EP4658281A1 - Suppresseurs de tauopathies - Google Patents

Suppresseurs de tauopathies

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Publication number
EP4658281A1
EP4658281A1 EP24702539.8A EP24702539A EP4658281A1 EP 4658281 A1 EP4658281 A1 EP 4658281A1 EP 24702539 A EP24702539 A EP 24702539A EP 4658281 A1 EP4658281 A1 EP 4658281A1
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EP
European Patent Office
Prior art keywords
tau
inhibitor
seq
disease
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24702539.8A
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German (de)
English (en)
Inventor
Patrik Verstreken
Roman PRASCHBERGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
Original Assignee
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
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Application filed by Katholieke Universiteit Leuven, Vlaams Instituut voor Biotechnologie VIB filed Critical Katholieke Universiteit Leuven
Publication of EP4658281A1 publication Critical patent/EP4658281A1/fr
Pending legal-status Critical Current

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • 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
    • 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
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • the invention relates to field of neurodegeneration, more particularly to Tau neurotoxicity.
  • the application identifies genes whose reduced expression suppresses pathological Tau mediated effects. These Tau suppressors are provided for use as a medicament in general, and for treating or inhibiting progression of tauopathies or symptoms of tauopathies in particular.
  • Tau pathology is associated with more than twenty neurodegenerative diseases, including Alzheimer's disease (Wang & Mandelkow 2016 Nat Rev Neurosci 17:5-21). Hyperphosphorylation or mutation of the microtubule-associated protein Tau is common to all of these diseases, collectively termed Tauopathies, and filamentous inclusions of hyperphosphorylated Tau are hallmark pathologies of Alzheimer's disease and other Tauopathies (Ballatore et al 2007 Nature Reviews Neuroscience 8:663-672).
  • Tau pathology is not merely a byproduct of other pathological pathways but is a key mediator of neurotoxicity itself (Roberson et al 2007 Science 316:750-754; Hutton et al 1998 Nature 393:702-705; Caffrey & Wade- Martins 2007 Neurobiol Dis 27:1-10; Le Guennec et al 2016 Molecular Psychiatry 1-7).
  • Tau is expressed in neurons and is bound to axonal microtubules.
  • mutations in Tau e.g.
  • RNA expression data of all vulnerable cell types and of all resilient cell types were compared to uncover molecular signatures associated with pathological tau-induced stress.
  • a non-obvious list of candidates genes in a heterozygous loss-of-function condition was found to partially overcome tau-induced effects in a pathogenic tau expressing model.
  • the invention relates to an inhibitor for use as a medicament, wherein the inhibitor statistically significantly reduces the functional expression of at least one gene of the list consisting of MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLOD3, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X and NLGN4Y.
  • the inhibitor is provided for use in (a method for) treating or inhibiting progression of a tauopathic disorder or for use in (a method for) treating or inhibiting a symptom of a tauopathic disorder.
  • the inhibitor is a genetic inhibitor, an antisense inhibitor, a transcript inhibitor, an RNA interference compound or an inhibitor of the RNAi technology.
  • the inhibitor is selected from the group consisting of an antisense oligonucleotide, a gapmer, a siRNA, a shRNA, an antisense oligonucleotide, a zinc-finger nuclease, a meganuclease, a TAL effector nuclease, a CRISPR-Cas effector, an antibody or a fragment thereof binding to any of the targets identified herein, an alpha-body, a nanobody, an intrabody, an aptamer, a DARPin, an affibody, an affitin, an anticalin, and a monobody.
  • the inhibitors herein disclosed are oligonucleotides that specifically bind to one of the genes selected from the list consisting of MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLOD3, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X and NLGN4Y and reduce the expression of said one of the genes through antisense or RNAi technology.
  • an oligonucleotide of 10 to 50 nucleotides in length comprising a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length which are at least 90% complementary to an equal length portion of nucleotides comprised in or being part of SEQ ID No.
  • said contiguous nucleotide sequence is at least 12, 14 or 16 nucleotides in length.
  • said contiguous nucleotide sequence is 100% complementary to a contiguous nucleotide sequence of equal length that is part of or comprised within the sequence set forth in any of SEQ. ID No. 51-63, 64-68, 69- 72, 73-81, 82-88, 96-98, 99-112, 89-95, 162-170, 153-161, 171-176, 177-184, 211-215, 216-221, 222-229, 230-232, 233-243, 244-246, 320, 321-325, 326-336, 311-319, 303-310, 337-343, 365-372, 373-378 or 379-382.
  • said oligonucleotide comprises one or more internucleoside linkage and/or one or more 2' sugar modified nucleosides. More particularly, the internucleoside linkage is a phosphorothioate internucleoside linkage and/or the 2' sugar modified nucleoside is selected from the group consisting of 2'-O-methyl-, 2'-O-methoxyethyl-, 2'-O-alkyl-, 2' -alkoxy, 2' -amino-, 2'-fluoro- and LNA nucleosides.
  • said oligonucleotide is a single stranded antisense oligonucleotide, an siRNA, a shRNA, a CRISPR gRNA or forms the guide strand of an siRNA or shRNA complex.
  • the oligonucleotide comprises a gapmer of formula 5'-F-G-F'-3', where region F and F' independently comprise between 1 and 8 nucleosides, of which 1 to 5 independently are 2' sugar modified nucleosides and define the 5' and 3' end of the F and F' region, and G is a region between 5 and 18 nucleosides for recruiting RNaseH.
  • said oligonucleotide is provided wherein the internucleoside linkages between one or more nucleosides of region F and/or F' and/or between F and G and/or between F' and G are phosphorothioate internucleoside linkages.
  • composition comprising the inhibitor according to the invention.
  • the tauopathic disorder may be selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy (PSP), progressive supranuclear palsy-parkinsonism (PSP-P), Richardson's syndrome, argyrophilic grain disease, corticobasal degeneration Pick's disease, frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), postencephalitic parkinsonism, Parkinson's disease complex of Guam, Guadeloupean parkinsonism, Huntington disease, Down's syndrome, dementia pugilistica, familial British dementia, familial Danish dementia, myotonic dystrophy, Hallevorden-Spatz disease, Niemann Pick type C, chronic traumatic encephalopathy, tangle-only dementia, white matter tauopathy with globular glial inclusions, subacute sclerosing panencephalitis, SLC9A6-related mental retardation, non-Guamanian motor neuron disease with neurofibrillary
  • the symptom of the tauopathic disorder may be selected from the group of mild cognitive impairment, dementia, cognitive decline, decline of motor function, oculomotor and bulbar dysfunction, synaptic dysfunction, neurotoxicity, neuronal degeneration, neuronal dysfunction, synapse loss, and amyloid deposition.
  • the synaptic dysfunction may be further specified as pre-synaptic dysfunction.
  • Figure 1A shows TAU expression levels across multiple regions and cell types of non-neurodegeneration human brains.
  • ACC anterior cingulate cortex. Arrowheads indicate vulnerable (dark red) and not reported as affected (green) example cell types from the text.
  • Human brains are from non- neurodegeneration control donors not known to carry a-synuclein or tau mutations.
  • Figure IB shows the percentile of mean TAU expression amongst the means of genes that are detected in at least 5% of a given cell type in the Allen Brain Map data (Allen Brain Map, 2021).
  • MTG middle temporal gyrus
  • CgG anterior cingulate gyrus
  • VIC primary visual cortex
  • A1C primary auditory cortex
  • Mlul upper limb region of primary motor cortex
  • Mllm lower limb region of primary motor cortex
  • Slum upper limb region of primary somatosensory cortex
  • Slim lower limb region of primary somatosensory cortex.
  • Figure 1C shows the percentile of mean TAU expression amongst the means of genes that are detected in at least 5% of a given cell type in the Agarwal et al. 2020 dataset.
  • SN substantia nigra
  • DaNs dopaminergic neurons
  • Ex excitatory neurons
  • In inhibitory neurons
  • ODC oligodendrocytes
  • OPC oligodendrocyte precursor cell.
  • FIG. 2A-B shows immuno-blots and quantification of tau expression in fly heads as compared to healthy human brains.
  • Weaker expressing QF2w (middle lanes) was used as compared to the stronger Q.F2 (Riabinina et al., 2015).
  • Only one tau splice isoform (0N4R) was expressed in the Drosophila models, which is contrasted by multiple tau bands in human brains.
  • Replicate values, mean and standard deviation (SD) are shown.
  • Figure 2C shows that the tau expression is maintained throughout the lifespan of the flies.
  • Figure 2D shows the ERG traces of repeat experiments (gray) and mean (red) for 45 day old flies are shown. Arrowheads indicate the loss of light-on and off transients in P301L tau expressing flies.
  • Figure 2F shows the automated quantification of vacuole area across entire brains normalized for brain size. From left to right: mini-w+, smGdP, a-syn[A53T], a-syn[dNAC], tau[P301L], tau[dVQIVYK]. Replicate values, median, IQR and whiskers at 1.5xlQR are shown, ns, p > 0.05; *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; Two-sample t-test and Bonferroni correction. Male flies were used throughout.
  • Figure 3A shows a scatter-plot and linear regression of mean normalized and log-transformed transgene levels across cell types of 5 day and 25 day old P301L tau vs. A53T a-synuclein models. Only cell types with a minimum of 50 cells and those that are not part of the large central clusters are shown.
  • Figure 3B shows the expression percentiles of a-synuclein[A53T] and tau[P301L] in neurons of male nSyb-QF2w/QUAS Drosophila model single-cell sequencing data as well as expression percentiles of the human MAPT (tau) and SNCA (a-synuclein) genes in neurons of the human substantia nigra and frontal gyrus from the Agarwal et al. 2020 dataset.
  • Figure 3C shows the quantification of optic lobes with dystrophic neuronal processes in C2/3 neurons co-expressing CD8::GFP and a-synuclein[A53T], tau[P301L] as well as smGdP control in 25 day old animals.
  • N number of optic lobes, noted at bottom of bars, ns, p > 0.05; ***p ⁇ 0.001; Fisher's exact test and Bonferroni correction.
  • Figure 3D shows the C2 CD8::GFP lamina projection width as measured in mid-lamina confocal slices at three equally spaced locations by a blinded observer. Replicate values, median, IQR and whiskers at 1.5xlQR are shown. *p ⁇ 0.05; ***p ⁇ 0.001; one-way ANOVA with Dunnett's multiple comparison test.
  • Figure 3E shows the quantification of Tml dendritic area. Replicate values, median, IQR and whiskers at 1.5xlQR are shown. *p ⁇ 0.05; ***p ⁇ 0.001; one-way ANOVA with Dunnett's multiple comparison test.
  • Figure 3G shows the maximum intensity projections of 25 day old a/p-Kenyon cell (KC) lobes which are the synaptic and dendritic areas of this cell-type.
  • Replicate values, median, IQR and whiskers at 1.5xlQR are shown, ns, p > 0.05; **p ⁇ 0.01; one-way ANOVA with Dunnett's multiple comparison test.
  • Figure 3H-I shows the CD8::GFP maximum intensity and synaptic area projections of 25 day old LClOa synaptic terminals. Replicate values, median, IQR and whiskers at 1.5xlQR are shown, ns, p > 0.05; *p ⁇ 0.05; one-way ANOVA with Dunnett's multiple comparison test.
  • Figure 4A-B show the cell-type proportions of neurons aged 45 days predicted to be vulnerable or resilient in the 5 and 25 day model. Replicate values, median, IQR and whiskers at 1.5xlQR are shown. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; Two-sample t-test and Bonferroni correction. Male flies of the genotypes as indicated were used.
  • Figure 4C shows the number of significantly up-/downregulated DEGs per neuron type are shown for nSyb-QF2w > QUAS-tau[P301L] vs. nSyb-QF2w > mini-w+ flies at 45 days as calculated with DESeq2 and FDR ⁇ 0.05.
  • Figure 4D shows the number of significantly up-/downregulated DEGs per neuron type are shown for nSyb-QF2w > QUAS- a-synuclein[A53T]/-tau[P301L] vs. nSyb-QF2w > mini-w+ flies at 25 days in Ll-4 and L5 neurons. DEG testing was performed with edgeR, with 5 days and 25 days combined and FDR ⁇ 0.05. Male flies of the indicated genotypes were used.
  • Figure 5A-B shows the grouped GO terms enriched in P301L tau resilient neurons as compared to vulnerable ones (A) or enriched in P301L tau vulnerable neurons as compared to resilient ones (B). Number of GO terms grouped under each parent term are shown in brackets. Top 10 parent terms per ontology are shown. BP, biological process; CC, cellular component; MF, molecular function.
  • Figure 6-7 show the ERG on-transient amplitude differences to P301L tau expression alone at 45 days ( Figure 6) or 25 days (Figure 7), after heterozygous loss of function alleles of predicted vulnerability genes have been crossed in.
  • Male flies of the indicated genotypes were used.
  • Individual flies, median, IQR and whiskers at 1.5xlQR are shown.
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • an indefinite or definite article is used when referring to a singular noun e.g. "a” or “an”, “the”, this includes a plural of that noun unless something else is specifically stated.
  • the terms first, second, third and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order.
  • the term "about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). For example, if it is stated that an inhibitor of MCTP1 reduces the expression of MCTP1 in a cell by at least about 60%, it is implied that the MCTP1 expression is reduced by a range of 50% to 70%.
  • nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid sequence” or “nucleic acid molecule” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of nucleic acids include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • the nucleic acid may comprise single stranded or double stranded DNA or RNA.
  • the nucleic acid may comprise any know type of modification, for example modified bases, a modified backbone, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analogue.
  • a nucleic acid can be up to about 100 nucleotides in length, in that case the nucleic acid is often referred to as an oligonucleotide, but can also be up to several 1000s of nucleotides in length.
  • a nucleic acid may comprise a promoter, one or more introns, one or more exons, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like.
  • the "coding sequence” is defined by the exons and is a nucleic acid sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
  • nucleotides refer to the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
  • nucleotides such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which are absent in nucleosides).
  • a nucleotide without a phosphate group is called a "nucleoside” and is thus a compound comprising a nucleobase moiety and a sugar moiety.
  • nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid.
  • Naturally occurring nucleobases of RNA or DNA comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Contiguous as used herein means next or together in sequence, hence the contiguous nucleotides or amino acids are linked nucleotides or amino acids (i.e. no additional nucleotides or amino acids are present between those that are linked).
  • complementary means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence.
  • the complementary sequence of the oligonucleotide has at least 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence.
  • the degree of “complementarity” is expressed as the percentage identity (or percentage homology) between the sequence of the oligomer (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of contiguous monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of monomers within the gap differs between the oligomer of the disclosure and the target region.
  • derivative refers to a chemical compound related structurally to a compound disclosed herein (e.g., an oligonucleotide of the present disclosure), e.g., having the same carbon skeleton, but chemically modified to introduce, e.g., a side chain or group, in one or more positions, and wherein the derivative possesses a biological activity (e.g., the capacity to reduce Syngr3 expression) that is substantially similar to a biological activity of the entity or molecule it is a derivative.
  • a biological activity e.g., the capacity to reduce Syngr3 expression
  • SEQ ID No. X refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ. ID No. X.
  • a gene defined in/by SEQ ID No. X consists of the nucleic acid sequence given in SEQ ID No. X.
  • a further example is nucleic acid sequence comprising SEQ ID No. X, which refers to a nucleic acid sequence longer than the nucleic acid sequence given in SEQ ID No. X but entirely comprising the nucleic acid sequence given in SEQ ID No. X (wherein the nucleic acid sequence given in SEQ ID No.
  • X can be located N-terminally or C-terminally in the longer nucleic acid sequence, or can be embedded in the longer nucleic acid sequence), or to a nucleic acid sequence consisting of the nucleic acid sequence given in SEQ ID No. X.
  • wild-type refers to a gene or gene product isolated from a naturally occurring source.
  • a wildtype gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
  • modified refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • Treatment refers to any rate of reduction or retardation of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no or zero progress of the disease or disorder (i.e. “inhibition” or “inhibition of progression”) or even in any rate of regression of the already developed disease or disorder.
  • Reduction or “reducing” as used herein refers to a statistically significant reduction, more particularly said statistically significant reduction is an at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% reduction compared to the control situation.
  • Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained.
  • the null hypothesis is the default assumption that nothing happened or changed, hence that there is no difference for example in expression of MCTP1 in the presence of an inhibitor compared to the expression of MCTP1 in the absence of said inhibitor.
  • an observed result has to be statistically significant, i.e. the observed p- value is less than the pre-specified significance level a.
  • the p-value of a result, p is the probability of obtaining a result at least as extreme, given that the null hypothesis were true.
  • a is 0.05.
  • a is 0.01.
  • a is 0.001.
  • the human Tau protein (alternatively known as microtubule-associated protein tau or MART; HGNC: 6893, NCBI Entrez Gene: 4137, Ensembl: ENSG00000186868) in the brain is a collection of six isoforms generated through alternative splicing.
  • the 6 isoforms lack or contain a different number of near-amino- terminal inserts ("N”: ON, IN or 2N) and lack or contain "R2", one of the 4 repeats in the microtubulebinding domain.
  • MAEPRQEFEVM EDHAGTYGLGDRKDQGGYTM HQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDV TAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIA TPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRT PPKSPSSAKSRLQ.TAPVPMPDLKNVKSKIGSTENLKHQ.PGGGKVQ.IINKKLDLSNVQ.SKCGSKDNIKHVPGGGSVQ.lv YKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDH GAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLA
  • RNA expression data of all vulnerable cell types and of all resilient cell types were compared to uncover molecular signatures associated with P301L tau-induced stress.
  • a non-obvious selection of the long list of 2000 candidate genes revealed a short list of 45 candidates of which heterozygous loss-of-function (LOF) effect in a pathogenic tau expressing fly model was tested.
  • LEF loss-of-function
  • About 18 genes partially overcame the tau-induced effects. Among them, both genes that have (e.g. Syngr) or have not already been linked to tauopathies.
  • HSP90AB1 refers to any of SEQ ID No. 1-4. In a particular embodiment, HSP90AB1 is SEQ ID No. 1.
  • HSP90AA1 or Heat Shock Protein 90 Alpha Family Class A Member 1 encodes the human stress-inducible 90-kDa heat shock protein alpha (HSP90A).
  • HSP90AA1 has 6 isoforms.
  • HSP90AA1 refers to any of SEQ. ID No. 5-10.
  • HSP90AA1 is SEQ ID No. 5.
  • TRPM3 or Transient Receptor Potential Cation Channel Subfamily M Member 3 (HGNC: 17992; NCBI Entrez Gene: 80036; Ensembl: ENSG00000083067; UniProtKB/Swiss-Prot: Q9HCF6) belongs to the family of transient receptor potential (TRP) channels. TRP channels are cation-selective channels important for cellular calcium signaling and homeostasis. The protein encoded by this gene mediates calcium entry, and this entry is potentiated by calcium store depletion. TRPM3 has 23 isoforms. In one embodiment, TRPM3 refers to any of SEQ ID No. 11-33. In a particular embodiment, TRPM3 is SEQ ID No. 11.
  • TRPM1 or Transient Receptor Potential Cation Channel Subfamily M Member 1 (HGNC: 7146; NCBI Entrez Gene: 4308; Ensembl: ENSG00000134160; UniProtKB/Swiss-Prot: Q7Z4N2) has 9 isoforms.
  • TRPM1 refers to any of SEQ ID No. 34-42.
  • TRPM1 is SEQ ID No. 34.
  • TRPM7 or Transient Receptor Potential Cation Channel Subfamily M Member 7 (HGNC: 17994; NCBI Entrez Gene: 54822; Ensembl: ENSG00000092439; UniProtKB/Swiss-Prot: Q96QT4) has 4 isoforms.
  • TRPM7 refers to any of SEQ ID No. 43-46.
  • TRPM1 is SEQ ID No. 43.
  • TRPM6 or Transient Receptor Potential Cation Channel Subfamily M Member 6 (HGNC: 17995; NCBI Entrez Gene: 140803; Ensembl: ENSG00000119121; UniProtKB/Swiss-Prot: Q9BX84) has 4 isoforms.
  • TRPM6 refers to any of SEQ ID No. 47-50.
  • TRPM1 is SEQ ID No. 47.
  • MCTP1 or Multiple C2 And Transmembrane Domain Containing 1 encodes for a calcium sensor which is essential for the stabilization of normal baseline neurotransmitter release and for the induction and long-term maintenance of presynaptic homeostatic plasticity.
  • MCTP1 has 13 isoforms.
  • MCTP1 refers to any of SEQ ID No. 51-63.
  • MCTP1 is SEQ ID No. 51.
  • MCTP2 or Multiple C2 And Transmembrane Domain Containing 2 (HGNC: 25636; NCBI Entrez Gene: 55784; Ensembl: ENSG00000140563; UniProtKB/Swiss-Prot: Q6DN12) has 5 isoforms.
  • MCTP2 refers to any of SEQ ID No. 64-68.
  • MCTP2 is SEQ ID No. 64.
  • SHANK1 or SH3 And Multiple Ankyrin Repeat Domains 1 encodes a member of the SHANK family of synaptic proteins that may function as molecular scaffolds in the postsynaptic density of excitatory synapses.
  • SHANK proteins contain multiple domains for protein-protein interaction, including ankyrin repeats, and an SH3 domain.
  • SHANK1 has 4 isoforms.
  • SHANK1 refers to any of SEQ. ID No. 69-72.
  • SHANK1 is SEQ ID No. 69.
  • SHANK2 or SH3 And Multiple Ankyrin Repeat Domains 2 (HGNC: 14295; NCBI Entrez Gene: 22941; Ensembl: ENSG00000162105; UniProtKB/Swiss-Prot: Q9UPX8) has 9 isoforms.
  • SHANK2 refers to any of SEQ ID No. 73-81.
  • SHANK2 is SEQ ID No. 73.
  • SHANK3 or SH3 And Multiple Ankyrin Repeat Domains 3 (HGNC: 14294; NCBI Entrez Gene: 85358; Ensembl: ENSG00000251322; UniProtKB/Swiss-Prot: Q9BYB0) has 7 isoforms.
  • SHANK3 refers to any of SEQ ID No. 82-88.
  • SHANK3 is SEQ ID No. 82.
  • PLOD1 or Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 1 has 3 isoforms.
  • PLOD1 refers to any of SEQ ID No. 96-98.
  • PLOD1 is SEQ ID No. 96.
  • PLOD2 (HGNC: 9082; NCBI Entrez Gene: 5352; Ensembl: ENSG00000152952; UniProtKB/Swiss-Prot: 000469) has 14 isoforms.
  • PLOD2 refers to any of SEQ ID No. 99-112.
  • PLOD2 is SEQ ID No. 99.
  • PLOD3 (HGNC: 9083; NCBI Entrez Gene: 8985; Ensembl: ENSG00000106397; UniProtKB/Swiss-Prot: 060568) has 7 isoforms.
  • PLOD3 refers to any of SEQ ID No. 89-95.
  • PLOD3 is SEQ ID No. 89.
  • SLC8A1-3 are members of the Solute Carrier Family 8 that mediate the exchange of one Ca 2+ ion against three to four Na + ions across the cell membrane, and thereby contribute to the regulation of cytoplasmic Ca 2+ levels and Ca 2+ -dependent cellular processes.
  • SLC8A1 or Solute Carrier Family 8 member Al has 19 isoforms.
  • SLC8A1 refers to any of SEQ ID No. 122-140.
  • SLC8A1 is SEQ ID No. 122.
  • SLC8A2 or Solute Carrier Family 8 member A2 has 4 isoforms.
  • SLC8A2 refers to any of SEQ ID No. 141-144.
  • SLC8A2 is SEQ ID No. 141.
  • SLC8A3 or Solute Carrier Family 8 member A3 (HGNC: 11070; NCBI Entrez Gene: 6547; Ensembl: ENSG00000100678; UniProtKB/Swiss-Prot: P57103) has 9 isoforms.
  • SLC8A3 refers to any of SEQ. ID No. 113-121.
  • SLC8A3 is SEQ ID No. 113.
  • DPP10 or Dipeptidyl Peptidase Like 10 (HGNC: 20823; NCBI Entrez Gene: 57628; Ensembl: ENSG00000175497; UniProtKB/Swiss-Prot: Q8N608) encodes a single-pass type II membrane protein that is a member of the S9B family in clan SC of the serine proteases. This protein has no detectable protease activity, most likely due to the absence of the conserved serine residue normally present in the catalytic domain of serine proteases. However, it does bind specific voltage-gated potassium channels and alters their expression and biophysical properties. DPP10 has 8 isoforms. In one embodiment, DPP10 refers to any of SEQ ID No. 145-152. In a particular embodiment, DPP10 is SEQ ID No. 145.
  • FARP1 or FERM, ARH/RhoGEF And Pleckstrin Domain Protein 1 encodes a protein containing a FERM (4.2, exrin, radixin, moesin) domain, a Dbl homology domain, and two pleckstrin homology domains. These domains are found in guanine nucleotide exchange factors and proteins that link the cytoskeleton to the cell membrane. The encoded protein functions in neurons to promote dendritic growth.
  • FARP1 has 9 isoforms.
  • FARP1 refers to any of SEQ ID No. 162-170.
  • FARP1 is SEQ ID No. 162.
  • FARP2 or FERM, ARH/RhoGEF And Pleckstrin Domain Protein 2 HGNC: 16460: NCBI Entrez Gene: 9855; Ensembl: ENSG00000006607; UniProtKB/Swiss-Prot: 094887
  • FARP2 has 9 isoforms.
  • FARP2 refers to any of SEQ ID No. 153-161.
  • FARP1 is SEQ ID No. 153.
  • POSTN or Periostin encodes a secreted extracellular matrix protein that functions in tissue development and regeneration, including wound healing, and ventricular remodeling following myocardial infarction.
  • the encoded protein binds to integrins to support adhesion and migration of epithelial cells. This protein plays a role in cancer stem cell maintenance and metastasis. Mice lacking this gene exhibit cardiac valve disease, and skeletal and dental defects.
  • Alternative splicing results in multiple transcript variants encoding different isoforms.
  • POSTN has 6 isoforms.
  • POSTN refers to any of SEQ ID No. 171-176.
  • POSTN is SEQ ID No. 171.
  • TGFBI or Transforming Growth Factor Beta Induced encodes an RGD-containing protein that binds to type I, II and IV collagens.
  • TGFBI plays a role in cell-collagen interactions and may be involved in endochondrial bone formation in cartilage. The protein is induced by transforming growth factor-beta and acts to inhibit cell adhesion.
