EP4669750A2 - Inhibitoren der synaptogyrin-3-expression - Google Patents

Inhibitoren der synaptogyrin-3-expression

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Publication number
EP4669750A2
EP4669750A2 EP24706719.2A EP24706719A EP4669750A2 EP 4669750 A2 EP4669750 A2 EP 4669750A2 EP 24706719 A EP24706719 A EP 24706719A EP 4669750 A2 EP4669750 A2 EP 4669750A2
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EP
European Patent Office
Prior art keywords
oligonucleotide
seq
aspects
present disclosure
antisense
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
EP24706719.2A
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English (en)
French (fr)
Inventor
Ana Rita SANTOS
Patrik Verstreken
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.)
Jay Therapeutics
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
Original Assignee
Jay Therapeutics
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
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Application filed by Jay Therapeutics, Katholieke Universiteit Leuven, Vlaams Instituut voor Biotechnologie VIB filed Critical Jay Therapeutics
Publication of EP4669750A2 publication Critical patent/EP4669750A2/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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 regions within the synaptogyrin-3 RNA sequence that are targetable by oligonucleotide inhibitors such as siRNA molecules.
  • the synaptogyrin-3 inhibitors disclosed herein 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). Under physiological conditions, Tau is expressed in neurons and is bound to axonal microtubules.
  • Synaptogyrin-3 mediates the association of Tau with synaptic vesicles in vitro and in vivo (W02019/016123).
  • Reduction of Drosophila Synaptogyrin or murine Synaptogyrin-3 levels in neurons from fly and mouse models of tauopathy reduced the association of Tau with synaptic vesicles, and subsequently rescued Tau-induced defects in vesicle mobility and neurotransmitter release.
  • the inventors of current application have found that some subsequences within the Synaptogyrin-3 mRNA transcript are significantly more accessible for oligonucleotides such as RNAi molecules and therefore are preferred target regions for designing oligonucleotides suitable for or capable of reducing the expression and/or activity of Synaptogyrin-3.
  • the borders of those identified target regions were determined by transcript-walking.
  • an oligonucleotide of 10 to 70 nucleotides in length comprising a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length, the contiguous nucleotide sequence being at least 90% complementary to an equal length portion of a target region within the Synaptogyrin-3 transcript as depicted in SEQ ID No. 1, wherein the target region is comprised between nucleobase 205 and 265, 255 and 348, 338 and 387, 369 and 433, 422 and 531, 603 and 656, 641 and 714, 717 and 768, 1150 and 1600, 1743 and 1868 or between nucleobase 1865 and 2026 of SEQ. ID No.
  • the oligonucleotide can bind to the synaptogyrin-3 mRNA transcript as set forth in SEQ ID No. 1. In another embodiment, said binding of the oligonucleotide to said synaptogyrin-3 mRNA transcript can reduce the expression and/or activity of synaptogyrin-3.
  • the oligonucleotide is a double stranded nucleic acid molecule, more particularly an RNAi molecule or an RNA duplex, even more particularly the RNAi molecule is an siRNA, a divalent siRNA or a shRNA.
  • the oligonucleotide is a single stranded nucleic acid molecule, more particularly the antisense portion of an RNAi molecule.
  • the sense and/or antisense strand of the oligonucleotide of the application comprises between 15 and 25 nucleotides in length, more particularly the antisense strand is 21 nucleotides in length.
  • the oligonucleotide of the application comprises at least one or at least two single stranded nucleotide overhang.
  • the oligonucleotide of the application is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 99% complementary or fully complementary (100% complementary) to an equal length portion of a target region selected from the group consisting of SEQ ID No. 2-4, 6-15, 17-18, 20-21, 23-25, 27-29, 31-41, 43- 45, 47-49, 51-57, 70-81, and 83-92.
  • the oligonucleotide of current application comprising a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length that shows at least 90% sequence identity to any of SEQ. ID No. 172-249.
  • the oligonucleotide of the application 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. Even more particularly, all oligonucleosides are modified with a phosphorothioate internucleoside linkage or with a 2'-O-methyl group.
  • an antisense oligonucleotide or RNAi molecule capable of reducing the level of synaptogyrin-3 mRNA, synaptogyrin-3 protein, synaptogyrin-3 activity, or a combination thereof in a cell by least 15% compared to a control situation in the absence of said antisense or RNAi molecule, wherein the antisense oligonucleotide or RNAi molecule nucleic acid sequence targets a subsequence of an mRNA encoding synaptogyrin-3 selected from the group consisting of SEQ ID NO. 5, 16, 19, 22, 26, 30, 42, 46, 50, 58, 59, 69, 82 and 93.
  • the oligonucleotides of the application including the antisense oligonucleotides and RNAi molecules herein disclosed, are provided a therapeutic, more particularly to treat or reduce the symptoms of tauopathies. Therefore, pharmaceutical composition comprising the oligonucleotides of the application are provided as well as methods to treat tauopathies in a subject in need thereof, wherein the methods comprising the step of administering any of the antisense oligonucleotide and RNAi molecules herein provided.
  • the oligonucleotide of the applications are also provided for use as a medicament, more particularly for use in treating or inhibiting progression of a tauopathic disorder or for use in treating or inhibiting a symptom of a tauopathic disorder.
  • Figure 1 illustrates the structure of an exemplary siRNA molecule of the application.
  • the molecule follows a 21/19 bp structure, where the sense strand is 19 nucleotides and the antisense strand is 21 nucleotides with 2 nucleotides overhanging on the 3' end.
  • the strands are 2'0Me (green)/2"F (blue) modified.
  • the red bar indicates a phosphorothioate modification, and "N" is complementary to the target mRNA.
  • Figure 2 A-B show Modification scheme 1 and 2 respectively and illustrate alternative architectures of the oligonucleotides of the present disclosure.
  • Figure 3 shows the different target regions in the synaptogyrin-3 mRNA transcript that have been identified herein, their start (5') and end (3') position according to SEQ ID No. 1, their sequence, as well as the sequences of the sense and antisense strands of the siRNA molecules herein disclosed.
  • Figure 4 shows the extended target regions within the synaptogyrin-3 mRNA transcript.
  • the current invention relates to oligonucleotides (“oligonucleotides of the present disclosure”) that specifically bind to Synaptogyrin-3 RNA and reduce the expression of Synaptogyrin-3, e.g. through antisense or RNAi technology.
  • the oligonucleotides of the present disclosure reduce Synaptogyrin-3 expression levels, Synaptopgyrin-3 activity (e.g., dopamine transporter activity), Synaptogyrin-3-mediated exocytosis, or a combination thereof.
  • the oligonucleotide of the present disclosure is 10 to 50, 10 to 40, or of 10 to 30 nucleotides in length, and comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length which are at least 90% complementary to an equal portion of a target region within the Synaptogyrin-3 transcript as depicted in SEQ. ID No. 1.
  • nucleobase 1 is between nucleobase 205 and 265, between nucleobase 255 and 348, between nucleobase 338 and 387, between nucleobase 369 and 433, between nucleobase 422 and 531, between nucleobase 603 and 656, between nucleobase 641 and 714, between nucleobase 717 and 768, between nucleobase 1150 and 1600, between nucleobase 1743 and 1868, or between nucleobase 1865 and 2026 of SEQ ID No. 1, wherein the endpoints are included.
