels relative to treatment with a scramble oligonucleotide.
• FIG. 16A is images of tau immunostaining of untreated (Ctrl) mouse motoneurons and motoneurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 11, with proximal and distal regions of the axon marked. Scale bars: 10 pm and 5 pm (inset).
• FIGs. 16B-16D are graphical illustrations quantifying the tau immunosignal in the somata (FIG. 16B) and proximal (FIG. 16C) and distal (FIG.
• FIG. 16D is images of tau immunostaining of untreated (Ctrl) mouse hippocampal neurons and hippocampal neurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 25, with distal regions of the axon marked. Scale bars: 10 pm and 5 pm (inset).
• 16F-16G are graphical illustrations quantifying the tau immunosignal in the somata (FIG. 16F) and distal axonal regions (FIG. 16G) of hippocampal neurons.
• FIGs. 17A-17E demonstrate that treatment with MAPT-ASO2 reduces axon growth relative to treatment with a scramble oligonucleotide.
• FIG. 17A is images showing the morphology of untreated (Ctrl) mouse motoneurons and motoneurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 7. Scale bar: 50 pm.
• FIG. 17D is images showing the morphology of untreated (Ctrl) mouse hippocampal neurons and hippocampal neurons treated with scramble oligonucleotide or MAPT-ASO2 at DIV 25. Scale bar: 50 pm.
• FIGs. 18A-18C demonstrate identification of additional ASOs for reducing axonal Afapt mRNA levels.
• FIG. 18A illustrates binding sites of MAPT-ASO1 to -ASO20 in he Map! 3' UTR.
• FIG. 18C illustrates the quantification of the Mapt FISH signal in the somata and axons of hippocampal neurons at DIV6. Data are shown as Tukey box plots.
• FIG. 19 are images of depletion of hnRNP R reducing senile plaque load in Alzheimer's disease mice.
• Coronal brain sections of 5xFAD;Hnrnpr+/+ and 5xFAD;Hnrnpr- /- mice were immunostained with antibody 6E10 to label A0 and an antibody against Ibal to label microglia.
• Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).
• DAPI 4',6-diamidino-2-phenylindole
• FIG. 20 are images of depletion of hnRNP R reducing phosphorylated tau in Alzheimer's disease mice.
• Coronal brain sections of 5xFAD;Hnrnpr+/+ and 5xFAD;Hnrnpr- /- mice were immunostained with antibody AT8 to label phosphorylated tau and an antibody against total tau. Nuclei were stained with DAPI.
• FIG. 21 is an illustration of an additional proposed mechanism for the depletion of hnRNP R through antisense oligonucleotides (ASOs) targeting the HNRNPR mRNA or inhibition of hnRNP R through small molecules, peptides and/or oligonucleotides, leading to reduced amounts of senile plaques and neurofibrillary tangles.
• ASOs antisense oligonucleotides
• the claimed method is based in part on the finding that the RNA-binding protein hnRNP R interacts with the 3 'UTR of Mapt mRNA in motoneurons and regulates its axonal localization, as shown in FIGs. 1 A and IB, which are further described below.
• the term “substantially” or “substantial”, is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.
• a surface that is “substantially” flat would either be completely at, or so nearly flat that the effect would be the same as if it were completely flat.
• any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range.
• reference herein to a range of “at least 50” or “at least about 50” includes whole numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc.
• reference herein to a range of “less than 50” or “less than about 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc.
• reference herein to a range of from “5 to 10” includes whole numbers of 5, 6, 7, 8, 9, and 10, and fractional numbers 5.1, 5.2, 5.3, 5,4, 5,5, 5.6, 5.7, 5.8, 5.9, etc.
• the tern “about” indicates that the value listed may be somewhat altered, as long as the alteration does not result in nonconformance of the process or structure to the illustrated embodiment.
• the term “about” can refer to a variation of ⁇ 0.1%, for other elements, the term “about” can refer to a variation of ⁇ 1% or ⁇ 10%, or any point therein.