  • TGFBI has 8 isoforms.
  • TGFBI refers to any of SEQ. ID No. 177-184.
  • TGFBI is SEQ ID No. 177.
  • RYR1 or Ryanodine Receptor 1 encodes a ryanodine receptor found in skeletal muscle.
  • the encoded protein functions as a calcium release channel in the sarcoplasmic reticulum but also serves to connect the sarcoplasmic reticulum and transverse tubule.
  • spliced transcripts encoding 8 different isoforms.
  • RYR1 refers to any of SEQ ID No. 190-197. In a particular embodiment, RYR1 is SEQ ID No. 190.
  • RYR2 or Ryanodine Receptor 2 encodes a ryanodine receptor found in cardiac muscle sarcoplasmic reticulum.
  • the encoded protein is one of the components of a calcium channel, composed of a tetramer of the ryanodine receptor proteins and a tetramer of FK506 binding protein IB proteins, that supplies calcium to cardiac muscle.
  • RYR2 has 5 isoforms.
  • RYR2 refers to any of SEQ ID No. 185-189.
  • RYR2 is SEQ ID No. 185.
  • RYR3 or Ryanodine Receptor 3 encodes a ryanodine receptor, which functions to release calcium from intracellular storage for use in many cellular processes.
  • the encoded protein is involved in skeletal muscle contraction by releasing calcium from the sarcoplasmic reticulum followed by depolarization of T-tubules.
  • RYR3 has 13 isoforms.
  • RYR3 refers to any of SEQ ID No. 198-210.
  • RYR3 is SEQ ID No. 198.
  • TRMT9B or TRNA Methyltransferase 9B encodes a tRNA methyltransferase predicted to be involved in tRNA wobble uridine modification.
  • TRMT9B has 5 isoforms.
  • TRMT9B refers to any of SEQ ID No. 211-215.
  • TRMT9B is SEQ ID No. 211.
  • ALKBH8 or AlkB Homolog 8 TRNA Methyltransferase also known as TRMT9A HGNC: 25189; NCBI Entrez Gene: 91801; Ensembl: ENSG00000137760; UniProtKB/Swiss-Prot: Q96BT7
  • TRMT9A HGNC: 25189; NCBI Entrez Gene: 91801; Ensembl: ENSG00000137760; UniProtKB/Swiss-Prot: Q96BT7
  • tRNA (uracil) methyltransferase activity HGNC: 25189; NCBI Entrez Gene: 91801; Ensembl: ENSG00000137760; UniProtKB/Swiss-Prot: Q96BT7
  • ALKBH8 has 6 isoforms.
  • ALKBH8/TRMT9A refers to any of SEQ ID No. 216-221.
  • ALKBH8/TRMT9A is SEQ ID No. 216.
  • ADCY4, ADCY2, ADCY7 and ADCY8 are members of the family of adenylate cyclases, which encode membrane-associated enzymes that catalyze the formation of the secondary messenger cyclic adenosine monophosphate (cAMP).
  • ADCY4 or Adenylate Cyclase 4 (HGNC: 235; NCBI Entrez Gene: 196883; Ensembl: ENSG00000129467; UniProtKB/Swiss-Prot: Q8NFM4) has 8 isoforms.
  • ADCY4 refers to any of SEQ. ID No. 222-229.
  • ADCY4 is SEQ ID No. 222.
  • ADCY2 or Adenylate Cyclase 2 (HGNC: 233; NCBI Entrez Gene: 108; Ensembl: ENSG00000078295; UniProtKB/Swiss-Prot: Q08462) has 3 isoforms.
  • ADCY2 refers to any of SEQ ID No. 230-232.
  • ADCY2 is SEQ ID No. 230.
  • ADCY7 or Adenylate Cyclase 7 (HGNC: 238; NCBI Entrez Gene: 113; Ensembl: ENSG00000121281; UniProtKB/Swiss-Prot: P51828) has 11 isoforms.
  • ADCY7 refers to any of SEQ ID No.
  • ADCY7 is SEQ ID No. 233.
  • ADCY8 or Adenylate Cyclase 8 (HGNC: 239; NCBI Entrez Gene: 114; Ensembl: ENSG00000155897; UniProtKB/Swiss-Prot: P40145) has 3 isoforms.
  • ADCY8 refers to any of SEQ ID No. 244-246.
  • ADCY8 is SEQ ID No. 244.
  • GRIA1-4 stands for Glutamate Ionotropic Receptor AMPA Type Subunit 1-4 are AMPA receptors and members of the ionotropic class of glutamate receptors. Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are activated in a variety of normal neurophysiologic processes. These receptors are heteromeric protein complexes with multiple subunits, each possessing transmembrane regions, and all arranged to form a ligand-gated ion channel. The classification of glutamate receptors is based on their activation by different pharmacologic agonists. This gene belongs to a family of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors.
  • AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate
  • GRIA1 (HGNC: 4571; NCBI Entrez Gene: 2890; Ensembl: ENSG00000155511; UniProtKB/Swiss-Prot: P42261) has 6 isoforms.
  • GRIA1 refers to any of SEQ ID No. 284-289.
  • GRIA1 is SEQ ID No. 284.
  • GRIA2 (HGNC: 4572; NCBI Entrez Gene: 2891; Ensembl: ENSG00000120251; UniProtKB/Swiss-Prot: P42262) has 32 isoforms.
  • GRIA2 refers to any of SEQ ID No.
  • GRIA2 is SEQ ID No. 252.
  • GRIA3 (HGNC: 4573; NCBI Entrez Gene: 2892; Ensembl: ENSG00000125675; UniProtKB/Swiss- Prot: P42263) has 5 isoforms.
  • GRIA3 refers to any of SEQ ID No. 247-251.
  • GRIA3 is SEQ ID No. 247.
  • GRIA4 (HGNC: 4574; NCBI Entrez Gene: 2893; Ensembl: ENSG00000152578; UniProtKB/Swiss-Prot: P48058) has 13 isoforms.
  • GRIA4 refers to any of SEQ ID No. 290-302.
  • GRIA4 is SEQ ID No. 290.
  • CALM1 and CALMS are members of the EF-hand calcium-binding protein family. Calcium-induced activation of calmodulin regulates and modulates the function of cardiac ion channels.
  • CALM1 or Calmodulinl (HGNC: 1442; NCBI Entrez Gene: 801; Ensembl: ENSG00000198668; UniProtKB/Swiss-Prot: P0DP23) has 9 isoforms.
  • CALM1 refers to any of SEQ ID No. 311-319.
  • CALM1 is SEQ. ID No. 311.
  • CALMS or Calmodulin3 has 8 isoforms.
  • CALMS refers to any of SEQ ID No. 303-310.
  • CALMS is SEQ ID No. 303.
  • IGLON5 or IgLON Family Member 5 (HGNC: 34550; NCBI Entrez Gene: 402665; Ensembl: ENSG00000142549; UniProtKB/Swiss-Prot: A6NGN9) encodes for Ig-Like Domain-Containing Protein 5 and refers to SEQ ID No. 320.
  • OPCML or Opioid Binding Protein/Cell Adhesion Molecule Like encodes a member of the IgLON subfamily in the immunoglobulin protein superfamily of proteins.
  • OPCML binds opioids in the presence of acidic lipids and is probably involved in cell contact.
  • OPCML has 5 isoforms.
  • OPCML refers to any of SEQ ID No. 321-325. In a particular embodiment, OPCML is SEQ ID No. 321.
  • NTM or Neurotrimin encodes a member of the IgLON (LAMP, OBCAM, Ntm) family of immunoglobulin (Ig) domain-containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules.
  • the encoded protein may promote neurite outgrowth and adhesion via a homophilic mechanism.
  • NTM has 11 isoforms. In one embodiment, NTM refers to any of SEQ ID No. 326-336. In a particular embodiment, NTM is SEQ ID No. 326.
  • NLGN1-3 or Neuroligin 1-3 encode members of a family of neuronal cell surface proteins. Members of this family may act as splice site-specific ligands for beta-neurexins and may be involved in the formation and remodeling of central nervous system synapses. Alternatively spliced transcript variants have been found for these genes.
  • NLGN1 (HGNC: 14291; NCBI Entrez Gene: 22871; Ensembl: ENSG00000169760; UniProtKB/Swiss-Prot: Q8N2Q7) has 6 isoforms.
  • NLGN1 refers to any of SEQ ID No. 344-349.
  • NLGN1 is SEQ ID No. 344.
  • NLGN3 (HGNC: 14289; NCBI Entrez Gene: 54413; Ensembl: ENSG00000196338; UniProtKB/Swiss-Prot: Q9NZ94) has 15 isoforms.
  • NLGN3 refers to any of SEQ ID No. 350-364.
  • NLGN3 is SEQ ID No. 350.
  • NLGN4X or Neuroligin 4 X-Linked (HGNC: 14287; NCBI Entrez Gene: 57502; Ensembl: ENSG00000146938; UniProtKB/Swiss-Prot: Q8N0W4) encodes a member of the type-B carboxylesterase/lipase protein family.
  • the encoded protein belongs to a family of neuronal cell surface proteins. Members of this family may act as splice site-specific ligands for beta-neurexins and may be involved in the formation and remodeling of central nervous system synapses.
  • NLGN4X has 7 isoforms. In one embodiment, NLGN4X refers to any of SEQ ID No. 337-343. In a particular embodiment, NLGN4X is SEQ ID No. 337.
  • NLGN4Y or Neuroligin 4 Y-Linked encodes a type I membrane protein that belongs to the family of neuroligins, which are cell adhesion molecules present at the postsynaptic side of the synapse, and may be essential for the formation of functional synapses. Alternatively spliced transcript variants have been found for this gene.
  • NLGN4Y has 8 isoforms.
  • NLGN4Y refers to any of SEQ. ID No. 365-372.
  • NLGN4Y is SEQ ID No. 365.
  • SLC38A11 or Solute Carrier Family 38 Member 11 (HGNC: 26836; NCBI Entrez Gene: 151258; Ensembl: ENSG00000169507; UniProtKB/Swiss-Prot: Q08AI6) is predicted to be involved in amino acid transmembrane transport.
  • SLC38A11 has 6 isoforms.
  • SLC38A11 refers to any of SEQ ID No. 373-378.
  • SLC38A11 is SEQ ID No. 373.
  • SLC38A2 or Solute Carrier Family 38 Member 2 is a symporter that cotransports neutral amino acids and sodium ions from the extracellular to the intracellular side of the cell membrane.
  • SLC38A2 has 4 isoforms.
  • SLC38A2 refers to any of SEQ ID No. 379-382.
  • SLC38A2 is SEQ ID No. 379.
  • the skilled person is aware of several means and methods to reduce the expression of target genes or the level of the proteins for which they code.
  • the nature of the inhibitor is not relevant for the invention as long as the transcript level of at least one of the genes selected from Table 1 or the level of one of the proteins for which said genes encode is statistically significantly reduced compared to a situation where the inhibitor is not present.
  • the concept of designing oligonucleotides to bind to specific sequences in target RNAs via Watson-Crick hydrogen bonding is well established as said antisense technique has been introduced already in the late 1970s.
  • an oligonucleotide of the present disclosure comprises an antisense oligonucleotide (ASO), e.g., an unconjugated or conjugated ASO.
  • an oligonucleotide of the present disclosure comprises a siRNA, e.g., an unconjugated or conjugated siRNA.
  • the inhibitors are genetic inhibitors or inhibitors of the inhibitory RNA technology or antisense inhibitors or inhibitors of the antisense technology.
  • the inhibitors are nucleic acid molecules that comprise a sequence complementary to a region in one of the sequences listed in SEQ ID No. 1-372, particularly SEQ ID No. SEQ ID No.
  • allelic variants refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.
  • the inhibitors are selected from the list consisting of an antisense oligonucleotide, a gapmer, an siRNA, a shRNA and a Crispr gRNA.
  • the application also provides inhibitors that reduce the protein level or the activity of one of the targets herein disclosed.
  • the generation of antibodies or fragments thereof that bind specific targets is a well-established method to reduce the protein level and/or activity of proteins.
  • the inhibitor of the present disclosure is an oligonucleotide, more particularly an ASO.
  • Antisense oligonucleotides or ASOs are small (generally, "'18-30 nucleotides or shorter, e.g. 12 to 20 nucleotides), synthetic, single-stranded nucleic acid polymers of diverse chemistries, which can be employed to modulate gene expression via various mechanisms.
  • ASOs can be subdivided into two major categories: RNase H competent and steric block ASOs.
  • the oligonucleotide of the present disclosure is RNase H competent.
  • the oligonucleotide of the present disclosure is a steric block ASO.
  • RNases H are a family of endonucleases that hydrolyze RNA residues in various nucleic acids, such as RNA-DNA-like duplexes.
  • RNA Hl canonical RNase H enzymes
  • RNase Hl is present in the nucleus, cytoplasm and in mitochondria.
  • the main function of RNase Hl is the clearance of R-Loops, particularly in GC rich genomic regions.
  • the endogenous RNase H enzyme RNaseHl also recognizes RNA-DNA heteroduplex substrates that are formed when DNA-based oligonucleotides are taken up by the cell and bind to complementary mRNA transcripts and subsequently catalyses the degradation of targeted RNA.
  • an ASO must contain at least 5 contiguous deoxynucleotide units, with optimal enzyme activity achieved with 8-10 contiguous deoxynucleotides. This approach has been widely used as a means of downregulating disease-causing or disease-modifying genes (Roberts et al 2020 Nature Reviews 19:673- 694).