  • the oligonucleotide of the present disclosure is 10 to 50, 10 to 40, or of 10 to 30 nucleotides in length, and comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length which are at least 90% complementary to an equal length portion of a target region within human Synaptogyrin-3, wherein the target region is selected from the list consisting of SEQ ID No. 5, 16, 19, 22, 26, 30, 42, 46, 50, 58, 59, 82 and 93.
  • the oligonucleotide of the present disclosure is complementary (full or partially complementary) to a target region of Synaptogyrin-3 selected from the group consisting of SEQ ID No. 2-4, SEQ ID No. 6-15, SEQ ID No. 17-18, SEQ ID No. 20-21, SEQ ID No. 23-25, SEQ ID No. 27-29, SEQ ID No. 31-41, SEQ ID No. 43-45, SEQ ID No. 47-49, SEQ ID No. 51-57, SEQ ID No. 70-81 and SEQ ID No. 83- 92.
  • the oligonucleotide of the application comprises or consists of 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides in length. In other aspects, the oligonucleotide of the application comprises or consists of 19, 20, 21 or more nucleotides in length and comprises or consists of the sequence selected from the list consisting of SEQ. ID No. 94-249, more particularly of SEQ ID No. 172-249.
  • the present disclosure also provides methods of treatment comprising the administration of the oligonucleotides of the present disclosure, or a combination thereof, to a subject in need thereof. Also provides are pharmaceutical compositions, pharmaceutical formulations, and kits and articles of manufacture comprising the oligonucleotides of the present disclosure. Also provided are methods of manufacture of the oligonucleotides of the present disclosure.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence”, is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
  • oligonucleotide of the present disclosure reduces expression the Syngr-3 transcript in a cell following administration of an oligonucleotide of the present disclosure by at least about 60%, it is implied that the Syngr-3 expression levels are reduced by a range of 50% to 70%.
  • nucleic acids refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
  • percent sequence identity or “percent identity” between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. gaps) that must be introduced for optimal alignment of the two sequences.
  • a matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.
  • sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402).
  • Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • BLAST-2 Altschul et al., 1996, Methods in Enzymology, 266:460-480
  • ALIGN ALIGN-2
  • Megalign Megalign
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch (J.
  • Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM 250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM 120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software.
  • the default parameters of the alignment software are used.
  • the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments.
  • One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org.
  • Another suitable program is MUSCLE, available from www.drive5.com/muscle/.
  • ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI (European Bioinformatics Institute).
  • the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity.
  • percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • nucleic acid molecule of the invention and “oligonucleotide of the present disclosure” and grammatical variants thereof are used interchangeably.
  • SEQ ID No. X refers to a biological sequence consisting of the sequence of nucleotides given in the SEQ. ID No. X.
  • SEQ ID No. X is interchangeable with SEQ ID NO: X.
  • SEQ ID No. 2-4 this is identical to a group consisting of SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
  • the target nucleic acid of the invention is a nucleic acid, e.g., an mRNA, encoding Synaptogyrin-3, more particularly human Synaptogyrin-3.
  • Synaptogyrin3 "Synaptogyrin3", “Synaptogyrin3”, “synaptogyrin-3”, “synaptogyrin- 3", “Syngr3", “Syngr-3", “SYNGR3” or “SYNGR-3” are interchangeably used and refer herein to Synaptogyrin-3 transcript if not otherwise specified.
  • the human nucleic acid sequence of Synaptogyrin-3 (hSyngr-3) is set forth in SEQ ID NO: 1; however also within the scope of the invention are nucleic acid sequence variants of Synaptogyin-3 as may exist due to allelic variation, e.g., a mRNA encoding a Synaptogyrin-3 allelic variant. Such variations are defined herein as "allelic variants of SEQ ID NO: 1".
  • 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)).
  • allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present disclosure.
  • non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.
  • the synaptogyrin-3 variant is a splice variant.
  • the synaptogyrin-3 variant is a post-translationally modified variant.
  • the synaptogyrin- 3 variant is a mutant synaptogyrin-3, e.g., a mutant comprising at least one nucleotide point mutation, deletion, or insertion.
  • the mutation is a silent mutation.
  • the synaptogyrin-3 variant is a mutant protein comprising at least one amino acid substitution, deletion, or insertion. In some aspects, the synaptogyrin-3 variant is a loss of function variant. In some aspects, the synaptogyrin-3 variant is a gain of function variant.
  • Specific to synaptogyrin-3 is referring to the fact that the nucleic acid molecule or oligonucleotide of the invention is acting at the level of synaptogyrin-3 and not at the level of another transcript. Specificity can be ascertained by e.g. determining the expression level of closely related RNA sequences.
  • 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 the synaptogyrin-3 transcript level in the presence of an antisense or RNAi molecule compared to the synaptogyrin-3 transcript level in the absence of said antisense or RNAi molecule.
  • 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 application discloses nucleic acid molecules, more particularly oligonucleotides, that comprise a sequence complementary (fully or partially) to a region of an mRNA encoding human Synaptogyrin-3, e.g. a mature mRNA as depicted in SEQ ID No. 1, or an mRNA encoding an allelic variant or isoforms thereof (e.g., any of the variants and isoform disclosed in the UniProtKB/Swiss-Prot 043761 entry).
  • SEQ. ID No. 1 represent the mature mRNA sequence of the human Synaptogyrin-3 gene (Gene ID: 9143; Sequence ID: NM_004209.6).
  • the mRNA is 2026 bp long, comprises 4 exons and encodes the human SYNAPTOGYRIN-3 protein (UniProtKB/Swiss-Prot: 043761).
  • the target mRNA is a pre- mRNA or a splice variant of a pre-mRNA.
  • the target sequence comprises an exon, an intron, or a combination thereof.
  • Synaptogyrin-3 coding sequence is underlined in the sequence below. "
  • SEQ ID No. 1 (Synaptogyrin-3 mRNA)
  • the nucleic acid molecule or the oligonucleotide of the present disclosure comprises of or consists of 1 oligonucleotide (e.g., an ASO oligomer or a shRNA).
  • the nucleic acid molecule or the oligonucleotide of the present disclosure comprises or consists of 2 oligonucleotides (e.g., a siRNA).
  • the 2 oligonucleotides are a sense oligonucleotide and an antisense oligonucleotide.
  • the sense oligonucleotide and the antisense oligonucleotide are connected by a loop.
  • 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).
  • oligomer or "oligonucleotide” in the context of the present disclosure are used interchangeably and refer to a molecule formed by covalent linkage of two or more nucleotides.
  • a single nucleotide (unit) can also be referred to as a monomer or unit.
  • the present disclosure provides a derivative of an oligonucleotide of the present disclosure which is a conjugate, e.g., a GalNAc conjugate.
  • 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 oligonucleotide (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 (e.g. nucleotides) in the oligomer (e.g. oligonucleotide), and multiplying by 100.
  • 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.