• the disclosed inhibitory compounds may be administered for either a prophylactic or therapeutic purpose either alone or with other immunosuppressive or anti-inflammatory agents.
• the immunosuppressive compound(s) are provided in advance of any inflammatory response or symptom (for example, prior to, at, or shortly after the time of an organ or tissue transplant but in advance of any symptoms of organ rejection).
• the disclosed inhibitory compounds may, in accordance with the invention, be administered in single or divided doses by the oral, parenteral or topical routes.
• a suitable oral dosage for a compound of formula I would be in the range of about 0.001 mg to about 10 g per day.
• a suitable dosage unit may contain from about 0.001 mg to about 250 mg of said compounds, whereas for topical administration, formulations containing about 0.001% to about 1% active ingredient are contemplated. It should be understood, however, that the dosage administration from patient to patient will vary and the dosage for any particular patient will depend upon the clinician's judgement, who will use as criteria for fixing a proper dosage the size and condition of the patient as well as the patient's response to the drug.
• the compounds of the present disclosure are to be administered by the oral route, they may be administered as medicaments in the form of pharmaceutical preparations which contain them in association with a compatible pharmaceutical carrier and/or excipient material.
• a compatible pharmaceutical carrier and/or excipient material can be an inert organic or inorganic carrier and/or excipient material suitable for oral administration.
• carrier and/or excipient materials are water, gelatin, talc, starch, magnesium stearate, gum arabic, vegetable oils, polyalkylene-glycols, petroleum jelly and the like.
• the pharmaceutical preparations can be prepared in a conventional manner and finished dosage forms can be solid dosage forms, for example, tablets, dragees, capsules, and the like, or liquid dosage forms, for example solutions, suspensions, emulsions and the like.
• the pharmaceutical preparations may be subjected to conventional pharmaceutical operations such as sterilization. Further, the pharmaceutical preparations may contain conventional adjuvants such as preservatives, stabilizers, emulsifiers, flavor-improvers, wetting agents, buffers, salts for varying the osmotic pressure and the like.
• Solid carrier and/or excipient material which can be used include, for example, starch, lactose, mannitol, methyl cellulose, microcrystalline cellulose, talc, silica, dibasic calcium phosphate, and high molecular weight polymers (such as polyethylene glycol).
• the inhibitory compounds can be administered in an aqueous or non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, antioxidants, preservatives, buffers or other solutes to render the solution isotonic with the blood, thickening agents, suspending agents or other pharmaceutically acceptable additives.
• Additives of this type include, for example, tartrate, citrate and acetate buffers, ethanol, propylene glycol, polyethylene glycol, complex formers (such as EDTA), antioxidants (such as sodium bisulfite, sodium metabisulfite, and ascorbic acid), high molecular weight polymers (such as liquid polyethylene oxides) for viscosity regulation and polyethylene derivatives of sorbitol anhydrides.
• complex formers such as EDTA
• antioxidants such as sodium bisulfite, sodium metabisulfite, and ascorbic acid
• high molecular weight polymers such as liquid polyethylene oxides for viscosity regulation and polyethylene derivatives of sorbitol anhydrides.
• Preservatives may also be added if necessary, such as benzoic acid, methyl or propyl paraben, benzalkonium chloride and other quaternary ammonium compounds.
• the compounds of this disclosure may also be administered as solutions for nasal application and may contain in addition to the compounds of this invention suitable buffers, tonicity adjusters, microbial preservatives, antioxidants and viscosity-increasing agents in an aqueous vehicle.