  • the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer.
  • a gapmer or gapmer oligonucleotide comprises at least three distinct structural regions: a 5'-flank, a gap and a 3'-flank or F-G-F' in the'5 -> 3' orientation.
  • the "gap" region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H.
  • the gap region is flanked by a 5' flanking region (F) comprising one or more sugar modified nucleosides, and by a 3' flanking region (F') comprising one or more sugar modified nucleosides.
  • the one or more sugar modified nucleosides in region F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e. are affinity enhancing sugar modified nucleosides).
  • the one or more sugar modified nucleosides in region F and F' are 2' sugar modified nucleosides, such as independently selected from LNA and 2'-MOE.
  • the 5' and 3' most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5' (F) or 3' (F') region respectively.
  • the flanks may further be defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5' end of the 5' flank and at the 3' end of the 3' flank.
  • Regions F-G-F' form a contiguous nucleotide sequence.
  • Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F- G-F'.
  • ASOs can also modulate gene expression by steric hindrance or occupancy-only mechanisms.
  • Steric block oligonucleotides are designed to bind to target transcripts with high affinity but do not induce target transcript degradation as they lack RNase H competence. Such oligonucleotides therefore comprise either nucleotides that do not form RNase H substrates when paired with RNA or a mixture of nucleotide chemistries such that runs of consecutive DNA-like bases are avoided.
  • Steric block oligonucleotides can mask specific sequences within a target transcript and thereby interfere with transcript RNA-RNA and/or RNA-protein interactions.
  • steric block ASOs The most widely used application of steric block ASOs is in the modulation of alternative splicing in order to selectively exclude or retain a specific exon(s) in order to disrupt the translation of the target gene.
  • ASOs can also be designed to interfere with maturation and stability of the RNA transcript or to block its interaction with the translation apparatus.
  • mRNA maturation can be modulated by inhibition of 5' cap formation, inhibition of mRNA splicing or activation of RNaseH (Chan et al 2006 Clin Exp Pharmacol Physiol 33:533-540; this reference also describes some of the software available for assisting in design of ASOs).
  • the inhibitor of the present disclosure is an oligonucleotide, more particularly the antisense portion of an RNAi duplex (double stranded RNA).
  • RNA interference is a mechanism by which double-stranded RNA triggers the loss of a homologous RNA molecule.
  • Short interfering RNA (siRNA) molecules are the effector molecules of RNAi and classically consist of a duplex of RNA molecules with a length of 21 nucleotides, i.e. 19 complementary bases and 2 terminal 3' overhangs.
  • One of the strands of the siRNA (the guide or antisense strand) is complementary to a target transcript, whereas the other strand is designated the passenger or sense strand.
  • siRNAs act to guide the Argonaute2 protein (AGO2), as part of the RNA-induced silencing complex (RISC), to complementary target transcripts. Complete complementarity between the siRNA and the target transcript results in cleavage of the target opposite position of the guide strand, catalysed by AGO2, leading to gene silencing.
  • AGO2 Argonaute2 protein
  • RISC RNA-induced silencing complex
  • the sense strand meets the formal definition of a drug delivery device: it is non-covalently bound, enhances the stability of the antisense strand and must be removed by the Ago2 loading complex before the pharmacophore, the antisense strand, is active.
  • siRNA design Numerous variations of the archetypal siRNA design have been developed in terms of reduced passenger strand activity and/or improved potency. These include Dicer substrate siRNAs, small internally segmented siRNAs, self-delivering siRNAs (asymmetric and hydrophobic), single-stranded siRNAs and divalent siRNAs.
  • the inhibitor of the present disclosure is the antisense portion of a shRNA.
  • Short hairpin RNAs are artificial RNA molecules that are transcribed as a single stranded RNA but because of internal complementarity form a loop or hairpin-like structure. The hairpin is subsequently processed to an siRNA and also leads to the degradation of mRNAs in a sequence-specific manner dependent upon complementary binding of the target mRNA.
  • shRNAs are slightly larger than siRNA molecules and, unlike siRNAs, are produced inside the cell in the nucleus.
  • RNAi-mediated duplex silencers are miRNAs and di-siRNAs.
  • microRNAs miRNAs
  • miRNAs are endogenous non-coding RNA molecules that trigger RNAi and that have been implicated in a multitude of physiological and pathophysiological processes.
  • miRNA hairpins embedded within long primary miRNA transcripts are sequentially processed by two RNase III family enzymes, DICER1 (Dicer) and DROSHA, which liberate the hairpin and then cleave the loop sequence, respectively.
  • the resulting duplex RNA is analogous to an siRNA and is then loaded into an Argonaute protein (for example, AG02) while one strand is discarded to generate the mature, single-stranded miRNA species.
  • siRNAs guide RISC to target sequences where they initiate gene silencing.
  • miRNAs typically bind with partial complementarity and induce silencing via slicer-independent mechanisms.
  • the inhibitor of the present disclosure is a di-siRNA.
  • Divalent siRNAs are recently developed RNA silencing agent alternatives and have been shown to support a potent, sustained gene silencing in the central nervous system of mice and non-human primates following a single injection into the cerebrospinal fluid (Alterman et al 2019 Nature Biotech 37, 884-894).
  • Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker.
  • the inhibitors herein disclosed are oligonucleotides of between 10 and 50 nucleotides in length and composed of 1 or 2 oligonucleotides.
  • the nucleic acid molecules of the invention comprise or consist of 8 to 70 nucleotides in length, 10 to 60 nucleotides in length, 12 to 50 nucleotides in length, 8 to 40 nucleotides in length, or from 9 to 35, from 10 to 30, from 11 to 22, from 12 to 20, from 13 to 18 or from 14 to 16 contiguous nucleotides in length.
  • the nucleic acid molecule of the invention is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.
  • contiguous nucleotides or “contiguous nucleotide sequence” as used herein refers to the uninterrupted region of the oligonucleotide which is complementary to the target nucleic acid.
  • the nucleic acid molecule described herein or the contiguous nucleotide sequence thereof comprises or consists of 24 or less nucleotides, such as 22 or less nucleotides, such as 20 or less nucleotides, such as 18 or less nucleotides, such as 14, 15, 16 or 17 nucleotides. It is to be understood that any range given herein includes the range endpoints. Accordingly, if a nucleic acid molecule is said to include from 10 to 30 nucleotides, both 10 and 30 nucleotides are included. In some embodiments, the contiguous nucleotide sequence comprises or consists of 8, 9, 10, 11, 12, 13, 14, 15,
  • the nucleic acid molecule of the invention is 14 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 15 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 16 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 17 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 18 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 19 nucleotides in length. In some embodiments, the nucleic acid molecule of the invention is 20 nucleotides in length.
  • the nucleic acid molecule(s) is typically for modulating the expression of MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLODS, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X or NLGN4Y as target nucleic acid in a mammal.
  • the nucleic acid molecule(s), such as siRNAs, shRNAs or antisense oligonucleotides is typically for inhibiting the expression of a target nucleic acid.
  • the nucleic acid molecule(s) or the oligonucleotide(s) of the invention is man-made and/or is chemically synthesized and/or is typically purified or isolated. Accordingly, the present disclosure provides a method of manufacturing the nucleic acid molecule(s) or the oligonucleotide(s) of the invention comprising chemically synthesizing the nucleic acid molecule(s) or the oligonucleotide(s) of the invention. In some aspects, the method comprises the conjugation of a delivery moiety, e.g., a GalNAc moiety.
  • a delivery moiety e.g., a GalNAc moiety.
  • siRNA and shRNA design programs are publicly available. Non-limiting examples are siDESIGN from ThermoScientific, siDirect (Naito et al), BLOCK-IT RNAi Designer from Invitrogen, siRNA Wizard from InvivoGen, shRNA design tool from Gene Link and shRNA design tool from transomic. Manufacturers of RNAi products also provide guidelines for designing siRNA/shRNA. siRNA sequences between 19-29 nucleotides (nt) are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing.
  • Ideal sites to target include AA dinucleotides and the 19 nt 3' of them in the target mRNA sequence.
  • siRNAs with 3' dlldll or dTdT dinucleotide overhangs are more effective.
  • Other dinucleotide overhangs could maintain activity but GG overhangs should be avoided.
  • siRNA designs with a 4-6 poly(T) tract acting as a termination signal for RNA pol III
  • the G/C content is advised to be between 35-55%.
  • shRNAs should comprise sense and antisense sequences (advised to each be 19-21 nt in length) separated by loop structure, and a 3' AAAA overhang.
  • Effective loop structures are suggested to be 3-9 nt in length. It is suggested to follow the sense-loop-antisense order in designing the shRNA cassette and to avoid 5' overhangs in the shRNA construct. Finally, several companies commercially offer premade siRNAs and shRNAs. Chemical modifications
  • the inhibitor of the present disclosure is an oligonucleotide comprising non-naturally occurring nucleotide analogues, e.g., nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.
  • nucleotide analogues e.g., nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.
  • the goals were to enhance the affinity for the target sequence (thereby increasing potency), assure effective distribution to peripheral tissues, enhance the duration of action by increasing resistance to degradation by nucleases, improve pharmacokinetic characteristics, reduce the class generic (chemically based) toxicities of the chemical classes widely used for therapeutics, and create designs that support multiple post-binding mechanisms, thereby broadening the utility of the technology.
  • the oligonucleotides of the present disclosure comprise one or more non- cleavable internucleotide linkages, e.g., phosphorothioate linkages.
  • the phosphodiester backbone of unmodified DNA and RNA oligonucleotides is highly susceptible to degradation by nucleases in vivo. So, to develop oligonucleotides for therapeutic applications, it was necessary to identify backbone modifications that reduce their susceptibility to nuclease degradation while not compromising other key characteristics such as RNase Hl activation and RNA binding too much.
  • PS phosphorothioate
  • PO phosphodiester
  • the oligonucleotides of the present disclosure comprise non-naturally occurring nucleotide analogues, e.g., nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.
  • Oligonucleotides are frequently modified at the ribose sugar, primarily with the aim of improving properties such as affinity and/or nuclease resistance.
  • modifications include those where the ribose ring structure is modified (e.g. locked nucleic acids or LNAs), where the sugar moiety is replaced by a non-sugar moiety (e.g. peptide nucleic acids or PNAs) or where the substituent groups on the ribose ring are altered to groups other than the hydrogen or 2' -OH group naturally found in DNA and RNA nucleosides.
  • Non-limiting examples of ring structure modifications are HNAs where ribose ring is replaced with a hexose ring, an UNA (unlocked nucleic acid) where an unlinked ribose ring lacks a bond between the C2 and C3 carbons or a Locked Nucleic Acid (LNA) where the C2' and C4' of the ribose sugar ring are linked by a methylene bridge (also referred to as a"2'-4' bridge”), which restricts or locks the conformation of the ribose ring.
  • HNA locked nucleic acid
  • LNA Locked Nucleic Acid
  • the locking of the conformation of the ribose (also referred to as Bridged Nucleic Acids or BNAs) is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule.
  • BNAs Bridged Nucleic Acids
  • Non limiting examples of LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy LNA such as (S)-6'- methyl-beta-D-oxy- LNA (ScET) and 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) or those disclosed in WO 1999/014226, WO 2000/66604, WO 1998/039352, WO 2004/046160, WO 2000/047599, WO 2007/134181 , WO 2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 2011/156202, WO 2008/154401 , WO 2009/067647, WO 2008/150729.
  • BN A modifications enhance both nuclease stability and the affinity of the oligonucleotide for target RNA, they have been incorporated into the flanking regions of gapmers to improve target binding. As such, cEt-flanking 3-10-3 gapmers are more efficacious than the MOE 5-10-5 equivalents.
  • BNAs are excluded from the DNA gap region because they are not compatible with RNase H-mediated cleavage. LNA modifications have also been utilized in steric block ASOs, such as miRNA inhibitors.
  • Non-limiting examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'- OMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl- RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA (2'-F), and 2'-F-ANA nucleoside.
  • These modifications increase oligonucleotide nuclease resistance by replacing the nucleophilic 2'-hydroxyl group of unmodified RNA, leading to improved stability in plasma, increased tissue half-lives and consequently prolonged drug effects.
  • oligonucleotides comprising or consisting of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred here generally as “DNA”), but also possibly ribonucleotides (referred here generally as “RNA”), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e., "nucleotide analogues", such as nucleotides having the ribose sugar modifications disclosed above.
  • DNA 2'-deoxynucleotides
  • RNA ribonucleotides
  • the oligonucleotide of the present disclosure comprises at least two nucleotide analogues. In some embodiments, the oligonucleotide of the present disclosure comprises from 3, 4, 5, 6, 7, or 8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In some embodiments, all the nucleotide analogues are the same. In some embodiments, some nucleotide analogs are different. In some embodiments, all the nucleotides in the oligonucleotide of the present disclosure are nucleotide analogues.
  • nucleotide analogues when all the nucleotides in the oligonucleotide of the present disclosure are nucleotide analogues, all the nucleotide analogues are the same. In some embodiments, when all the nucleotides in the oligonucleotide of the present disclosure are nucleotide analogues, some of the nucleotide analogues are different.
  • the oligonucleotide of the present disclosure is a conjugate, e.g., a GalNAc conjugate.
