Composition and method for regulating alpha-synuclein expression

Abstract

An SNCA antisense oligonucleotide capable of regulating the expression of SNCA target nucleic acids is provided herein. A method of using the same is also provided herein.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 618,693 filed on January 8, 2024, which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application includes a sequence list electronically submitted in XML format, the entire sequence list incorporated herein by reference. The XML copy created on January 7, 2025, is named DNL-047-01-WO_SeqListing.XML and has a size of 152 kb. Background Technology

[0005] Neurodegenerative diseases, including Parkinson's disease (PD), are emerging as a significant and growing health problem worldwide. PD is a complex, progressive neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra, causing motor dysfunction, tremors, rigidity, and postural instability. Although the exact etiology of PD has not yet been clearly identified, there is growing evidence suggesting that the abnormal aggregation and accumulation of alpha-synuclein protein plays a key role in the pathogenesis of the disease.

[0006] Alpha-synuclein (alpha-synuclein, aSyn, or SNCA) is closely involved in regulating synaptic function and neurotransmitter release. However, under pathological conditions, SNCA undergoes misfolding and aggregation to form insoluble fibers known as Lewy bodies, which is a representative neuropathological feature of other neurodegenerative disorders such as PD, multiple system atrophy (MSA), Lewy body dementia, pure autonomic failure (PAF), and REM sleep behavior disorder (RBD) (collectively referred to as synucleinopathy). Abnormal aggregation of SNCA is believed to contribute to neuronal dysfunction and apoptosis, ultimately leading to the clinical signs observed in affected individuals.

[0007] Current PD treatment strategies focus primarily on alleviating symptoms and enhancing dopaminergic neurotransmission, but these approaches do not address the underlying mechanisms that drive disease progression. Therefore, there is an urgent need for innovative therapeutic interventions that directly target the pathological processes associated with SNCAs and their aggregation.

[0008] Some embodiments provide an SNCA antisense oligonucleotide (SNCA ASO) comprising a nucleic acid sequence comprising at least nine consecutive nucleobases of a sequence selected from any one of SEQ ID NOs 1 to 15 and 18 to 23, at least one modified nucleoside linkage, and / or at least one modified sugar. In some embodiments, the SNCA ASO is 10 to 25 nucleotides in length.

[0009] Some embodiments provide an SNCA ASO comprising a nucleobase sequence of any one of SEQ ID NOs 24 to 46, at least one modified nucleoside linkage, and / or at least one modified sugar. In some embodiments, the SNCA ASO is 10 to 25 nucleotides in length.

[0010] Some embodiments provide an SNCA ASO comprising a nucleic acid sequence described in any one of SEQ ID NOs. 1–15 and 18–23, comprising at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO is 10 to 25 nucleotides in length.

[0011] Some embodiments provide an SNCA ASO having a length of 16 to 20 nucleotides comprising any one of the nucleobase sequences of SEQ ID NOs 24 to 38 and 41 to 46, at least one modified nucleoside linkage, and / or at least one modified sugar.

[0012] Some embodiments provide an SNCA ASO having a length of 18 to 20 nucleotides comprising any one of the nucleobase sequences of SEQ ID NOs 1 to 15, 18 to 38 and 41 to 46, at least one modified nucleoside linkage and / or at least one modified sugar.

[0013] Some embodiments provide an SNCA ASO having a length of 16 nucleotides comprising any one of the nucleobase sequences of SEQ ID NOs 24–38 and 41–46, at least one modified nucleoside linkage, and / or at least one modified sugar.

[0014] Some embodiments provide an SNCA ASO gapmer comprising a gap segment having a nucleobase sequence of any one of SEQ ID NOs 24 to 46, at least one modified nucleoside linkage, and / or at least one modified sugar. In some embodiments, the SNCA ASO gapmer is composed of 16 nucleotide lengths, wherein the 5' and 3' wing segments are each composed of 3 nucleotide lengths.