  • the nucleic acid molecule described above or the contiguous nucleotide sequence thereof comprises or consists of less than 60 nucleotides, less than 59 nucleotides, less than 58 nucleotides, less than 57 nucleotides, less than 56 nucleotides, less than 55 nucleotides, less than 54 nucleotides, less than 53 nucleotides, less than 52 nucleotides, less than 51 nucleotides, less than 50 nucleotides, less than 49 nucleotides, less than 48 nucleotides, less than 47 nucleotides, less than 46 nucleotides, less than 45 nucleotides, less than 44 nucleotides, less than 43 nucleotides, less than 42 nucleotides, less than 41 nucleotides, less than 40 nucleotides, less than 39 nucleotides, less than 38 nucleotides, less than 37 nucleotides, less than 36 nucle
  • any range given in current application 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.
  • the contiguous nucleotide sequence comprises or consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 1 , 28, 29 or 30 contiguous nucleotides in length.
  • the nucleic acid molecule of the invention is 14 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 15 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 16 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 17 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 18 nucleotides in length.
  • the nucleic acid molecule of the invention is 19 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 20 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 21 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 22 nucleotides in length. In some aspects, the nucleic acid molecule of the invention is 24 nucleotides in length.
  • the nucleic acid molecule(s) is typically for modulating the expression of Synaptogyrin-3 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 oligonucleotides of the application are provided as being capable of reducing the level of Synaptogyrin-3 mRNA transcript (and thus indirectly SYNGR3 protein) in a cell, wherein the reduction is determined by comparison to a control situation, i.e. the level of Synaptogyrin-3 mRNA transcript in the same cell or same cell type grown in the same conditions but in the absence of the oligonucleotide of the application.
  • the additional nucleotides are complementary to the contiguous nucleotide sequence and are capable of forming a stem loop (hairpin) structure by hybridizing to the contiguous nucleotide sequence.
  • the additional nucleotides are 1 to 5 phosphodiester linked nucleotides.
  • all the nucleotides of the oligonucleotide form the contiguous nucleotide sequence.
  • RNAi agent or "RNA interference (RNAi) molecule” refers to any molecule inhibiting RNA expression or translation via the RNA reducing silencing complex (RISC) in a cell's cytoplasm, where the RNAi molecule interacts with the catalytic RISC component argonaut.
  • RISC RNA reducing silencing complex
  • a small interfering RNA (siRNA) is typically a double-stranded RNA complex comprising a passenger (sense) and a guide (antisense) oligonucleotide (strand), which when administered to a cell, results in the incorporation of the guide (antisense) strand into the RISC complex (si R ISC) resulting in the RISC associated inhibition of translation or degradation of complementary RNA target nucleic acids in the cell.
  • the sense strand is also referred to as the passenger strand, and the antisense strand as the guide strand.
  • a small hairpin RNA is a single nucleic acid molecule which forms a stem loop (hairpin) structure that is able to degrade mRNA via RISC.
  • RNAi nucleic acid molecules may be synthesized chemically (typical for siRNA complexes) or by in vitro transcription, or expressed from a vector.
  • shRNA molecules are generally between 40 and 70 nucleotides in length, such as between 45 and 65 nucleotides in length, such as 50 and 60 nucleotides in length, and interacts with the endonuclease known as Dicer which is believed to processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs which are then incorporated into an RNA-induced silencing complex (RISC).
  • Dicer the endonuclease known as Dicer which is believed to processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs which are then incorporated into an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the guide (antisense) strand of an siRNA (or antisense region of a shRNA) is 17-25 nucleotide in length, such as 19-23 nucleotides in length and complementary to the target nucleic acid or target sequence.
  • RNAi may be mediated by longer dsRNA substrates which are processed into siRNAs within the cell (a process which is thought to involve the dsRNA endonuclease DICER), such as miRNAs.
  • the nucleic acid molecule of the invention or the oligonucleotide of the disclosure is an antisense oligonucleotide (ASO), such as single stranded antisense oligonucleotide, such as a high affinity modified antisense oligonucleotide interacting with RNase H.
  • ASO antisense oligonucleotide
  • single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than 50% across of the full length of the oligonucleotide.
  • the single stranded antisense oligonucleotide of the invention does not contain RNA nucleosides, since this will decrease nuclease resistance.
  • the antisense oligonucleotide of the invention comprises one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides.
  • the nucleosides which are not modified are DNA nucleosides.
  • the oligonucleotide e.g. the therapeutic antisense oligonucleotide, shRNA or siRNA
  • the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack.
  • modified internucleoside linkage is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. Increased resistance of the oligonucleotide towards nucleases compared to a phosphodiester linkage is particular advantage for therapeutic oligonucleotides.
  • Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art.
  • SVPD snake venom phosphodiesterase
  • Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages.
  • At least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.
  • all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof are nuclease resistant internucleoside linkages.
  • nucleosides which link the oligonucleotide of the invention to a non- nucleotide functional group, such as a conjugate may be phosphodiester.
  • the modified internucleoside linkage is phosphorothioate.
  • all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof are phosphorothioate.
  • the use of fully phosphorothioate modified oligonucleotides or contiguous nucleotide sequences is often used in antisense oligonucleotides, although in siRNAs partial phosphorothioate modifications may be preferred as fully phosphorothioate modifications have been reported to limit RNAi activity, particularly when used in the guide (antisense) strand.
  • Phosphorothioate modifications may be incorporated into the 5' and 3' ends of an antisense strand of a siRNA without unduly limiting RNAi activity.
  • the RNAi molecules of the invention comprise one or more phosphorothioate internucleoside linkages.
  • phosphorothioate internucleoside linkages may reduce the nuclease cleavage in RICS and it is therefore advantageous that not all internucleoside linkages are modified.
  • Phosphorothioate internucleoside linkages can advantageously be place in the 3' and/or 5' end of the RNAi molecule, in particular in part of the molecule that is not complementary to the target nucleic acid (e.g. the sense strand or passenger strand in an siRNA molecule).
  • the region of the RNAi molecule that is complementary to the target nucleic acid e.g.
  • the antisense or guide strand in a siRNA molecule may however also be modified in the first 2 to 3 internucleoside linkages in the 3' and/or 5' terminal.
  • the oligonucleotides of the invention may be chemically modified by incorporating high affinity nucleosides such as 2' sugar modified nucleosides, such as 2' -4' bicyclic ribose modified nucleosides, including LNA and cET or 2' substituted modifications like of 2'-O-alkyl-RNA, 2'-O- methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA.
  • 2' sugar modified nucleosides such as 2' -4' bicyclic ribose modified nucleosides, including LNA and cET or 2' substituted modifications like
  • siRNA complexes See for example WO 2002/044321 which discloses 2'-O-Methyl modified siRNAs, W02004083430 which discloses the use of LNA nucleosides in siRNA complexes, known as siLNAs, and W02007107162 which discloses the use of discontinuous passenger strands in siRNA such as siLNA complexes.
  • the oligonucleotides of the invention may comprise one or more of the above described chemically modified sugar nucleosides and may comprise one or more of the above described phosphorothioate internucleoside linkages.
  • 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' dUdU 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.
  • any of the oligonucleotides of present disclosure are provided, wherein the oligonucleotide is an RNAi molecule such as a siRNA, shRNA or di-siRNA comprising at least one nucleotide variant (e.g., an LNA unit).