• suitable buffers tonicity adjusters
• microbial preservatives antioxidants
• viscosity-increasing agents in an aqueous vehicle.
• agents used to increase viscosity are polyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone, polysorbates or glycerin.
• Microbial preservatives added may include benzalkonium chloride, thimerosal, chloro-butanol or phenylethyl alcohol.
• the compounds provided in this disclosure can be administered topically or by suppository.
• hnRNP R knockout mice were generated (Hnrnpr-!-) and their motoneurons were cultured in microfluidic chambers (Fig. 1C).
• hnRNP R knockout mice were generated (Hnrnpr-!-) and their motoneurons were cultured in microfluidic chambers (Fig. 1C).
• qPCR quantitative PCR
• Fig. 1A shows hnRNP R binding sites along the Mapt pre-mRNA revealed by individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP).
• Fig. IB shows the co-immunoprecipitation of Mapt mRNA with an anti-hnRNP R antibody from mouse motoneurons.
• Fig. 1C is a schematic of microfluidic chamber for compartmentalized neuron cultures.
• Fig. ID is a quantitative PCR of Mapt from somatodendritic and axonal RNA of Hnrnpr+I+ and -I- mouse motoneurons. A statistical analysis was performed using a two-way ANOVA with Sidak’s multiple comparisons test. ***p ⁇ 0.001.
• FISH Fluorescent in situ hybridization
• FIG. 2A depicts fluorescent in situ hybridization (FISH) results of Mapt mRNA in motoneurons cultured from Hnrnpr+/+ and -I- mice.
• Fig. 2B shows quantification of the FISH signal. A statistical analysis was performed using a Mann-Whitney test. ****p ⁇ 0.0001.
• Fig. 3 A shows tau immunostaining of motoneurons cultured from Hnrnpr+/+ and -I- mice, with proximal (P) and distal (D) regions of the axon marked.
• Fig. 3B is the quantification of tau immunosignal from the immunostaining. A statistical analysis was performed using a Mann-Whitney test. **p ⁇ 0.01; n.s., not significant.
• Fig. 4 shows that in the diseased state, MAPT mRNA is transported into axons by hnRNP R where it is locally translated into tau protein, giving rise to NFTs.
• hnRNP R interacts with hundreds of RNAs including the abundant noncoding RNA 7SK, however, its widespread depletion could have detrimental side-effects. Therefore, the use of small molecules, peptides, ASOs or combinations thereof can be used to inhibit the association between MAPT 3'UTR and hnRNP R. These small molecules, peptides, ASOs or combinations thereof anneal to the region of the MAPT 3'UTR which hnRNP R otherwise binds to, in order to block the interaction between hnRNP R with MAPT. As a result of these treatments, transport of MAPT into axons and local synthesis of tau protein should be reduced, thereby alleviating tau pathology.
• Fig. 4 shows a proposed mechanism of the invention, in which ASOs are being used to block hnRNP R from binding XoMAPT mRNA, thereby preventing its axonal localization and local synthesis of tau. As a result, formation of tau fibrils is reduced.
• ASOs have been successfully delivered to the central nervous system and used therapeutically to correct the splicing defect underlying the motoneuron disorder spinal muscular atrophy. Jablonka, S. and M. Sendtner, Developmental regulation of SMN expression: pathophysiological implications and perspectives for therapy development in spinal muscular atrophy. Gene Ther, 2017. 24(9): p. 06-513.
• Two binding sites of the MAPT mRNA are bound by either a small molecule, peptide, or ASO, the mRNA binding is blocked.
• Two mRNA sites associated with the hnRNP R binding protein are: MAPT-S1 : 5’-TTTGGCTCGGGACTTCAAAA-3’ and MAPT-S2: 5’- ATTTC ATCTTTCC AAATTGA-3 ’ .
• ASOs can be generated that bind to these two mRNA sites to prevent the association with the hnRNP R binding protein.
• Fig. 5 shows the design of ASOs to inhibit hnRNP R binding to MAPT.
• MAPT-ASO1 5’- TTTTGAAGTCCCGAGCC AAA-3’ and MAPT-ASO2: 5’-