[0015] In some embodiments, the SNCA ASO is conjugated to a targeting ligand to form a conjugate. The targeting ligand may be, but is not limited to, a molecule that specifically binds to the transferrin receptor (TfR) or a molecule expressed on the lumen of the blood-brain barrier. The molecule may be, but is not limited to, an anti-TfR antibody or its TfR-binding fragment or an Fc polypeptide modified to bind to TfR (incorporated herein by reference as WO2023279099).

[0016] Some embodiments provide a pharmaceutical composition comprising any SNCA ASO described herein or a conjugate comprising an SNCA ASO described herein and a pharmaceutically acceptable carrier or diluent.

[0017] Some embodiments are methods for generating neurons with reduced alpha-synuclein expression, comprising the step of delivering to the neuron the SNCA ASO described herein or a conjugate comprising the SNCA ASO described herein, wherein the SNCA ASO is endogenous SNCAA method for reducing gene expression levels is provided. Neurons may be brain cells, deep brain cells, or spinal cord cells, but are not limited thereto. In some embodiments, a method for generating oligodendrocytes with reduced alpha-synuclein expression comprises the step of delivering to oligodendrocytes SNCA ASO as described herein or a conjugate comprising SNCA ASO as described herein, wherein the SNCA ASO is endogenous SNCA A method for reducing gene expression levels is provided herein.

[0018] Some embodiments are methods for modifying neurons to reduce alpha-synuclein expression, comprising the step of delivering to the neuron the SNCA ASO described herein or a conjugate comprising the SNCA ASO described herein, wherein the SNCA ASO is endogenous SNCA A method for reducing gene expression levels is provided. Neurons may be brain cells, deep brain cells, or spinal cord cells, but are not limited thereto. In one embodiment, a method for modifying oligodendrocytes to reduce alpha-synuclein expression comprises the step of delivering to a neuron the SNCA ASO described herein or a conjugate comprising the SNCA ASO described herein, wherein the SNCA ASO is endogenous SNCA A method for reducing gene expression levels is provided herein.

[0019] Some embodiments are methods for modifying a neuron to reduce alpha-synuclein expression, comprising the step of delivering to the neuron SNCA ASO described herein or a conjugate comprising SNCA ASO described herein to the neuron, wherein the SNCA ASO in the cell SNCAA method is provided for specifically reducing the expression level of a transcript. Neurons may be brain cells, deep brain cells, or spinal cord cells, but are not limited thereto. In one embodiment, a method for modifying oligodendrocytes to reduce alpha-synuclein expression comprises the step of delivering to a neuron the SNCA ASO described herein or a conjugate comprising the SNCA ASO described herein to the neuron, wherein the SNCA ASO in the cell SNCA A method for specifically reducing the expression level of a transcript is provided herein.

[0020] Some embodiments are methods for delivering SNCA ASO to the central nervous system (CNS) of a human subject requiring SNCA ASO delivery, comprising the step of administering to the subject a composition described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand), wherein the SNCA ASO is endogenous SNCA Provides a method to reduce gene expression levels.

[0021] Delivering the described SNCA ASO to neurons or oligodendrocytes may be used to treat neurodegenerative disorders. Delivering the described SNCA ASO to neurons may be used to treat alpha-synuclein-associated neurodegenerative disorders. Neurodegenerative disorders may be Parkinson's disease, but are not limited thereto. Delivering the described SNCA ASO to oligodendrocytes may be used to treat alpha-synuclein-associated neurodegenerative disorders (i.e., synucleinopathy). Neurodegenerative disorders may be multiple system atrophy, Lewy body dementia, pure autonomic failure (PAF), and REM sleep behavior disorder (RBD), but are not limited thereto.

[0022] Some embodiments provide a method for delivering an SNCA ASO to a human subject's CNS cell, comprising the step of administering a composition described herein (e.g., an SNCA ASO or an SNCA ASO conjugated to a targeting ligand) to the subject, wherein the composition is administered by intrathecal injection.