  • the oligonucleotide of the present disclosure further comprises at least one non-nucleotide or non-polynucleotide moiety covalently (e.g., a GalNac moiety) attached to said oligonucleotide directly or via a linker positioned between the contiguous nucleotide sequence and the non-nucleotide or non-polynucleotide moiety.
  • the present disclosure provides oligonucleotides of the present disclosure comprising 16 to 22 contiguous oligonucleotides in length comprising a contiguous sequence of 16 nucleotides in length which is 100% complementary to a human synaptogyrin-3 target sequence selected from the group consisting of SEQ ID No. 2-93, wherein oligonucleotide is an RNAi molecule such as a siRNA, shRNA or di-siRNA comprising at least one nucleotide variant (e.g., an LNA unit), and wherein the RNAi molecule targets the synaptogyrin-3 transcript as set forth in SEQ ID No. 1.
  • RNAi molecule such as a siRNA, shRNA or di-siRNA comprising at least one nucleotide variant (e.g., an LNA unit)
  • the oligonucleotide of the present disclosure comprises, consists, or consists essentially of an RNAi molecule binding to the human synaptogyrin-3 transcript as set forth in SEQ. ID No. 1, the RNAi molecule comprising one or more sequences selected from the group consisting of SEQ ID No. 94- 249, more particularly of SEQ ID No. 172-249.
  • the oligonucleotide of the present disclosure comprises an RNAi molecule comprising one or more sequences selected from the group consisting of SEQ ID No. 94-249, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleobase substitutions.
  • the oligonucleotide of the present disclosure comprises an RNAi molecule comprising a sequence which is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID No. 94-249.
  • the oligonucleotide of the present disclosure comprises an RNAi molecule comprising a sequence which is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID No. 172-249.
  • the oligonucleotide of the present disclosure comprises a sequence that overlaps with 9, 10, 11, 12, 13, 14, 15, or 16 nucleobase subsequence from a sequence selected from the group consisting of SEQ ID No. 172-249.
  • the oligonucleotide of the present disclosure comprises at least one non-cleavable internucleoside linkage, e.g., a phosphorothioate linkage.
  • all the internucleoside linkages in an oligonucleotide of the present disclosure are non-cleavable, e.g., phosphorothioates linkages.
  • the non-cleavable internucleoside linkages e.g., a phosphorothioate linkages
  • the oligonucleotide of the present disclosure comprises nucleotide analogues. In some aspects, the oligonucleotide of the present disclosure comprises affinity enhancing nucleotide analogues.
  • the nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides independently or dependently selected from the group consisting of 2'-O-alkyl-RNA units, 2'-0Me-RNA units, 2'-amino-DNA units, and 2'-fluoro-DNA units.
  • the oligonucleotide of the present disclosure is an siRNA, di-siRNA, shRNA, RNA duplex or the antisense strand from an RNA duplex.
  • the oligonucleotide of the present disclosure comprises one or more locked nucleic acids (LNA).
  • the LNA oligonucleotide comprises a wing on each side (5' and 3') of 2 to 4 nucleotide analogues, preferably LNA analogues.
  • every LNA unit in the oligonucleotide is a beta-D-oxy LNA unit or every LNA unit in the oligonucleotide is an alpha- L-oxy-LNA unit.
  • the sequence of the oligonucleotide comprises at least one phosphorothioate, phosphorodithioate, or boranophosphate internucleoside linkage.
  • one or more of the internucleoside linkages comprises a chiral center in the R conformation and/or in the S conformation.
  • the oligonucleotide comprising an LNA can form a duplex with a human synaptogyrin-3 target sequence selected from the group consisting of SEQ.
  • the oligonucleotide of the present disclosure is an RNAi molecule conjugate comprising an RNAi molecule covalently attached to non-nucleotide or non-polynucleotide moiety, which can be attached to the 5' end, 3' end, or both.
  • the non-nucleotide or non-polynucleotide moiety is a targeting moiety that is attached to the 5' -end or to the 3' -end of the RNAi molecule.
  • the targeting moiety is linked to the RNAi molecule via a linker.
  • the targeting moiety comprises a carbohydrate conjugate moiety comprising a carbohydrate selected from the group consisting of galactose, lactose, N-acetylgalactosamine (GalNAc), mannose, mannose-6-phosphate, and combinations thereof.
  • the carbohydrate conjugate moiety is not a linear carbohydrate polymer.
  • the carbohydrate conjugate moiety is a carbohydrate group comprising 1, 2, 3, or 4 carbohydrate moieties.
  • the carbohydrate moieties are identical or non-identical.
  • the carbohydrate conjugate moiety comprises at least one asialoglycoprotein receptor targeting conjugate moiety.
  • the asialoglycoprotein receptor targeting conjugate moiety comprises a monovalent, divalent, trivalent, or tetravalent GalNAc cluster.
  • each GalNAc in the GalNAc cluster is attached to a branch point group via a spacer.
  • the branch point group comprises di-lysine.
  • the spacer comprises a PEG spacer.
  • the linker comprises a C6 to C12 amino alkyl group or a biocleavable phosphate nucleotide linker comprising between 1 to 6 nucleotides.
  • the targeting moiety targets the oligonucleotide of the present disclosure to the central nervous system (CNS). In some aspects, the targeting moiety allow the oligonucleotide of the present disclosure to permeate through the blood-brain-barrier (BBB).
  • CNS central nervous system
  • BBB blood-brain-barrier
  • the oligonucleotide of the application more particularly the RNAi molecule of the application is a double stranded nucleic acid.
  • the RNAi molecule is a siRNA.
  • the RNAi molecule of the present disclosure is a di-siRNA.
  • the RNAi molecule of the present disclosure is a shRNA.
  • the antisense oligomer portion of an oligonucleotide of the present disclosure is an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the antisense oligomer portion of an oligonucleotide of the present disclosure is multimeric.
  • the antisense oligomer portion of an oligonucleotide of the present disclosure is a multimeric ASO, e.g., it can comprise several concatenated antisense oligomers of the present disclosure.
  • the antisense oligomer portion of an oligonucleotide of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 concatenated antisense oligomers.
  • the concatenated oligomers are connected via cleavable linkers interposed between each ASO unit in the ASO multimer.
  • the antisense oligomer portion of an oligonucleotide of the present disclosure can target a target region in the synaptogyrin-3 mRNA selected from the group consisting of SEQ ID No. 2- 93.
  • the antisense oligomer portion of an oligonucleotide of the present disclosure comprises a complementarity region that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of a target region in the synaptogyrin-3 mRNA selected from the group consisting of SEQ. ID No. 2-93.
  • the oligonucleotides of the present disclosure are capable of modulating the expression of the synaptogyrin-3 gene by specifically targeting a targeting region in a synaptogyrin-3 RNA, e.g., a mRNA.
  • the oligonucleotide of the present disclosure is capable of down-regulating expression of the synaptogyrin-3 gene by binding to such target region.
  • the oligonucleotide of the present disclosure can affect (reduce or inhibit) the expression of synaptogyrin-3, e.g., in a mammalian subject such a human, by binding to a specific target region in a synaptogyrin-3 RNA, e.g., an mRNA.