  • the delivery potential of ASOs and siRNAs can be enhanced through direct covalent conjugation of various moieties that promote intracellular uptake, target the drug to specific cells/tissues or reduce clearance from the circulation.
  • Non-limiting examples are lipids, peptides, aptamers, antibodies and sugars.
  • Bioconjugates constitute distinct, homogeneous, single-component molecular entities with precise stoichiometry, meaning that high-scale synthesis is relatively simple and their pharmacokinetic properties are well defined.
  • bioconjugates are typically of small size meaning that they generally exhibit favourable biodistribution profiles. For example, conjugating ASOs or siRNAs to the sugar moiety GalNAc results in more productive delivery to hepatocytes without a meaningful shift in distribution to other tissues and results in 15-30 fold increases in potency for RNA targets in those cells.
  • Exosomes are heterogeneous, lipid bilayer- encapsulated vesicles approximately 100 nm in diameter that are generated as a result of the inward budding of the multivesicular bodies. Exosomes are thought to be released into the extracellular space by all cells, where they facilitate intercellular communication via the transfer of their complex macromolecular cargoes. Exosomes present numerous favourable properties in terms of oligonucleotide drug delivery of which crossing biological membranes, such as the blood-brain-barrier (BBB) is highly relevant for treatments of CNS disorders.
  • BBB blood-brain-barrier
  • RNA knock-out can be a gene knockdown or the gene can be knocked out by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer.
  • a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer.
  • Zinc-finger nucleases Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target desired DNA sequences, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of the endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
  • Other technologies for genome customization that can be used to knock out genes are meganucleases and TAL effector nucleases (TALENs, Cellectis bioresearch).
  • TALENs TAL effector nucleases
  • a TALEN is composed of a TALE DNA binding domain for sequence-specific recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB).
  • the DNA binding domain of a TALEN is capable of targeting with high precision a large recognition site (for instance 17bp).
  • Meganucleases are sequence-specific endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes).
  • CRISPR/Cas system Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA-guided genome engineering.
  • CRISPR interference is a genetic technique which allows for sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway.
  • CRISPR-Cas editing system can also be used to target RNA. It has been shown that the Class 2 type Vl-A CRISPR-Cas effector C2c2 can be programmed to cleave single stranded RNA targets carrying complementary protospacers (Abudayyeh et al 2016 Science 353/science.aaf5573). C2c2 is a single-effector endoRNase mediating ssRNA cleavage once it has been guided by a single crRNA guide toward the target RNA.
  • the invention disclosed herein can also be applied to develop gRNAs specifically reducing the expression of any of the genes selected from the listed consisting of MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLODS, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X, NLGN4Y, HSP90AB1, HSP90AA1, TRPM3, TRPM1, TRPM7, TRPM6, SLC8A1, SLC8A2, SLC8A3, DPP10, RYR1, RYR2, RYR3, GRIA1, GRIA2, GRIA3, GRIA4, CALM1, CALM2, CALM3, NLGN1, NLGN2 and NLGN3, more particular consisting of MCTP1, MCTP2, SHANK1, SHANK2, SHANKS
  • any of the oligonucleotides herein provided is a gRNA or CRISPR gRNA, more particularly a gRNA or CRISPR gRNA is provided with at least 90%, at least 95% or 100% complementary to a region within a sequence selected from SEQ. ID No. 1-372, particularly SEQ ID No.
  • Ribozymes are another type of molecules that can be used to modulate expression of a target gene. They are RNA molecules capable of catalyzing specific biochemical reactions, in the current context capable of targeted cleavage of nucleotide sequences. Examples of ribozymes include the hammerhead ribozyme, the Varkud Satellite ribozyme, Leadzyme and the hairpin ribozyme.
  • Interfering with structure can be achieved by e.g. binding moieties that shield e.g. the binding site on the protein of interest for a ligand of interest.
  • binding moieties that shield e.g. the binding site on the protein of interest for a ligand of interest.
  • Non-limiting examples are (monoclonal) antibodies or antigenbinding fragments thereof, alpha-bodies, nanobodies, intrabodies (antibodies binding and/or acting to intracellular target; this typically requires the expression of the antibody within the target cell, which can be accomplished by gene therapy), aptamers, DARPins, affibodies, affitins, anticalins, monobodies, phosphatases (in case of phosphorylated target) and kinases (in case of a phosphorylatable target).
  • antibody refers to any naturally occurring format of antibody or antigenbinding protein the production of which is induced by an immune system (immunoglobulins or IgGs). It is clear, however, that not all antibodies are naturally occurring as e.g. some antigens are problematic in the sense that they are poor or not at all immunogenic, or are not recognized by the immune system (e.g. self-antigens); artificial tricks may be required to obtain antibodies against such antigens (e.g. knock-out mice: e.g. Declercq et al. 1995, J Biol Chem 270:8397-8400; DNA immunization for e.g. transmembrane antigens; e.g.
  • "Conventional” antibodies comprise two heavy chains linked together by disulfide bonds and two light chains, one light chain being linked to each of the heavy chains by disulfide bonds.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (three or four constant domains, CHI, CH2, CH3 and CH4, depending on the antibody class).
  • Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end; the constant domains of the light chains each align with the first constant domains of the heavy chains, and the light chain variable domains each align with the variable domains of the heavy chains.
  • Ig new antigen receptors IgNARs
  • CNAR constant domains
  • VNAR variable domain
  • the complementary determining region 3 (CDR3) of camel antibodies and shark antibodies is usually longer (comprising about 16-21 amino acids, and about 16-27 amino acids, respectively) than the CDR3 of mouse VH region (comprising about 9 amino acids) (Muyldermans et al. 1994, Prot Eng 7, 1129-1135; Dooley & Flajnik 2005, Eur J Immunol 35, 936-945). Without the light chain, these heavy-chain antibodies bind to their antigens by one single domain, the variable antigen binding domain of the heavy-chain immunoglobulin, referred to as Vab (camelid antibodies) or V-NAR (shark antibodies).
  • Vab variable antigen binding domain of the heavy-chain immunoglobulin
  • V-NAR shk antibodies
  • Vab smallest intact and independently functional antigenbinding fragment Vab is referred to as nano-antibody or nanobody (Muyldermans 2001, J Biotechnol 74, 277-302).
  • Multivalent (etc. divalent, trivalent, tetravalent and pentavalent) Vab and/or V-NAR domains may be preferred in some instances due to their potentially higher cellular intake and retention and may be made by recombinant technology or by chemical means, such as described in WO 2010/033913.
  • the variable domains of the light and/or heavy chains are involved directly in binding the antibody to the antigen.
  • variable domains of naturally occurring light and heavy chains have the same general structure: four framework regions (FRs) connected by three complementarity determining regions (CDRs) (see e.g. Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD).
  • the CDRs in a light or heavy chain are held in close proximity by the FRs and contribute to the formation of the antigen binding site.
  • An antibody, or antibody fragment as described hereafter, may also be part of a multivalent and/or multispecific antigen binding molecule.
  • An overview of e.g. available bispecific formats (around 100) is provided in Brinkmann & Kontermann 2017 (mAbs 9:182-212).
  • antibody fragment refers to any molecule comprising one or more fragments (usually one or more CDRs) of an antibody (the parent antibody) such that it binds to the same antigen to which the parent antibody binds.
  • Antibody fragments include Fv, Fab, Fab', Fab'-SH, single-chain antibody molecules (such as scFv), F(ab') 2, single variable VH domains, and single variable VL domains (Holliger & Hudson 2005, Nature Biotechnol 23, 1126-1136), Vab and V-NAR.
  • the term further includes microantibodies, i.e. the minimum recognition unit of a parent antibody usually comprising just one CDR (Heap et al.
  • Any of the fragments can be incorporated in a multivalent and/or multispecific larger molecule, e.g. mono- or bi-specific Fab 2, mono- or tri-specific Fab 3, bis-scFv (mono- or bispecific), diabodies (mono- or bispecific), triabodies (e.g. trivalent monospecific), tetrabodies (e.g. tetravalent monospecific), minibodies and the like (Holliger & Hudson 2005, Nature Biotechnol 23, 1 126-1136). Any of the fragments can further be incorporated in e.g. V-NAR domains of shark antibodies or VhH domains of camelid antibodies (nanobodies). All these are included in the term "antibody fragment".
  • Alphabodies are also known as Cell-Penetrating Alphabodies and are small 10 kDa proteins engineered to bind to a variety of antigens.
  • DNA/RNA/XNA aptamers are single stranded and typically around 15-60 nucleotides in length although longer sequences of 220 nt have been selected; they can contain nonnatural nucleotides (XNA) as described for antisense RNA.
  • XNA nonnatural nucleotides
  • a nucleotide aptamer binding to the vascular endothelial growth factor (VEGF) was approved by FDA for treatment of macular degeneration.
  • Variants of RNA aptamers are aptmers are composed entirely of an unnatural L-ribonucleic acid backbone.
  • a Spiegelmer of the same sequence has the same binding properties of the corresponding RNA aptamer, except it binds to the mirror image of its target molecule.
  • Peptide aptamers consist of one (or more) short variable peptide domains, attached at both ends to a protein scaffold, e.g. the Affimer scaffold based on the cystatin protein fold.
  • a protein scaffold e.g. the Affimer scaffold based on the cystatin protein fold.
  • a further variation is described in e.g. WO 2004/077062 wherein e.g. 2 peptide loops are attached to an organic scaffold. Phage-display screening of such peptides has proven to be possible in e.g. WO 2009/098450.
  • DARPins stands for designed ankyrin repeat proteins. DARPin libraries with randomized potential target interaction residues, with diversities of over 10 12 variants, have been generated at the DNA level. From these, DARPins can be selected
  • Affitins or nanofitins, are artificial proteins structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as peptides, proteins, viruses, and bacteria.
  • Anticalins are derived from human lipocalins which are a family of naturally binding proteins and mutation of amino acids at the binding site allows for changing the affinity and selectivity towards a target of interest. They have better tissue penetration than antibodies and are stable at temperatures up to 70 °C.
  • Monobodies are synthetic binding proteins that are constructed starting from the fibronectin type III domain (FN3) as a molecular scaffold.
  • the inhibitors of the application for use (in a method) according to the invention can be defined as those inhibitors specific to any of the genes selected from the list consisting of MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLOD3, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X, NLGN4Y, HSP90AB1, HSP90AA1, TRPM3, TRPM1, TRPM7, TRPM6, SLC8A1, SLC8A2, SLC8A3, DPP10, RYR1, RYR2, RYR3, GRIA1, GRIA2, GRIA3, GRIA4, CALM1, CALM2, CALM3, NLGN1, NLGN2 and NLGN3 or selected from MCTP1, MCTP2, SHANK1,
  • nucleic acid molecules or oligonucleotides according to the present invention may exist in the form of their pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the nucleic acid molecules or oligonucleotides of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
  • Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluene sulfonic acid, salicylic acid, methane sulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
  • Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide.
  • the chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described by Bastin (2000 Organic Process Research & Development 4:427-435) or in Ansel (1995 In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed., pp. 196 and 1456-1457).
  • the pharmaceutically acceptable salt of the nucleic acid molecules or oligonucleotides provided herein may be a sodium salt.
  • the pharmaceutically acceptable salt is a sodium or a potassium salt.
  • the invention provides pharmaceutical compositions comprising any of the nucleic acid molecules or oligonucleotides described herein or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.
  • a pharmaceutically acceptable diluent includes phosphate- buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to sodium and potassium salts.
  • the pharmaceutically acceptable diluent is sterile phosphate buffered saline.
  • the nucleic acid molecules or oligonucleotides of the application are used in the pharmaceutically acceptable diluent at a concentration of 1-100 nanomolar (nM), 2- 50 nm, 5-150 nM, 10-250 nM, 25-500 nM, 50-750 nM, 0.1-1 micromolar (pM), 0.5-10 pM, 2-50 pM, 5- 250 pM or 10-500 pM solution.
  • Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see e.g. Langer (1990 Science 249:1527-1533).
  • Non-limiting examples of pharmaceutically acceptable diluents, carriers, adjuvants, suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are provided in W02007/031091.
  • the nucleic acid molecules or oligonucleotides of the application or salts thereof may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including but not limited to route of administration, extent of disease, or dose to be administered.
  • Pharmaceutical compositions comprising any of the nucleic acid molecules or oligonucleotides of the application or salts thereof may be sterilized by conventional sterilization techniques or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more particularly between 5 and 9 or between 6 and 8, most particularly between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the nucleic acid molecules or oligonucleotides of the application or salts thereof, such as in a sealed package of tablets or capsules.
  • the composition in solid form can also be packaged in a container for a flexible quantity.
  • Tauopathies are a diverse group of disorders all having in common their association with prominent accumulation of intracellular tau protein.
  • the tau protein is abundantly expressed in the central nervous system.
  • the group of tauopathies is growing as recently Huntington disease (Fernandez-Nogales et al 2014 Nat Med 20:881-885) and chronic traumatic encephalopathy (CTE; McKee et al 2009 J Neuropathol Exp Neurol 68,709-735) were added.
  • tauopathic disorders are divided in predominant Tau pathologies, tauopathies associated with amyloid deposition and tauopathies associated with another pathology (Williams et al 2006 Intern Med J 36:652-660).
  • Predominant Tau pathologies include progressive supranuclear palsy (PSP), progressive supranuclear palsy-parkinsonism (PSP-P), Richardson's syndrome, argyrophilic grain disease, corticobasal degeneration, Pick's disease, frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), post-encephalitic parkinsonism, Parkinson's disease complex of Guam, and Guadeloupean parkinsonism.