[0023] Some embodiments provide a method for delivering an SNCA ASO to CNS cells of a human subject, comprising the step of administering the SNCA ASO described herein to the subject, wherein the SNCA ASO is conjugated to a molecule or delivery vehicle that facilitates the transport of the SNCA ASO across the blood-brain barrier. The SNCA ASO may be conjugated to any molecule or delivery vehicle known in the art that facilitates transport across the blood-brain barrier. Such molecules and delivery vehicles include, but are not limited to, brain shuttles (e.g., brain shuttles disclosed in WO2018210898, WO2015101588, WO2023056388, and WO2014033074, each incorporated herein by reference). CNS cells may be brain cells, deep brain cells, spinal cord cells, neurons, and oligodendrocytes, but are not limited thereto.

[0024] Some embodiments provide a method for treating a human subject requiring treatment for an alpha-synuclein-associated neurodegenerative disorder (i.e., synucleinopathy), comprising the step of administering to the human subject a composition described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand).

[0025] Some embodiments provide a method for treating Parkinson's disease comprising the step of administering a composition described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand) to a human subject requiring treatment.

[0026] Some embodiments are SNCAA method for reducing messenger ribonucleic acid (mRNA) expression in a human subject requiring reduction of mRNA expression comprises the step of administering to a human subject a composition described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand).

[0027] Some embodiments provide compositions described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand) for use in treating human subjects requiring treatment for alpha-synuclein-associated neurodegenerative disorders.

[0028] Some embodiments provide compositions described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand) for use in treating human subjects requiring treatment for Parkinson's disease.

[0029] Some embodiments are SNCA The present invention provides a composition described herein (e.g., SNCA ASO or SNCA ASO conjugated to a targeting ligand) for use in reducing expression in human subjects where reduction of mRNA expression is required.

[0030] Some embodiments are SNCA Provides a use of SNCA ASO in the manufacture of a drug to reduce expression in human subjects requiring reduction of mRNA expression. Brief explanation of the drawing

[0031] Fig. 1 (Panel A) shows SNCA knockdown in the brain using 25 μg or 50 μg ICV injection, (Panel B) shows SNCA knockdown in the spinal cord using 25 μg or 50 μg ICV injection, and (Panel C) shows glial cell activation in the brain. Fig. 2 represents the ED50 curve in the brain (A) and the ED50 curve in the spinal cord (B). Fig. 3...represents the total ASO concentration in the brain after injection of 25 μg or 50 μg ICV. Specific details for implementing the invention

[0032] Antisense oligonucleotides are short, single-stranded synthetic nucleic acid polymers that can be used to regulate gene expression. They can target pre-mRNA, mRNA, or non-coding RNA to induce degradation, regulate splicing, or interfere with protein translation. SNCA RNA transcripts (e.g., SNCA Targeting mRNA, such as pre-mRNA or mature mRNA SNCA Antisense oligonucleotides that reduce gene expression levels are described herein.

[0033] SNCA The gene encodes the presynaptic neuron protein alpha-synuclein (also referred to herein as aSyn or SNCA). Alpha-synuclein is primarily expressed in neurons and is abundant at presynaptic terminals. Structurally, it is characterized by three distinct regions: an amphiphilic N-terminal domain, a central hydrophobic region known as the non-amyloid beta component (NAC), and an acidic C-terminal domain. The N-terminal domain is involved in membrane binding, while the NAC region plays a crucial role in alpha-synuclein aggregation. Functionally, alpha-synuclein is involved in synaptic vesicle dynamics and the regulation of neurotransmitter release. However, under pathological conditions, alpha-synuclein undergoes misfolding to form oligomers and insoluble fibers, which contribute to the development of neurodegenerative diseases. SNCA is associated with several neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), PAF, and RBD. In one embodiment, human SNCAThe gene has the sequence identified in GENBANK accession number NG_011851.1. In one embodiment SNCA The pre-mRNA has the sequence of SEQ ID NO. 48 (Ensembl ENST00000394991). An embodiment of the mRNA transcript is provided in SEQ ID NO. 49 (refseq NM_000345).