  • the oligonucleotide of the present disclosure can affect the expression of synaptogyrin-3 in a human cell, by binding to a specific target region in a synaptogyrin-3 RNA, e.g., an mRNA.
  • the RNA is an mRNA, such as pre-mRNA.
  • the RNA is a mature mRNA.
  • the oligonucleotide according to the present disclosure is preferably capable of hybridizing to the target nucleic acid.
  • the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 5' end of a synaptorgyrin-3 target region comprising or consisting of a sequence set forth in SEQ ID No. 2- 93. In some aspects, the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 3' end of a synaptorgyrin-3 target region comprising or consisting of a sequence set forth in SEQ ID No. 2-93.
  • the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 5' end and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 3' end of a synaptogyrin-3 target region comprising or consisting of a sequence set forth in SEQ ID No. 2-93.
  • the extended target region overlaps with 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of a synaptogyrin-3 target region comprising or consisting of a sequences set forth in SEQ ID No. 2-93.
  • the nucleotides extending beyond the 5' end and/or the 3' end of a sequence set forth in SEQ ID No. 2-93 is complementary (partially or fully complementary) to a corresponding sequence in the mRNA transcript of SEQ ID No. 1.
  • the present disclosure also provides antisense oligonucleotides that are complementary, e.g., fully complementary, to these target sequences.
  • the present disclosure provides a target sequence comprising a 21-mer sequence selected from SEQ ID No.
  • a target sequence comprising (i) a sequence selected from the group consisting of SEQ. ID No.
  • the additional 5' and/or 3' nucleotides are complementary (partially or fully complementary) to a corresponding sequence in the mRNA transcript of SEQ ID No. 1.
  • the present disclosure also provides antisense oligonucleotides that are complementary, e.g., fully complementary, to these target sequences.
  • the target region comprises or consists of a corresponding target sequence region derived from the sequence of a mutant or allelic variant of a human synaptogyrin-3 gene encoding the mRNA transcript of SEQ ID No. 1.
  • the target region can be a subsequence present in another synaptogyrin-3 mRNA transcript variant encoding human synaptogyrin-3.
  • the target region comprises or consists of a corresponding target sequence region derived from the sequence of a paralog or ortholog of the human synaptogyrin-3 gene encoding the mRNA of SEQ ID No. 1.
  • the target region is within an exon. In some aspects, the target region comprises the junction between and intron and an exon.
  • the oligonucleotides of the present disclosure bind to the target nucleic acid (e.g., a subsequence of an mRNA transcript wherein the subsequence is selected from the group consisting of SEQ ID No. 2-93 and the effect on synaptogyrin-3 expression and/or activity level is at least about 10% to about 20% reduction in synaptogyrin-3 expression and/or activity level compared to the normal or control synaptogyrin-3 expression level (e.g., the synaptogyrin-3 expression level of a cell, animal or human treated with saline) and/or normal or control synaptogyrin-3 activity level (e.g.
  • the target nucleic acid e.g., a subsequence of an mRNA transcript wherein the subsequence is selected from the group consisting of SEQ ID No. 2-93
  • the effect on synaptogyrin-3 expression and/or activity level is at least about 10% to about 20% reduction
  • the reduction in synaptogyrin-3 expression and/or activity is at least about 10%, about least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% compared to the normal or control expression and/or activity level.
  • the reduction in synaptogyrin-3 expression and/or activity is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% compared to the normal or control synaptogyrin-3 expression and/or activity level.
  • the synaptogyrin-3 expression level and/or protein level and/or activity level after the administration of an oligonucleotide of the present disclosure is less than about 2%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, or less than about 80% of the synaptogyrin-3 expression level and/or protein level and/or activity level prior to the administration of an oligonucleotide of the present disclosure.
  • the synaptogyrin-3 expression level and/or protein level and/or activity level after the administration of an oligonucleotide of the present disclosure is about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20%, to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or about 75% to about 80% of the synaptogyrin-3 expression level and/or protein level and/or activity level prior to the administration of an oligonucleotide of the present disclosure.
  • the present disclosure therefore provides an in vitro or in vivo method of down-regulating or inhibiting the expression of synaptogyrin-3 protein and/or mRNA transcript in a cell which is expressing synaptogyrin-3 protein and/or mRNA, said method comprising administering an oligonucleotide of the present disclosure, e.g., as a pharmaceutical composition of the present disclosure to said cell to down- regulate or inhibit the expression of synaptogyrin-3 protein and/or mRNA in said cell.
  • the cell is a mammalian cell such as a human cell.
  • the oligonucleotides of the present disclosure can be multimers comprising, e.g., 2, 3, 4, 5, 6, or more concatenated oligonucleotides disclosed herein, which can optionally be connected by spacers or linkers comprising nucleotide or non-nucleotide units interposed between each oligonucleotide in the multimer.
  • the oligonucleotides of the present disclosure can comprise or consist of a contiguous nucleotide sequence of a total of at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 contiguous nucleotides in length.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 172, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No. 173, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 174, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 175, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 176, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 177, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 178, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 179, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 180, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 181, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 182, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 183, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No. 184, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 185, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 186, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 187, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 188, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 189, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 190, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 191, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 192, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 193, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 194, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 195, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 196, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 197, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 198, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No. 199, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 200, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 201, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 202, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 203, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 208, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 210, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 212, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 214, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 216, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 218, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 220, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 222, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 224, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 226, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No. 228, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 230, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 232, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer.ln some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 234, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 236, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 238, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 240, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ. ID No. 242, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No. 248, or a 12 to 21 contiguous nucleotides subsequence thereof, e.g., a 16-mer. In some aspects, the oligonucleotide of the present disclosure comprises a single stranded oligomer comprising an antisense sequence set forth in SEQ ID No.
  • the nucleic acid of the present disclosure is a duplex (e.g., a siRNA or an shRNA) comprising a sense strand and an antisense strand which respectively comprise or consist of the sense and antisense sequences set forth in SEQ ID No. 96 and 174.
  • the nucleic acid of the present disclosure is a duplex (e.g., a siRNA or an shRNA) comprising a sense strand and an antisense strand which respectively comprise or consist of the sense and antisense sequences set forth in SEQ ID No. 97 and 175.
  • the nucleic acid of the present disclosure is a duplex (e.g., a siRNA or an shRNA) comprising a sense strand and an antisense strand which respectively comprise or consist of the sense and antisense sequences set forth in SEQ. ID No. 98 and 176.
  • the nucleic acid of the present disclosure is a duplex (e.g., a siRNA or an shRNA) comprising a sense strand and an antisense strand which respectively comprise or consist of the sense and antisense sequences set forth in SEQ ID No. 99 and 177.
  • a gRNA or CRISPR gRNA provided herein has 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% complementary to an equal length of a target region of synaptogyrin-3 as depicted in SEQ ID No. 1, wherein the target region is selected from any of SEQ. ID No. 5, 16, 19, 22, 26, 30, 42, 46, 50, 58, 59, 69, 82 or 93, more particularly the target region is selected from any of SEQ ID No. 2-4, 6-15, 17-18, 20-21, 23-25, 27-29, 31-41, 43-45, 47-49, 51-57, 70-81, and 83-92.