  • PSP progressive supranuclear palsy
  • PSP-P progressive supranuclear palsy-parkinsonism
  • Richardson's syndrome argyrophilic grain disease
  • corticobasal degeneration corticobasal degeneration
  • Pick's disease frontotemporal dementia with par
  • Tauopathic disorders associated with amyloid deposition include Alzheimer's disease, Down's syndrome, dementia pugilistica, familial British dementia and familial Danish dementia.
  • Tauopathic disorders associated with another pathology include myotonic dystrophy, Hallevorden-Spatz disease, and Niemann Pick type C.
  • 4R tauopathies include progressive supranuclear palsy (PSP), corticobasal degeneration, tangle predominant dementia, and argyrophilic grain disease.
  • 3R tauopathies include Pick disease
  • 3R+4R tauopathies include Alzheimer's disease (Dickson et al 2011 J Mol Neurosci 45:384-389; Murray et al 2014 Alzheimer's Res Ther 6:1). The tau protein is discussed herein in more detail further below.
  • tauopathies include tangle-only dementia, white matter tauopathy with globular glial inclusions, subacute sclerosing panencephalitis, SLC9A6-related mental retardation, non-Guamanian motor neuron disease with neurofibrillary tangles, neurodegeneration with brain iron accumulation, Gerstmann- Straussler-Scheinker disease, frontotemporal lobar degeneration, diffuse neurofibrillary tangles with calcification, chronic traumatic encephalopathy, amyotrophic lateral sclerosis of Guam, amyotrophic lateral sclerosis and parkinsonism-dementia complex, prion protein cerebral amyloid angiopathy, and progressive subcortical gliosis (Murray et al 2014 Alzheimer's Res Ther 6:1; Spillantini & Goedert 2013 Lancet Neurol 12:609-622).
  • Symptoms of tauopathic disorders include clinical or pathological symptoms such as mild cognitive impairment, dementia, cognitive decline (e.g. apathy, impairment in abstract thought), decline of motor function (causing e.g. postural instability, tremor or dystonia), oculomotor and bulbar dysfunction. Criteria for diagnosing dementia are outlined in e.g. the Diagnostic and Statistical Manual of Mental Disorders (DSM) or in the International Classification of Disease (ICD) and are subject to regular updates. The type of clinical symptoms depends on which region of the brain is affected by the tauopathy and explains why Alzheimer's disease is mainly a dementing disease and why Parkinson's disease is mainly affecting movement.
  • DSM Diagnostic and Statistical Manual of Mental Disorders
  • ICD International Classification of Disease
  • any of the inhibitors, nucleic acid molecules or oligonucleotides herein described is applicable for use as a medicament.
  • any of the nucleic acid molecules or oligonucleotides herein described is provided for use in (a method for) treating or inhibiting progression of a tauopathic disorder or for use in (a method for) treating or inhibiting a symptom of a tauopathic disorder.
  • nucleic acid molecules or oligonucleotides of the invention are inhibitors of human MCTP1, MCTP2, SHANK1, SHANK2, SHANK3, PLOD1, PLOD2, PLOD3, FARP1, FARP2, POSTN, TGFBI, TRMP9B, ALKBH8, ACDY4, ACDY2, ACDY7, ACDY8, IGLON5, OPCML, NTM, CALM1, CALM3, SLC38A11, SLC38A2, NLGN4X, NLGN4Y, HSP90AB1, HSP90AA1, TRPM3, TRPM1, TRPM7, TRPM6, SLC8A1, SLC8A2, SLC8A3, DPP10, RYR1, RYR2, RYR3, GRIA1, GRIA2, GRIA3, GRIA4, CALM1, CALM2, CALM3, NLGN1, NLGN2 or NLGN3 expression.
  • any of the nucleic acid molecules or oligonucleotides herein described is administered to a subject in need thereof (a subject suffering of or displaying a tauopathy or symptom thereof) in an effective amount, i.e. in an amount sufficient to treat or to inhibit progression of a tauopathic disorder or a symptom of a tauopathic disorder.
  • an effective amount of the therapeutic compound is administered to a subject in need thereof.
  • An "effective amount" of an active substance in a composition is the amount of said substance required and sufficient to elicit an adequate response in treating, preventing, inhibiting (progression of) the intended or targeted medical indication. It will be clear to the skilled artisan that such response may require successive (in time) administrations with the composition as part of an administration scheme.
  • the effective amount may vary depending on the nature of the compound, the route of administration of the compound (crossing of the blood-brain barrier and the cell membrane are potential barriers to be taken by oligonucleotides as described herein), the health and physical condition of the individual to be treated, the age of the individual to be treated (e.g. dosing for infants may be lower than for adults) the taxonomic group of the individual to be treated (e.g. human, non-human primate, primate, etc.), the capacity of the individual's system to respond effectively, the degree of the desired response, the formulation of the active substance, the treating doctor's assessment and other relevant factors.
  • the effective amount further may vary depending on whether it is used in monotherapy or in combination therapy. Determination of an effective amount of a compound usually follows from pre-clinical testing in a representative animal or in vitro model (if available) and/or from dose-finding studies in early clinical trials.
  • any of the inhibitors, nucleic acid molecules or oligonucleotides described herein is provided for use in (a method for) treating or inhibition progression of a tauopathic disorder
  • the tauopathic disorder is selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy (PSP), progressive supranuclear palsy-parkinsonism (PSP-P), Richardson's syndrome, argyrophilic grain disease, corticobasal degeneration Pick's disease, frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), post-encephalitic parkinsonism, Parkinson's disease complex of Guam, Guadeloupean parkinsonism, Huntington disease, Down's syndrome, dementia pugilistica, familial British dementia, familial Danish dementia, myotonic dystrophy, Hallevorden-Spatz disease, Niemann Pick type C, chronic traumatic encephalopathy, tangle-only dementia, white matter tauopathy with globular glial inclusions, sub
  • any of the inhibitors, nucleic acid molecules or oligonucleotides described herein is thus likewise applicable for use in (a method for) treating or inhibition progression of a symptom of tauopathic disorder selected from the group of mild cognitive impairment, dementia, cognitive decline, decline of motor function, oculomotor and bulbar dysfunction, synaptic dysfunction, neurotoxicity, neuronal degeneration, neuronal dysfunction, synapse loss, and amyloid deposition.
  • a symptom of tauopathic disorder selected from the group of mild cognitive impairment, dementia, cognitive decline, decline of motor function, oculomotor and bulbar dysfunction, synaptic dysfunction, neurotoxicity, neuronal degeneration, neuronal dysfunction, synapse loss, and amyloid deposition.
  • a symptom of tauopathic disorder selected from the group of mild cognitive impairment, dementia, cognitive decline, decline of motor function, oculomotor and bulbar dysfunction, synaptic dysfunction, neurotoxicity, neuronal degeneration, neuronal dysfunction, synapse loss, and amyloid deposition
  • the level of inhibition can be about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the level of inhibition can be at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • reducing e.g., reducing the expression of the MCTPl gene transcript and/or MCTPl protein level or MCTPl activity refers to the oligonucleotide of the present disclosure (e.g., an ASO or siRNA) disclosed herein reducing the expression of the MCTPl gene transcript and/or MCTPl protein level and/or activity in a cell, a tissue, or a subject.
  • oligonucleotide of the present disclosure e.g., an ASO or siRNA
  • the term “reducing” refers to complete inhibition (100% inhibition or non-detectable level) of the gene transcript or the protein level and/or activity. In other aspects, the term “reducing” refers, e.g., to at least about 25%, to at least about 30%, to at least about 35%, to at least about 40%, to at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least 95% or at least 99% inhibition of the gene transcript and/or the protein expression and/or activity in a cell, a tissue, or a subject.
  • a mammal includes all mammals, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like).
  • domestic animals e.g., dogs, cats and the like
  • farm animals e.g., cows, sheep, pigs, horses and the like
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like.
  • the application also provides methods of treating or inhibiting progression of a symptom of a tauopathic disorder, the method comprises the step of administering any of the nucleic acid molecules or oligonucleotides herein described to a subject in need thereof.
  • Treatment refers to any rate of reduction or retardation of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no/zero progress of the disease or disorder (i.e. "inhibition” or “inhibition of progression”) or even in any rate of regression of the already developed disease or disorder.
  • Tauopathies are in general progressive disorders, and progression may imply propagation of pathological tau protein (Asai et al 2015 Nat Neurosci 18:1584-1593; deCalumble et al 2012 Neuron 73:685-697).
  • Reduction or “reducing” in terms of progress of a disease as used herein refers to a statistically significant reduction. More particularly, a statistically significant reduction upon administering the inhibitor of the invention compared to a control situation wherein the inhibitor is not administered. In a particular embodiment, said statistically significant reduction in progress of a disease is an at least 25%, 30%, 35%, 40%, 45% or 50% reduction compared to the control situation.
  • Magnetic resonance imaging (MRI) in itself allows for radiologic determination of brain atrophy.
  • Midbrain atrophic signs such as the Hummingbird or Penguin silhouette are for instance indicators of progressive supranuclear palsy (PSP).
  • PSP progressive supranuclear palsy
  • Determination of tau protein content in the cerebrospinal fluid (CSF) may also serve as an indicator of tauopathies.
  • the ratio between the 33 kDa/55 kDa tau-forms in CSF was e.g. found to be reduced in a patients with PSP (Borroni et al 2008 Neurology 71:1796-1803).
  • tauopathies benefits from the existence of Tau imaging ligands detectable by positron emission tomography (PET), and include the radiotracers 2-(l-(6-((2-[ 18 F]fluoroethyl) (methyl) amino)-2-naphthyl)ethylidene) malononitrile ([ 18 F]FDDNP), 2-(4-aminophenyl)-6-(2-
  • the inhibitors, nucleic acid molecules or the oligonucleotides of the present invention may be administered via intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraventricular, intraocular, or intrathecal administration. In some embodiments, the administration is via intrathecal administration.
  • Administration means to give a composition comprising a composition disclosed herein to a subject via a pharmaceutically acceptable route.
  • Inhibition of expression of the target gene can be also achieved through the creation of transgenic organisms expressing one of the oligonucleotides of the invention (e.g. siRNA), or by administering said inhibitor to the subject.
  • the nature of the inhibitor siRNA, shRNA, gapmer, etc.
  • whether the effect is achieved by incorporating the oligonucleotide into the subject's genome or by administering the inhibitor is not vital to the invention, as long as said oligonucleotide or inhibitor reduces the level of the target transcripts.
  • oligonucleotide construct can be delivered, for example as an expression plasmid, which when transcribed in the cell, produces the oligonucleotide that is complementary to at least a unique portion of the cellular target RNA.
  • oligonucleotide inhibitors such as siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing these inhibitors from a plasmid include, for example the U6 or Hl RNA polymerase III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • Non-limiting examples are neuronal-specific promoters, glial cell specific promoters, the human synapsin 1 gene promoter, the Hb9 promotor or the promoters disclosed in US7341847B2.
  • the recombinant plasmids comprising any of the inhibitors, nucleic acid molecules or oligonucleotides of the invention can also comprise inducible or regulatable promoters for expression of the inhibitor, nucleic acid molecule or oligonucleotide in a particular tissue or in a particular intracellular environment.
  • the inhibitor, nucleic acid molecule or oligonucleotide expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly, e.g. in brain tissue or in neurons. Nucleic acid molecules or oligonucleotides can also be expressed intracellularly from recombinant viral vectors.
  • the recombinant viral vectors comprise sequences encoding the nucleic acid molecules or oligonucleotides of the invention and any suitable promoter for expressing them.
  • the nucleic acid molecules or oligonucleotides will be administered in an "effective amount" which is an amount sufficient to cause a statistically significant reduction of the target transcript.
  • an effective amount of a nucleic acid molecule or oligonucleotide targeting target transcripts comprises an intracellular concentration of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or lesser amounts of inhibitor can be administered.
  • shRNAs for example can be introduced into the nuclei of target cells using a vector (e.g. bacterial or viral) that optionally can stably integrate into the genome.
  • shRNAs are usually transcribed from vectors, e.g. driven by the Pol III U6 promoter or Hlpromoter. Vectors allow for inducible shRNA expression, e.g.
  • Plasmid DNA or dsRNA can be delivered to a cell by means of transfection (lipid transfection, cationic polymer-based nanoparticles, lipid or cell-penetrating peptide conjugation) or electroporation.
  • Viral vectors include lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
  • the inhibitors of the present disclosure are administered across the blood-brain barrier.
  • the blood-brain barrier (BBB) is a protective layer of tightly joined cells that lines the blood vessels of the brain which prevents entry of harmful substances (e.g. toxins, infectious agents) and restricts entry of (non-lipid) soluble molecules that are not recognized by specific transport carriers into the brain.
  • harmful substances e.g. toxins, infectious agents
  • non-lipid soluble molecules that are not recognized by specific transport carriers into the brain.
  • the BBB often is to some degree affected or broken down in case of a tauopathic disorder, it may be needed to rely on a means to enhance permeation of the BBB for a candidate drug for treating a tauopathic disorder to be able to enter the affected brain cells.
  • the oligonucleotides of the present disclose are formulated, conjugated, or carried by vectors, polymers, cells, or devices, to name a few alternatives, that allow the oligonucleotides to cross the BBB.