[0034] The SNCA ASO described herein can be used to treat Asyn-associated disorders, e.g., Parkinson's disease.

[0035] The singular forms 'a', 'an', and 'the' used herein include plural referents unless the content explicitly indicates otherwise.

[0036] When the terms 'approximately' and 'roughly' as used herein are used to modify quantities specified in numerical values ​​or ranges, they indicate that not only the numerical values ​​but also reasonable deviations from values ​​known to a person skilled in the art, e.g., ±20%, ±10%, or ±5%, are within the intended meaning of the listed values.

[0037] The term 'antisense oligonucleotide (ASO)' refers to a single strand of a selected target polynucleotide sequence, for example, a DNA-like or RNA-like molecule that is complementary or partially complementary to mRNA [e.g., a molecule comprising modified nucleoside linkage(s), modified nucleobase(s), and / or modified sugar(s) as described herein]. By binding to a complementary target sequence, the ASO may alter or regulate gene expression through a number of mechanisms, including, for example, by splicing alteration (exon exclusion or exon inclusion); by mobilization of RNA degradase H leading to targeted degradation; through translational inhibition; and by short RNA inhibition.

[0038] As used herein, the terms 'SNCA antisense oligonucleotide', 'SNCA ASO', or 'SNCA targeting antisense oligonucleotide' refer to SNCA targeting nucleic acids [e.g., RNA transcripts, e.g. SNCA mRNA (for example, SNCA pre-mRNA or SNCA By binding (hybridizing) to mature mRNA or cDNA in a sequence-specific manner SNCA It refers to an ASO that can reduce gene expression.

[0039] As used herein, 'transferrin receptor' or 'TfR' refers to transferrin receptor protein 1. Sequences of transferrin receptor protein 1 in other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term 'transferrin receptor' also encompasses exemplary reference sequences encoded by the gene at the transferrin receptor protein 1 chromosomal locus, e.g., allelic variants of the human sequence. The full-length transferrin receptor protein comprises a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0040] With respect to two or more polypeptide sequences, the term 'identical' or 'identical' percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, or are identical for a specified region or an indicated region as measured by using a sequence comparison algorithm or by manual alignment and visual inspection when compared and aligned to the maximum correspondence for a comparison window.

[0041] For the sequence comparison of polypeptides, a single amino acid sequence typically serves as a reference sequence against which candidate sequences are compared. Alignment can be performed using various methods available to a person skilled in the art, for example, by visual alignment, or by using publicly available software that employs algorithms known to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by a person skilled in the art. For the purposes of this application, the standard protein BLAST algorithm is used to align two protein sequences as a basic parameter when comparing polypeptide sequences.

[0042] When used in the context of identifying a given nucleotide residue in a nucleic acid sequence, the terms 'corresponding to,' 'determined by reference,' or 'numbered by reference' refer to the residue positions of the specified reference sequence when the given nucleotide sequence is maximally aligned with and compared to the reference sequence. The nucleic acid sequence aligned with the reference sequence does not need to be the same length as the reference sequence.

[0043] As used herein, the terms ‘nucleic acid’ and ‘polynucleotide’ refer to single-stranded or double-stranded deoxyribonucleotides or ribonucleotides and polymers thereof, composed of monomers (nucleotides) containing a sugar moiety, a phosphate, and a nucleobase. Unless specifically limited, the terms encompass both modified nucleic acids and unmodified nucleic acids.