  • the gRNA comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides in length comprising a sequence selected from the group consisting of SEQ ID No. 172-249.
  • 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.
  • 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' and OH group naturally found in DNA and RNA nucleosides.
  • Non-limiting examples of ring structure modifications are HNAs (hexitol nucleic acids) where the 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.
  • HNAs hexitol nucleic acids
  • UNA unlocked 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, and WO 2008/150729, all of which are herein incorporated by reference in their entireties.
  • 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.
  • all the nucleotides in the oligonucleotide of the present disclosure are nucleotide analogues, i.e., the oligonucleotide of the present disclosure (e.g., an ASO, siRNA, or shRNA) is fully modified.
  • the oligonucleotide of the present disclosure e.g., an ASO, siRNA, or shRNA
  • all the nucleotide analogues are the same.
  • some of the nucleotide analogues are different.
  • all nucleotides in an oligonucleotide of the present disclosure are 2' modified.
  • all nucleotides in an oligonucleotide of the present disclosure are 2'-Fluoride and 2'-O-Methyl nucleotides.
  • all nucleotides in an oligonucleotide of the present disclosure are 2'-Fluoride and 2'-O-methyl nucleotides in an alternating pattern.
  • all nucleotides in a duplex of the present disclosure are 2'-Fluoride and 2'-O- Methyl nucleotides in an alternating pattern, wherein all or substantially all of the 2'-Fluoride modified nucleotides in the sense strand are complementary to all or substantially all of the 2'-O-methyl modified nucleotides in the antisense strand.
  • a duplex oligonucleotide of the present disclosure e.g., siRNA, or shRNA
  • the nucleotide overhang is a dinucleotide overhang.
  • nucleic acid or oligonucleotide of the present disclosure comprises a modification motif set forth in FIGURE 2A or 2B.
  • a nucleic acid or oligonucleotide of the present disclosure comprises a modification motif (e.g., the pattern of distribution of nucleotide analogs along the sense and antisense sequences, internucleoside linkages, conjugate moieties, etc.) disclosed in U.S. Pat. Nos. 8,110,674; 8,420,799; 8,809,516; 9,222,091; 9,708,615; 10,273,477; 9,290,760; 10,233,448; or 9,796,974; U.S. Appl. Publ. No. 2018 and 0258427A1; or Int'l Publ. WO2018098328A1, all of which are herein incorporated by reference in their entireties.
  • a modification motif e.g., the pattern of distribution of nucleotide analogs along the sense and antisense sequences, internucleoside linkages, conjugate moieties, etc.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one phosphoryl DMI amidate diester internucleoside linkage (PN).
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one 8-oxo-deoxyadenosine.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one phosphoramidite internucleoside linkage.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one phosphoramidate internucleoside linkage.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one pseudouridine.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one isouridine. See, e.g., WO2022/099159 and WO2021/071858, which are herein incorporated by reference in their entireties.
  • a nucleic acid or oligonucleotide of the present disclosure comprises at least one glycol nucleic acid (GNA).
  • a nucleic acid or oligonucleotide of the present disclosure e.g., an ASO, siRNA, or shRNA
  • a nucleic acid or oligonucleotide of the present disclosure comprises a loop.
  • a nucleic acid or oligonucleotide of the present disclosure comprises a cleavable loop.
  • the oligonucleotide of the present disclosure is a conjugate, e.g., a GalNAc conjugate.
  • ASOs and RNAi molecules such as 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.
  • 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.
  • 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
  • the oligonucleotide(s) of the invention such as the RNAi molecule(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.
  • the present disclosure also provides a method for designing or manufacturing an oligonucleotide of the present disclosure (e.g., an ASO or a siRNA) capable of inhibiting a human synaptogyrin-3 (hSYNGR3) gene transcript and/or hSYNGR3 protein expression and/or activity in a cell, a tissue, or a subject, wherein the oligonucleotide of the present disclosure is complementary (partially or fully complementary) to any of the target regions of the application as described above.
  • the complementary sequence of the oligonucleotide of the present disclosure comprises or consists of a subsequence of a nucleotide sequence set forth in SEQ ID No.
  • the complementary sequence of the oligonucleotide of the present disclosure partially overlaps of a nucleotide sequence set for in SEQ ID No. 94-249, more particularly in SEQ ID No. 172-249.
  • the complementarity is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% complementary.
  • manufacturing refers to chemically synthesizing, e.g., using solid phase synthesis, an oligonucleotide of the present disclosure.
  • manufacturing further comprises chemically attaching or conjugating a moiety such a delivery moiety (e.g., a GalNAc moiety), and/or a targeting moiety.
  • a delivery moiety e.g., a GalNAc moiety
  • the present disclosure also provides a method of manufacturing an oligonucleotide of the present disclosure, the method comprising chemically synthesizing the oligonucleotide of the present disclosure using sequential solid phase oligonucleotide synthesis.
  • the present disclosure provides a method of manufacturing an 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 nonpolynucleotide moiety) covalently to the oligonucleotide disclosed herein.
  • the conjugate moiety e.g., a non-nucleotide or non-polynucleotide moiety, for example a carbohydrate conjugate moiety such as a GalNAc moiety
  • the conjugate moiety is attached to an oligonucleotide disclosed herein directly or via a linker positioned between the oligonucleotide sequence and the conjugate moiety.
  • the non-nucleotide or non-polynucleotide moiety is a liver targeting moiety that is attached to the 5' -end or to the 3' -end of an oligonucleotide disclosed herein.
  • the liver targeting moiety is linked to the oligonucleotide via a linker.
  • the liver targeting moiety comprises a carbohydrate conjugate moiety comprising a carbohydrate selected from the group consisting of galactose, lactose, N-acetylgalactosamine (GalNAc), mannose, mannose-6-phosphate, and combinations thereof.
  • the carbohydrate conjugate moiety is not a linear carbohydrate polymer.
  • the carbohydrate conjugate moiety is a carbohydrate group comprising 1, 2, 3, or 4 carbohydrate moieties. In some aspects, all the carbohydrate moieties are identical. In some aspects, at least one carbohydrate moiety is different (non-identical) with respect to the other carbohydrate moieties. In some aspects, the carbohydrate conjugate moiety comprises at least one asialoglycoprotein receptor targeting conjugate moiety. In some aspects, the asialoglycoprotein receptor targeting conjugate moiety comprises a monovalent, divalent, trivalent, or tetravalent GalNAc cluster. In some aspects, each GalNAc in the GalNAc cluster is attached to a branch point group via a spacer.
  • the branch point group comprises di-lysine.
  • the spacer comprises a PEG spacer.
  • the linker comprises a C6 to C12 amino alkyl group or a biocleavable phosphate nucleotide linker comprising between 1 to 6 nucleotides.