  • Drugs can be directly injected into the brain (invasive strategy) or can be directed into the brain after BBB disruption with a pharmacological agent (pharmacologic strategy).
  • an inhibitor of the present disclosure can be directly injected into the brain, e.g., using a needle or a catheter.
  • an inhibitor of the present disclosure can be directed into the brain by BBB disruption with a pharmacological agent.
  • Invasive means of BBB disruption are associated with the risk of hemorrhage, infection or damage to diseased and normal brain tissue from the needle or catheter.
  • Direct drug deposition may be improved by the technique of convection-enhanced delivery. Accordingly, in some embodiments, an inhibitor of the present disclosure can be administered via convection- enhanced delivery.
  • a therapeutic protein e.g. a neurotrophic factor or nerve growth factor, or a proteinaceous inhibitor as described herein
  • a therapeutic protein e.g. a neurotrophic factor or nerve growth factor, or a proteinaceous inhibitor as described herein
  • an inhibitor of the present disclosure can be administered, e.g., by implantation of genetically modified cells (e.g., stem cells), recombinant vectors (e.g., viral vectors), delivery devices (e.g., pumps such as osmotic pumps), or incorporation in a polymer.
  • Pharmacologic BBB disruption has the drawback of being non-selective and can be associated with unwanted effects on blood pressure and the body's fluid balance. This is circumvented by targeted or selective administration of the pharmacologic BBB disrupting agent.
  • intra-arterial cerebral infusion of an antibody (bevacizumab) in a brain tumor was demonstrated after osmotic disruption of the BBB with mannitol (Boockvar et al. 2011, J Neurosurg 114:624-632); other agents capable of disrupting the BBB pharmacologically include bradykinin and leukotriene C4 (e.g. via intracarotid infusion; Nakano et al. 1996, Cancer Res 56:4027-4031).
  • the inhibitors of the present disclosure are formulated in combination with a pharmacologic BBB disrupting agent.
  • the inhibitors of the present disclosure are administered in combination with a pharmacologic BBB disrupting agent.
  • the pharmacologic BBB disrupting agent is administered prior to the administration of the inhibitor of the present disclosure.
  • the pharmacologic BBB disrupting agent is administered concurrently to the administration of the inhibitor of the present disclosure.
  • the pharmacologic BBB disrupting agent is administered subsequently to the administration of the inhibitor of the present disclosure.
  • the pharmacologic BBB disrupting agent comprises mannitol, bradykinin, leukotriene C4, or a combination thereof.
  • BBB transcytosis and efflux inhibition are other strategies to increase brain uptake of drugs supplied via the blood.
  • Using transferrin or transferrin-receptor antibodies as carrier of a drug is one example of exploiting a natural BBB transcytosis process (Friden et al. 1996, J Pharmacol Exp Ther 278:1491-1498). Exploiting BBB transcytosis for drug delivery is also known as the molecular Trojan horse strategy.
  • the inhibitor of the present disclosure are conjugated to carrier, e.g., transferrin or a transferrin-receptor antibody.
  • the inhibitors of the present disclosure are conjugated or formulated to transverse the BBB via transcytosis.
  • BBB efflux pumps or ATP-binding cassette (ABC) transporters
  • BCRP/ABCG2 breast cancer resistance protein
  • Pgp/MDRl/ABCBl P-glycoprotein
  • the inhibitors of the present disclosure can be formulated in combination with a compound that can block an ABC transporter, a compound that can block P-glycoprotein, or a combination thereof.
  • Kumar et al (2007 Nature 448:39-43) demonstrated uptake of siRNAs in the brain after coupling to a 29- amino acid peptide derived from rabies virus glycoprotein (RVG) which is specifically binding the acetylcholine receptor.
  • RVG rabies virus glycoprotein
  • the inhibitors of the present disclosure are conjugated to RVG.
  • Therapeutic drugs can alternatively be loaded in liposomes to enhance their crossing of the BBB, an approach also known as liposomal Trojan horse strategy.
  • the inhibitors of the present disclosure are formulated in liposomes, e.g., liposomes for use in a liposomal Trojan horse strategy.
  • a more recent and promising avenue for delivering therapeutic drugs to the brain consists of (transient) BBB disruption by means of ultrasound, more particularly focused ultrasound (FUS; Miller et al. 2017, Metabolism 69:S3-S7).
  • this technique has, often in combination with realtime imaging, the advantage of precise targeting to a diseased area of the brain.
  • Therapeutic drugs can be delivered in e.g. microbubbles e.g. stabilized by an albumin or other protein, a lipid, or a polymer.
  • Therapeutic drugs can alternatively, or in conjunction with microbubbles, be delivered by any other method, and subsequently FUS can enhance local uptake of any compound present in the blood (e.g. Nance et al.
  • Microbubbles with a therapeutic drug load can also be induced to burst (hyperthermic effect) in the vicinity of the target cells by means of FUS, and when driven by e.g. a heat shock protein gene promoter, localized temporary expression of a therapeutic protein can be induced by ultrasound hyperthermia (e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574).
  • the inhibitors of the present disclosure are formulated for FUS-mediated delivery.
  • the inhibitors of the present disclosure are formulated for intracellular administration. Besides the need to cross the BBB, drugs targeting disorders of the central nervous system, such as the inhibitors described herein, may also need to cross the cellular barrier. Although most antisense oligonucleotides are readily taken up by neurons and glia after reaching the nervous system, it can be advantageous to use facilitators of intracellular drug uptake.
  • CPPs cell-penetrating proteins or peptides
  • TPDs Protein Transduction Domains
  • CPPs include the TAT peptide (derived from HIV-1 Tat protein), penetratin (derived from Drosophila Antennapedia -Antp), pVEC (derived from murine vascular endothelial cadherin), signal-sequence based peptides or membrane translocating sequences, model amphipathic peptide (MAP), transportan, MPG, polyarginines; more information on these peptides can be found in Torchilin 2008 (Adv Drug Deliv Rev 60:548-558) and references cited therein.
  • the TAT peptide was e.g. used to shuffle a tau-fragment into neuronal cells (Zhou et al. 2017).
  • CPPs can be coupled to carriers such as nanoparticles, liposomes, micelles, or generally any hydrophobic particle. Coupling can be by absorption or chemical bonding, such as via a spacer between the CPP and the carrier. To increase target specificity an antibody binding to a target-specific antigen can further be coupled to the carrier (Torchilin 2008, Adv Drug Deliv Rev 60:548-558)
  • CPPs have already been used to deliver payloads as diverse as plasmid DNA, oligonucleotides, siRNA, peptide nucleic acids (PNA), proteins and peptides, small molecules and nanoparticles inside the cell (Stalmans et al. 2013, PloS One 8:e71752).
  • the present disclosure also provides a method of manufacturing an inhibitor, more particularly an oligonucleotide of the present disclosure, the method comprising chemically synthesizing the inhibitor or oligonucleotide of the present disclosure using sequential solid phase oligonucleotide synthesis.
  • the present disclosure provides a method of manufacturing an inhibitor or oligonucleotide of the present disclosure comprising a conjugate moiety, wherein the method comprises covalently attaching the conjugate moiety (e.g., at least one non-nucleotide or non-polynucleotide moiety) covalently to an antisense oligomer disclosed herein.
  • the conjugate moiety e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety
  • the conjugate moiety is attached to an antisense oligomer disclosed herein directly or via a linker positioned between the antisense oligomer sequence and the conjugate moiety.
  • covalently attaching the conjugate moiety e.g., a non-nucleotide or non- polynucleotide moiety, such as a GalNAc moiety
  • covalently attaching the conjugate moiety comprises: (i) chemically synthesizing the antisense oligomer; and, (ii) adding by chemical synthesis or conjugation the conjugate moiety to the antisense oligomer to yield an oligonucleotide conjugate.
  • adding by chemical synthesis or conjugation the conjugate moiety (e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety) to the antisense oligomer to yield an oligonucleotide conjugate comprises: (i) incorporating by chemical synthesis or conjugation at least one conjugate moiety (e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety) to the antisense oligomer; (ii) incorporating by chemical synthesis or conjugation at least one linker to the antisense oligomer or conjugate moiety (e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety); (iii) incorporating by chemical synthesis or conjugation at least one branching point to the antisense oligomer or conjugate mo
  • At least one linker is interposed between the antisense oligomer and a branching point;
  • at least one branching point is interposed between a linker and a conjugate moiety (e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety);
  • at least one, two, or three conjugate moieties e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety
  • at least one polymer spacer e.g., a PEG spacer
  • kits and products of manufacture comprising one or more compositions (e.g., an inhibitor more particularly an oligonucleotide of the present disclosure or pharmaceutical compositions comprising the inhibitor or oligonucleotide of the present disclosure) described herein.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein.
  • the kit or product of manufacture comprises, e.g., a first container comprising a first pharmaceutical composition comprising an inhibitor or oligonucleotide of the present disclosure, a second container containing a solvent, and optionally an instruction for use.
  • the kit or product of manufacture comprises a container comprising an inhibitor or oligonucleotide of the present disclosure and optionally an instruction for use.
  • the kit contains a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. In some aspects, the kit further comprises instructions to administer a composition of the present disclosure according to any method disclosed herein. In some aspects, the kit is for use in the treatment of a medical indication disclosed herein. In some aspects, the kit is a diagnostic kit.
  • biomolecules e.g., proteins, genes
  • database accession numbers disclosed herein refer to the database version that in effect on August 10, 2022.
  • the nucleic acid sequences of genes identified by name as well as their official names and alternative names correspond to those in the version of the Genbank database active on August 10, 2022, and are herein incorporated by reference.
  • the amino acid sequences of proteins identified by name or translation products of genes identified by name as well as their official and alternative names correspond to those in the version of the UniProt database active on January 23, 2023 and are herein incorporated by reference.
  • Example 1 Cell-type specific a-sy nuclein and tau expression levels do not account for differential vulnerability in mutation carriers
  • a-synuclein and tau are higher in the cerebellum as compared to the primary sites of vulnerability, i.e. substantia nigra and frontal cortex, respectively ( Figure 1A).
  • a-synuclein or tau expression levels in specific brain regions do not correlate with known vulnerable regions.
  • median a-synuclein and tau expression in substantia nigra and ventral tegmental area dopaminergic neurons are within the top decile of cell types (data not shown). Furthermore, median a-synuclein expression in CA3 hippocampal neurons that substantially degenerate in a-synuclein mutation carriers (Spira et al., 2001), ranks high (5th) across 265 cell types (data not shown). However, also many other cell types not reported as affected in a-synuclein and tau mutation carriers express high levels of a-synuclein and tau, such as spinal cord excitatory neurons or cranial nerve nuclei (data not shown).
  • the preferentially vulnerable CA2 and CA3 neurons express high levels of a-synuclein in the human hippocampus (Franjic et al., 2022) (data not shown). Tau expression is similarly high across different neuron types of the entorhinal cortex, subiculum and hippocampus (data not shown). Across multiple human cortical areas a-synuclein expression is higher in excitatory compared to inhibitory neurons (data not shown). Tau is expressed highly throughout excitatory and inhibitory neurons and expression levels are similar across brain areas, providing no direct explanation why frontal and temporal cortices are preferentially vulnerable in tau mutant patients (Figure IB; data not shown).
  • Example 2 Drosophila models of a-synuclein and tau mutations show differential neuronal vulnerability
  • transgenic flies with an empty backbone insertion mini-w+
  • a fluorophore-dead GFP smGdP
  • a-synuclein as well as tau mutants where the main aggregation-prone regions are deleted
  • NAC and PHF6 VQIVYK respectively; von Bergen et al., 2000, 2001; Bodies et al., 2001; Rodriguez et al., 2015; Sawaya et al., 2007).
  • ERGs electro-retinogram
  • vacuolization and the regional distribution is markedly different compared to A53T a-synuclein-expressing flies.
  • vacuoles are present throughout the brain; in A53T a-synuclein they mainly cluster in the optic lobes. This further suggests that different neurons are affected in A53T a-synuclein vs. P301L tau brains.
  • Example 3 Differential transcriptomic impact to pathogenic a-synuclein and tau across >200 neuron types
  • RNA sequencing To identify neuron types differentially vulnerable to a-synuclein and tau we performed brain wide singlecell RNA sequencing. We sampled A53T a-synuclein, P301L tau, mini-w+ controls and smGdP at 5 and 25 days of age in six repeat experiments. Cells from all genotypes and experimental dates intermix homogenously, suggesting that all cell types are formed across conditions. After quality control filtering, we retain 143,013 cells, evenly distributed between the conditions.
  • edgeR Robot et al., 2009
  • TmY5a, Tm3, a/b-KC, TmY8, T2a, Tml neurons are impacted significantly stronger than expected upon A53T a-synuclein expression. They are cholinergic or glutamatergic excitatory neurons, from the optic lobes - where the vast majority of identified cell-types reside - as well as from the learning and memory centre (a/b-KC).
  • Tm3, C2, T2, C3, Tml, T3 and Mil5 are impacted significantly stronger than expected upon P301L tau expression, independent of the covariates cell and gene number as well as transgene levels.
  • C2 and C3 neurons are GABAergic, Mil5 neurons dopaminergic and the rest cholinergic neurons.
  • Neurons with significantly smaller transcriptomic deregulation than expected in response to A53T a-synuclein or P301L tau expression are cholinergic, GABAergic and glutamatergic neurons from the optic, antennal lobes as well as of yet unresolved identity.