• Blocking axonal Mapt transport was investigated for reduction of axonal tau protein production, providing a therapeutic strategy for protection of neurons against NFT formation and spreading.
• Example 2 motoneurons treated with MAPT-ASO1 or MAPT-ASO2 and cultured for 6 DIV showed reduced axonal Mapt mRNA levels compared to untreated motoneurons as detected by FISH (FIGs. 11A-11C). A similar reduction in axonal Mapt was also detectable in cultured hippocampal neurons (FIGs. 11D-11F). Importantly, Mapt levels in the cell bodies of MAPT-ASO-treated neurons were unchanged (FIGs. 1 IB, HE).
• MAPT-ASO2 more efficiently reduces axonal tau relative to MAPT-ASO1
• MAPT-ASO2Scr a scrambled version of it
• Cy3 -labeled MAPT-ASO2 and Scr were efficiently taken up by motoneurons (FIG. 13 A) and hippocampal neurons (FIG. 13B).
• MAPT-ASO2 significantly downregulated axonal Mapt levels in motoneurons (FIGs. 14A- 14C) and hippocampal neurons (FIGs. 14D-14F). Puro-PLA was then used to assess the axonal translation of tau.
• Motoneurons treated with MAPT-ASO2 revealed a reduced Puro-PLA signal for tau in axons compared to Scr-treated and untreated motoneurons (FIGs. 15A-15C). Tau synthesis in cell bodies was unaffected by MAPT-ASO2 treatment (FIG. 15B).
• tau protein levels were reduced in axons of MAPT-ASO2-treated motoneurons (FIGs. 16A-16D) and hippocampal neurons (FIGs. 16E-16G) compared Scr- treated neurons.
• axon lengths of motoneurons subjected to MAPT-ASO2 treatment were reduced compared to Scr treatment while survival was unaffected (FIGs. 17A-17C).
• Reduced axon growth was also detectable for hippocampal neurons exposed to MAPT-ASO2 (FIGs. 17D-17E).
• MAPT-ASO2 treatment can reduce axonal levels of Mapt, resulting in less axonal tau due to lowered local translation.
• Additional MAPT-ASOs were designed along the Mapt 3' UTR in regions that contain hnRNP R iCLIP hits and that are conserved between mouse and human (FIGs. 18A- 18B). These ASOs were screened by fluorescene in situ hybridization (FISH) in hippocampal neurons for their potential to reduce axonal Mapt mRNA levels. Several candidates were identified that lowered axonal Mapt levels >50% in MAPT-ASO-treated relative to untreated hippocampal neurons (FIGs. 18B-18C). Two of these MAPT-ASOs (19 and 20) were shortened versions of MAPT-ASO2 with a length of 18 and 16 nucleotides, respectively.
• FISH fluorescene in situ hybridization
• This example is directed to a method whereby the number of SPs and NFTs is reduced through depleting the RNA-binding protein hnRNP R.
• This method is based on the observation that, when hnRNP R is missing, brains of 5xFAD mice, an AD mouse model overexpressing mutant human amyloid precursor protein (APP) and presenilin 1 (PS 1), exhibit reduced numbers of SPs.
• APP human amyloid precursor protein
• PS 1 presenilin 1
• 5xFAD mice show widespread deposition of SPs in cortex and hippocampus, accompanied by activated microglia revealed by Ibal immunostaining.
• 5xFAD mice homozygous for a hnRNP R knockout allele (5xFAD;Hnrnpr-/ ⁇ ) have reduced SP deposition and less microglial activation.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Neurology (AREA)
• Neurosurgery (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Psychiatry (AREA)
• Hospice & Palliative Care (AREA)
• Epidemiology (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (5)

Applications Claiming Priority (4)

Publications (1)

Family

ID=85685250

Family Applications (1)

Country Status (6)

Citations (3)

• 2023
  • 2023-03-13 WO PCT/EP2023/056392 patent/WO2023217437A1/en not_active Ceased
  • 2023-03-13 EP EP23711444.2A patent/EP4522217A1/en active Pending
  • 2023-03-13 JP JP2024564846A patent/JP2025517123A/ja active Pending
  • 2023-03-13 CA CA3246379A patent/CA3246379A1/en active Pending
  • 2023-03-13 US US18/865,204 patent/US20250304958A1/en active Pending
  • 2023-03-13 AU AU2023267136A patent/AU2023267136A1/en active Pending

Patent Citations (3)

Non-Patent Citations (16)

Also Published As

Similar Documents

Legal Events