  • 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 oligonucleotide; and, (ii) adding by chemical synthesis or conjugation the conjugate moiety to the oligonucleotide 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 oligonucleotide 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 oligonucleotide; (ii) incorporating by chemical synthesis or conjugation at least one linker to the oligonucleotide 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 oligonucleotide or conjug
  • At least one linker is interposed between the oligonucleotide and a branching point;
  • at least one branching point is interposed between a linker and a conjugate moiety (e.g., a nonnucleotide 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
  • a conjugate moiety e.g., a non-nucleotide or non-polynucleotide moiety, such as a GalNAc moiety
  • 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 between about 2 and 100 nM, between about 5 and 500 nM, between about 20 and 750 nM, between about 0.05 and 10 pM, between about 1 and 500 pM, between about 2 and 750 pM, between about 0.01 and 1 mM, between about 0.5 and 10 mM or between about 50 and 300 mM 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, which is herein incorporated by reference in its entirety.
  • 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.
  • the present disclosure provides oligonucleotides that are capable of inhibiting the expression and/or activity of synaptogyrin-3 to reduce binding between synaptogyrin-3 and (the N-terminal sequence of) the tau protein. Therefore in a further aspect, any of the nucleic acid molecules or oligonucleotides described in current application is provided for use as a medicament. More particularly for use to treat tauopathies.
  • the present disclosure also provides methods to treatment, prevent, or ameliorate a symptom or sequelae of a disease or disorder disclosed herein comprising administering an effective of any of the nucleic acid molecules or oligonucleotides described herein, or a combination thereof, to a subject in need thereof.
  • 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 and 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.
  • 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
  • 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.
  • any of the nucleic acid molecules or oligonucleotides described herein is provided for use in (a method for) treating or inhibition progression of a tauopathic disorder wherein 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, subacute
  • any of the 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 present disclosure also provides a method of treating or inhibiting progression of a tauopathic disorder or treating or inhibiting a symptom of a tauopathic disorder in a subject in need thereof, the method comprising administering comprising administering an effective amount of an oligonucleotide of the present disclosure to the subject.
  • 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).
  • the present disclosure provides an in vitro method of reducing expression levels and/or activity of synaptogyrin-3 in a cell comprising administering an effective amount of an oligonucleotide of the present disclosure to the cell. Also provided is a method of reducing expression levels and/or activity of synaptogyrin-3 in a subject in need thereof comprising administering an effective amount of an oligonucleotide of the present disclosure to the subject. Also provided is method of reducing synaptogyrin-3 levels in a subject in need thereof comprising administering to said subject an effective amount of an oligonucleotide of the present disclosure.
  • the nucleic acid molecules or oligonucleotides of the present disclosure can also be used for diagnostic purposes.
  • 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).
  • In vivo imaging techniques of neurodegeneration have become available. Such techniques can clearly support the clinical diagnosis of neurodegenerative diseases in general and of tauopathies in particular.
  • In vivo diagnosis of 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 present disclosure provides nucleic acid molecules or oligonucleotides of the present disclosure which are conjugated to a detectable moiety, for example, a radiotracer, a fluorescent moiety (e.g., a fluorescent protein), or any detectable moiety known in the art.
  • a detectable moiety for example, a radiotracer, a fluorescent moiety (e.g., a fluorescent protein), or any detectable moiety known in the art.
  • methods for the diagnosis or prognosis of tauopathic disorders methods to monitor the efficacy of a treatment, methods to select a patient for treatment, or methods to select a subject for a clinical trial or to exclude a subject from a clinical trial comprising administering a nucleic acid molecule or oligonucleotides of the present disclosure.
  • oligonucleotides of the disclosure By using the oligonucleotides of the disclosure, inhibition of synaptogyrin-3 is obtained at the expression level.
  • the administration of an oligonucleotide of the present disclosure can reduce the level of mRNA encoding synaptogyrin-3, which in turn would result in a lower protein expression level of synaptogyrin-3.
  • such reduction of expression levels of synaptogyrin-3 can result in a reduction in synaptogyrin-3 activity.
  • partial inhibition of synaptogyrin-3 activity is sufficient to restore pathological Tau-induced presynaptic dysfunction.
  • the administration of an oligonucleotide of the disclosure can result in a reduction in synaptogyrin-3 mRNA level of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% with respect to control conditions (e.g., prior to the administration of the oligonucleotide of the present disclosure).
  • the administration of an oligonucleotide of the disclosure can result in a reduction in synaptogyrin-3 protein level of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% with respect to control conditions (e.g., prior to the administration of the oligonucleotide of the present disclosure).
  • the administration of an oligonucleotide of the disclosure can result in a reduction in synaptogyrin-3 activity level of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% with respect to control conditions (e.g., prior to the administration of the oligonucleotide of the present disclosure).
  • the skilled person is familiar with multiple ways of determining the level of synaptogyrin-3 in a cell and hence to determine a reduction of the Syngr-3 transcript level compared to a control.
  • a non-limiting example is quantitative reverse transcriptase (RT)-PCR.
  • RT reverse transcriptase
  • the Syngr-3 levels have been determined using a TaqMan assay.
  • nucleic acid molecules or the oligonucleotides of the present disclosure can be administered via intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraventricular, intraocular, or intrathecal administration. In some embodiments, the administration is via intrathecal administration.
  • administering means to give a composition comprising a composition disclosed herein to a subject via a pharmaceutically acceptable route.
  • SYNGR-3 gene inactivation i.e. inhibition of expression of the target gene
  • SYNGR-3 gene inactivation 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, ASO, etc) and whether the effect is achieved by incorporating the oligonucleotide into the subject's genome or by administering the oligonucleotide is not vital to the invention, as long as said oligonucleotide reduces the level of Syngr-3 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 Syngr-3 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 targeted against Syngr-3 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. Nonlimiting 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 nucleic acid molecules or oligonucleotides of the invention can also comprise inducible or regulatable promoters for expression of the nucleic acid molecule or oligonucleotide in a particular tissue or in a particular intracellular environment.
  • the 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 Syngr-3 transcript.
  • an effective amount of a nucleic acid molecule or oligonucleotide targeting Syngr-3 transcripts comprises an intracellular concentration of from about 0.2 nanomolar (nM) to about 100 nM, preferably from about 1 nM to about 10 nM, more preferably from about 2 nM to about 20 nM, more preferably from about 2.5 nM to about 50 nM, even more preferably from about 5 nM to about 75 nM or from about 10 nM to about 150 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. relying on the Tet-on and Tet-off inducible systems commercially available, or on a modified U6 promoter that is induced by the insect hormone ecdysone.
  • a Cre-Lox recombination system has been used to achieve controlled expression in mice.
  • Synthetic shRNAs can be chemically modified to affect their activity and stability.
  • 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 oligonucleotides 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 oligonucleotide of the present disclosure can be directly injected into the brain, e.g., using a needle or a catheter.
  • an oligonucleotide 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 aspects, an oligonucleotide 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 synaptogyrin-3 inhibitor as described herein
  • a therapeutic protein e.g. a neurotrophic factor or nerve growth factor, or a proteinaceous synaptogyrin-3 inhibitor as described herein
  • implantation of genetically modified stem cells by recombinant viral vectors, by means of osmotic pumps, or by means of incorporating the therapeutic drug in a polymer (slow release; can be implanted locally).
  • an oligonucleotide 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.
  • genetically modified cells e.g., stem cells
  • recombinant vectors e.g., viral vectors
  • delivery devices e.g., pumps such as osmotic pumps
  • 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 oligonucleotides of the present disclosure are formulated in combination with a pharmacologic BBB disrupting agent.