  • Tml and Tm3 neurons appear preferentially deregulated both upon A53T a-synuclein and P301L tau expression
  • neuron types where the responses in the A53T a-synuclein versus P301L tau versus smGdP conditions diverge, such as C2, C3, T2, T3, Tm2, a/b-KC and TmY5a neurons.
  • Tml neurons which we predict to be vulnerable to A53T a-synuclein and in P301L tau, we note increased density of dendritic proliferations in both conditions versus control, as measured by increased CD8::GFP signal in the medulla ( Figure 3E).
  • P301Ltau-expressing Tml neurons but not A53T a-synuclein-expressing Tml neurons, frequently display dystrophic neurites with swollen segments ( Figure 3F).
  • Tml neurons are structurally affected in A53T a-synuclein and P301L tau, with additional defects in tau, consistent with the higher number of DEGs in Tml neurons of P301L tau expressing animals.
  • KC a/p-Kenyon cells
  • KC a/p-Kenyon cells
  • KC a/p-Kenyon cells
  • Figure 3G a/p-Kenyon cells
  • LClOa neurons which we predict to be resilient to both P301L tau and A53T a-synuclein do not display synaptic hyperintensities or synaptic loss
  • Figure 3H-I a/p-Kenyon cells
  • P301L tau resilient neurons exhibit higher expression of 'axon guidance' and 'synapse organization' terms (Figure 5A), supporting the idea that genes involved in the development or remodeling of these compartments might be able counteract tau-induced synaptic loss.
  • P301L vulnerable neurons are enriched for ion channel and Ca2+-related GO terms, consistent with a role of neuronal excitability and altered Ca2+ homeostasis in neurodegenerative diseases (Fu et al., 2018; Saxena and Caroni, 2011; Sulzer and Surmeier, 2013) (Figure 5B).
  • BMPR1B+ layer 2 entorhinal cortex neurons
  • pQUASTattB To create the Drosophila models, we first generated pQUASTattB by transferring the '5xQUAS-hsp70 promoter-MCS-SV40 polyA signal' cassette from pQUAST (Addgene #24349, (Potter et al., 2010)) into pUASTattB (Bischof et al., 2013) via BamHI digestion and T4 ligation (NEB). pQUASTattB or pUASTattB were linearized with EcoRI-Xhol to enable the insertion of transgenes via Gibson assembly (HiFi DNA Assembly, NEB).
  • V5-tagged, fluorophore-dead GFP (smGdP::V5; to enable downstream experiments with GFP based reporters together with this line) was amplified from pJFRC206 (with primers smGFP::v5_hifi_F/R from Addgene #63168, (Nern et al., 2015)); human wild-type and A53T mutant a- synuclein coding sequence was custom synthesized (IDT, sequences below) and tau[P301L] 0N4R amplified from previously reported constructs with tau_hifi_F/R (Zhou et al., 2017).
  • a-synuclein NACore 68-GAVVTGVTAVA-78 and PHF6 tau 306-VQIVYK-311 deletions were introduced with site- directed mutagenesis with asyn_del_NAC_F/R and tau_del_VQIVYK_F/R (von Bergen et al., 2000; Rodriguez et al., 2015).
  • nSyb-QF2w.JK22C was generated by microinjection of pattB-synaptobrevin-7ml-QFBDADMl-hsp70 (Addgene #46116) into theJK22C landing site (BestGene). Flies were maintained on standard corn meal and molasses food. For all experiments, male flies of control and experimental genotypes were raised in parallel on the same batch of food in a temperature and light-controlled incubator at 25°C and 12-hour light-dark cycle.
  • Electroretinograms were recorded as previously described (Slabbaert et al., 2016). Briefly, flies were immobilized on glass slides with 5 s fix UV glue (JML). Glass electrodes (borosilicate, 1.5 mm outer diameter, Hilgenberg) were filled with 3 M NaCI. One electrode was positioned in the thorax as a reference while another was placed on the fly eye for recording. Responses to repetitive light stimuli were measured using Axosope 10.7 (Molecular Devices) and analyzed with Clampfit 10.7 (Molecular Devices) and Igor Pro 6.37 (WaveMetrics).
  • ethoscope Profiling of sleep and activity behaviour of 5, 25 and 45 day old male flies was performed using the fly behaviour video recording platform ethoscope (Geissmann et al., 2017). Briefly, flies were sorted into glass tubes (65 mm length, 5 mm external and 3 mm internal diameter) with standard corn meal and molasses food on one end and closed with cotton wool on the other. Twenty individual animals per ethoscope were recorded for at least 4 consecutive days at 2 frames per second with infrared light in incubators at 25°C with 12 hour light-dark conditions. The position of each animal was saved at each time point in SQLite files and subsequently analysed with R (v3.6.3) and rethomics with adjusted R packages behavr, scopr and sleepr (vO.3.99).
  • the behaviour of individual flies was determined with the automatic behaviour annotator at a resolution of 10 s. Thus, in 10 s intervals flies were scored as moving or asleep. Sleep was defined following the 5-min rule, in which immobility bouts longer than 5 min were counted as sleep bouts, including the first 5 min. A fly was scored as immobile if the distance between 2 consecutive frames in the 10 s interval was less than 0.27 mm. Flies that died during the monitoring as well as the first day of recording due to habituation were excluded from further analysis.
  • the sleep amount per light condition (sleep_fraction_day) and per dark condition (sleep_fraction_night) were calculated.
  • the mean bout length as well as the number of sleep bouts were scored during the dark condition.
  • Morning and evening anticipation were calculated as described previously (Valadas et al., 2018).
  • the velocity_if_awake was assessed based on 10 s interval annotation 'moving'.
  • the cumulative distance within the 10 s interval was calculated and summed per 24 h to determine the travelled distance (total_distance) for each animal.
  • the means of the activity and sleep parameters were calculated for each genotype, scaled and hierarchically clustered based on correlation distance and average-linkage with the R pheatmap package.
  • EWCE Expression Weighted Celltype Enrichment
  • Drosophila genes up- or downregulated with a Iog2-fold change >2 or ⁇ 2 and a Benjamini-Hochberg corrected p- value ⁇ 0.05 in vulnerable vs. resilient neurons were mapped to human neuron types by converting them to their human orthologous genes with DIOPT at a minimum score of 5 (Hu et al., 2011).
  • AD vulnerability genes differential gene expression testing was carried out between vulnerable cell types (entorhinal cortex L2 and CAI neurons) and resilient ones (CA2, CA3 and dentate gyrus neurons) in the healthy human Franjic et al. 2022 dataset.
  • MAST was used based on log2(CPM+l) and number of genes as a covariate (Finak et al., 2015).
  • Resulting DEGs with Benjamini-Hochberg corrected p-value of ⁇ 0.05 and Iog2-fold change > 0.5 were intersected with human orthologs of Drosophila tau vulnerability/resilience genes, converted with DIOPT at a minimum score of 5 (Hu et al., 2011).
  • mutants that were predicted or confirmed null alleles, such as deletions encompassing large portions of the gene as well as MiMIC, Trojan Gal4 and CRIMICS lines that had insertions in an early exon or in an early intron in the same orientation as the target gene (Venken et al., 2011; Diao et al., 2015; Lee et al.; 2018). All mutants had white eyes, therefore not interfering with ERG recordings.
  • ERGs were recorded at 25 and 45 days of age and analyzed in R (v3.6.3), with the readABF (vl.0.2), tidyverse (vl.3.1) and multcomp (vl.4.19) packages.
  • nSyb-QF2w > mini-w+ and nSyb-QF2w > QUAStau[P301L] flies were aged 45 +/- 1 day of age in parallel and their brains including lamina and retina were dissected.
  • Library preparations for single cell RNAseq was performed using Chromium v3.1 Next GEM chemistry (lOx Genomics). However, for single cell encapsulation, custom microfluidics as described previously (De Rop et al., 2022) was used instead of the standard lOx GEM generation chips.
  • the custom HyDrop chip provided an improved encapsulation efficiency of >75% and better control of droplet formation.
  • Cell count and viability (>96%) of the samples were assessed using FLUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems). Both genotypes of the same batch were processed on the same experimental day.
  • the workflow of the HyDrop chip allowed combining two lOx reactions into a single run (De Rop et al., 2022), which enabled us to target for 20 000 cell recovery per genotype and experimental date.
  • the rest of the single cell RNAseq libraries were prepared as per manufacturer's recommendations and detailed before. For quality assessment and cell number estimation of finished libraries we performed shallow sequencing on a NextSeq 2000 sequencer (Illumina).
  • Deeper sequencing (targeted sequencing saturation of 80%), were performed on the MGISEQ-2000 sequencing platform (MGI Hong Kong SAR sequencing facility).
  • the lOx libraries need to undergo a conversion step using MGIEasy Universal DNA Library Prep kit. Briefly, the final lOx sequencing libraries were circularized using the splint-ligation step and the circularized libraries were converted to single stranded DNA copies. DNA nanoballs were prepared from the circularized ssDNA using Rolling Circle Amplification (RCA). DNB libraries generated were then flown through the patterned flow cell of the MGISEQ.-2000RS High-Throughput Sequencing kit.
  • RCA Rolling Circle Amplification
  • a custom sequencing recipe of paired end reads of 28 bps (read 1), 100 bps (read 2) and 10 bps (index 1) was used for sequencing.
  • the 45 day dataset was jointly clustered with the 5 and 25 day datasets as detailed above.
  • Leiden resolution 3 was chosen as the base resolution and cluster labels were transferred to the 45 day cells if >80% of that cluster had a unique label in the 5 and 25 day dataset. Otherwise higher resolutions were explored to split the respective cluster.
  • Differential gene expression testing was carried out with DESeq2 as described above. Differential abundances were calculated based on cell-type fractions of the cell-types that were used for the P301L tau vulnerability classification at 5 and 25 days.

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Abstract

L'invention concerne le domaine de la neurodégénérescence, plus particulièrement la neurotoxicité de Tau. L'invention identifie des gènes dont l'expression réduite supprime les effets pathologiques induits par Tau. Ces suppresseurs de Tau sont fournis pour une utilisation en tant que médicament en général, et pour le traitement ou l'inhibition de la progression de tauopathies ou de symptômes de tauopathies en particulier.
EP24702539.8A 2023-01-30 2024-01-30 Suppresseurs de tauopathies Pending EP4658281A1 (fr)

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EP23153997 2023-01-30
PCT/EP2024/052131 WO2024160756A1 (fr) 2023-01-30 2024-01-30 Suppresseurs de tauopathies

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Family Cites Families (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3756313B2 (ja) 1997-03-07 2006-03-15 武 今西 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体
JP4236812B2 (ja) 1997-09-12 2009-03-11 エクシコン エ／エス オリゴヌクレオチド類似体
ES2234563T5 (es) 1999-02-12 2018-01-17 Daiichi Sankyo Company, Limited Nuevos análogos de nucleósidos y oligonucleótidos
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
WO2004046160A2 (fr) 2002-11-18 2004-06-03 Santaris Pharma A/S Conception antisens
EP1452868A2 (fr) 2003-02-27 2004-09-01 Pepscan Systems B.V. Procédé pour sélectionner un médicament d'intérêt potentiel
US7341847B2 (en) 2003-04-02 2008-03-11 Agency For Science, Technology And Research Promoter construct for gene expression in neuronal cells
WO2007031091A2 (fr) 2005-09-15 2007-03-22 Santaris Pharma A/S Composes antagonistes d'arn de modulation de l'expression de p21 ras
WO2007090071A2 (fr) 2006-01-27 2007-08-09 Isis Pharmaceuticals, Inc. Analogues d'acides nucleiques bicycliques modifies en position 6
US7666854B2 (en) 2006-05-11 2010-02-23 Isis Pharmaceuticals, Inc. Bis-modified bicyclic nucleic acid analogs
AU2007249349B2 (en) 2006-05-11 2012-03-08 Isis Pharmaceuticals, Inc. 5'-Modified bicyclic nucleic acid analogs
CA2688321A1 (fr) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. Analogues d'acides nucleiques bicycliques pontes par aminomethylene n-substitue
DK2173760T4 (en) 2007-06-08 2016-02-08 Isis Pharmaceuticals Inc Carbocyclic bicyclic nukleinsyreanaloge
AU2008272918B2 (en) 2007-07-05 2012-09-13 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
WO2009067647A1 (fr) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Analogues d'acide nucléique alpha-l-bicyclique carbocyclique
EP2653545A1 (fr) 2008-02-05 2013-10-23 Bicycle Therapeutics Limited Procédés et compositions
WO2010033913A1 (fr) 2008-09-22 2010-03-25 Icb International, Inc. Anticorps, analogues et leurs utilisations
DK2356129T3 (da) 2008-09-24 2013-05-13 Isis Pharmaceuticals Inc Substituerede alpha-L-bicykliske nukleosider
EP2580228B1 (fr) 2010-06-08 2016-03-23 Ionis Pharmaceuticals, Inc. 2' amino- et 2' thio-nucléosides bicycliques substitués et composés oligomères préparés à partir de ces derniers
EP4036234A1 (fr) * 2017-07-17 2022-08-03 Vib Vzw Méthode de dépistage des inhibiteurs de la synaptogyrine-3
KR20220140176A (ko) * 2021-04-09 2022-10-18 경북대학교 산학협력단 혈관신생 촉진용 약학적 조성물

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