  • the oligonucleotides 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 oligonucleotide of the present disclosure.
  • the pharmacologic BBB disrupting agent is administered concurrently to the administration of the oligonucleotide of the present disclosure.
  • the pharmacologic BBB disrupting agent is administered subsequently to the administration of the oligonucleotide 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 oligonucleotides of the present disclosure are conjugated to carrier, e.g., transferrin or a transferrin-receptor antibody.
  • the oligonucleotides of the present disclosure are conjugated or formulated to transverse the BBB via transcytosis.
  • oligonucleotides 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.
  • oligonucleotides 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 oligonucleotides of the present disclosure are formulated in liposomes, e.g., liposomes for use in a liposomal Trojan horse strategy.
  • oligonucleotides of the present disclosure are formulated for intranasal delivery.
  • 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 oligonucleotides of the present disclosure are formulated for FUS-mediated delivery. Intracellular drug administration
  • the oligonucleotides 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 synaptogyrin-3 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 commonly used CPP is the transduction domain of TAT termed TATp.
  • 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).
  • kits and products of manufacture comprising one or more compositions (e.g., an oligonucleotide of the present disclosure or pharmaceutical compositions comprising an 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 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 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 February 1, 2023.
  • 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 February 1, 2023, 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 February 1, 2023, and are herein incorporated by reference.
  • a bioinformatic analysis was performed to identify potential screening candidate siRNAs targeting human SYNGR3 mRNA (NCBI gene ID: 9143).
  • the bioinformatical approach assumed a canonical siRNA structure. Positions 2 and 18 (5' -3') of the sense and antisense strand were used for the specificity calculations. Positions 1-19 (5' -3') of the antisense strand were used to assess cross reactivity and human SNP analysis.
  • Target specificity in human, rhesus monkey, cynomolgus monkey and mouse was performed to identify siRNAs with lowest sequence complementary to any non-target transcript.
  • Our analysis considered the likelihood of unintended downregulation of any other transcript by full or partial complementarity of a siRNA strand (up to 4 mismatches in within position 2-18; it was based on the number and position of the mismatches and described the predicted most likely off-target(s) for antisense and sense strand of each siRNA.
  • siRNAs can function in a miRNA-like manner via base-pairing with complementary sequences within the 3'-UTR of mRNA molecules. To circumvent that siRNAs would act via functional miRNA binding sites, we avoided siRNA strands, that contained natural miRNA seed regions (position 2-7).
  • NCBI DB SNP Analysis of human SNP database was performed to identify siRNAs targeting regions with known SNPs. When data was available, we also included positions of SNPs within the target sequence as well as minor allele frequency. All target sites with abundant SNPs were excluded.
  • siRNA activity prediction was done based on canonical siRNA design.
  • siRNAs molecules were fully modified with 2'-Fluoride and 2'-O-methyl as shown in FIGURE 1.
  • SH-SY5Y cells ATCC, CRL-2266
  • Dharmafect-4 0.5 pl/well; Horizon Discovery
  • siRNAs were added to cells at final concentrations of 20 nM and 2 nM and assay plates were incubated for 24h at 37°C / 5% CO2 in a humidified incubator.
  • bDNA branched DNA
  • the bDNA assay provides a unique and powerful tool for reliable quantification of nucleic acid molecules.
  • the bDNA assay directly measures nucleic acid molecules at physiological levels by boosting the reporter signal, rather than replicating target sequences as the means of detection, and hence avoids the errors inherent in the extraction and amplification of target sequences.
  • Probes for Syngr3 were custom and made by ThermoFisher Scientific, Assay ID: DRAAACA). Addition of a fluorescent reporter generates a signal directly proportional to the amount of target RNA present in the sample.
  • a first target region is built up by the siRNA molecules R2000060, R2000061 and R2000064.
  • a second target region by R2000072, R2000073, R2000075, R2000076, R2000077, R2000080, R2000081, R2000083, R2000084 and R2000085.
  • a third target region by R2000095 and R2000097, a fourth target region by R2000099 and R2000100 and a fifth target region by R2000104, R2000105 and R2000106.
  • a sixth target region comprises oligonucleotides R2000123, R2000124 and R2000125.
  • a seventh target region is formed by R2000130, R2000132, R2000133, R2000134, R2000135, R2000136, R2000137, R2000138, R2000140, R2000141 and R2000142.
  • An eight target region by R2000156, R2000157 and R2000158 and finally two larger target regions defined by R2000175, R2000176, R2000177, R2000179, R2000180, R2000181, R2000182, R2000183, R2000187 and R2000190, and defined by R2000191-R2000221.
  • siRNAs molecules From the siRNA molecules that make up the target regions, we further selected 48 siRNAs molecules to be tested in a dose-response curve. The selection was based on activity, target sequence binding, and cross-reactivity.
  • SH-SY5Y cells were incubated for 24 hours with the siRNAs to be tested. Media was removed and the SH- SY5Y cells (sourced from ATCC, CRL-2266) were lysed by addition of 150pl lysis mixture (1 volume lysis mixture, 2 volumes nuclease-free water) per 96-well and by subsequent incubation at 53°C for at least 60 minutes. Upon release of the target RNA, several oligonucleotide probes were incubated to allow binding to Syngr3 (and GAPDH as a control). Probes for Syngr3 were custom and made by ThermoFisher Scientific, Assay ID: DRAAACA). During this incubation, the probes cooperatively hybridize to the Syngr3.
  • 50pl working probe set hsSYNGR3 (gene target, Synaptogyrin 3 from Homo sapiens) and 90pl working probe set hsGAPDH (endogenous control, Glyceraldehyde-3-phosphate dehydrogenase from Homo sapiens), and 50pl (for hsSYNGR3) and lOpI (for hsGAPDH) of cell lysate were then added to the capture plates provided by the manufacturer. Capture plates were incubated at 53°C for approximately 16-20 hours. The next day, the capture plates were washed 3 times with at least 300pl of lx Wash Buffer (nuclease-free water, wash buffer component 1 and wash buffer component 2).
  • lOOpI of pre-amplifier working reagent was added to both hsSYNGR3 and hsGAPDH capture plates, which were sealed with clear adhesive foil and incubated for 1 hour at 53°C. Following incubation, the wash step was repeated, then lOOpI amplifier working reagent was added to both hsSYNGR3 and hsGAPDH capture plates. After 1 hour incubation at 53°C, the wash and dry steps were repeated, and lOOpI label probe was added per 96-well to all capture plates. Capture plates were incubated for 53°C for 1 hour. The plates were then washed with lx wash buffer and dried, and then 100 .l substrate was added to the capture plates sealed by adhesive aluminum foil. Following 30 minutes of incubation in the dark, luminescence was read using 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jugesheim, Germany).
  • hsSYNGR3 mRNA level was normalized to the hsGAPDH mRNA level.
  • the activity of a given hsSYNGR3 targeting siRNA was expressed as percent hsSYNGR3 mRNA concentration (normalized to hsGAPDH mRNA) in treated cells, relative to the hsSYNGR3 mRNA concentration (normalized to hsGAPDH mRNA) averaged across control wells.

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