[0044] As used herein, the term 'nucleobase' refers to a nitrogen-containing compound that connects to a sugar moiety to form a nucleoside and can eventually become a component of a nucleotide. The ability of nucleosides to form base pairs and stack on top of each other leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleosides can be naturally occurring [i.e., adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)] or modified (e.g., 5-methylcytosine). Thus, nucleobase moiety can be represented by a corresponding letter code for each nucleobase, e.g., A, T, G, C, or U, where each letter may optionally include a functionally equivalent modified nucleobase (e.g., based on Watson-Crick base pairing ability).

[0045] As used herein, the term 'nucleoside' refers to a compound comprising a nucleobase and a sugar moiety (e.g., deoxyribose or ribose, or a modified variant thereof). The term nucleoside includes both modified nucleosides and unmodified nucleosides.

[0046] As used herein, the term 'nucleotide' refers to a compound comprising a nucleobase, a sugar moiety, and one or more phosphate groups. The term nucleotide includes both modified nucleotides and unmodified nucleotides.

[0047] As used herein, the term 'nucleoside linkage' refers to a covalent bond between two nucleosides in an oligonucleotide. Nucleosides may be linked through natural [i.e., phosphodiester (PO) bonds] or modified bonds.

[0048] The terms 'chemical alteration', 'alteration', or 'altered' may refer to a chemical change of a compound compared to its natural counterpart. For example, a nucleobase, sugar moiety, or linkage between nucleosides may be chemically altered.

[0049] The terms 'nucleotide sequence', 'nucleic acid sequence', and 'nucleic acid strand' refer to a sequence of bases (purines and / or pyrimidines, or synthetic derivatives thereof) in a DNA or RNA polymer, which may be single-stranded or double-stranded containing synthetic, unnatural, or modified nucleotides and / or backbone modifications (e.g., modified oligomers) that may optionally be incorporated into the DNA or RNA polymer. The terms 'oligo', 'oligonucleotide', and 'oligomer' may be used interchangeably and refer to such sequences of purines and / or pyrimidines. For example, an oligonucleotide may comprise a chemically modified or unmodified nucleic acid molecule (RNA or DNA) having a length of about, for example, less than 200 nucleotides (e.g., about 100 or less than 50 nucleotides). The oligonucleotide may be, for example, single-stranded DNA or RNA (e.g., ASO), double-stranded DNA or RNA including double-stranded DNA or RNA having a hairpin loop (e.g., small interfering RNA [siRNA]), or a DNA / RNA hybrid. In one embodiment, the oligonucleotide has a length ranging from about 5 to about 60 nucleotides or from about 10 to about 50 nucleotides. In another embodiment, the oligonucleotide has a length ranging from about 5 to about 30 nucleotides or from about 12 to about 30 nucleotides. In another embodiment, the oligonucleotide has a length ranging from about 10 to about 25 nucleotides. In another embodiment, the oligonucleotide has a length ranging from about 16 to about 20 nucleotides.

[0050] The terms 'modified oligo', 'modified oligonucleotide', or 'modified oligomer' may be used interchangeably and refer to such sequences containing synthetic, unnatural, or altered bases, sugars, and / or skeletal modifications.

[0051] 'Modified nucleotide' is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide) or a deoxyribonucleotide (2'-H nucleotide). A modified nucleotide may comprise one or more of a modified nucleobase, a modified ribose (sugar) moiety, and a link between the modified nucleoside and another nucleoside. A modified nucleotide includes a nucleotide mimic. A modified nucleoside includes a nucleoside without a nucleobase and a nucleoside in which ribose is substituted with a non-sugar moiety (e.g., a sugar substitute, e.g., a morpholino or peptide nucleic acid).

[0052] '2'-deoxyribose nucleosides' are nucleosides containing a 2'-deoxyribose sugar moiety. In naturally occurring DNA, 2'-deoxyribose nucleosides contain ribose having a β-D ribosyl arrangement.

[0053] '2'-substituted modified nucleoside' or '2'-substituted nucleoside' or '2'-modified nucleoside' is a nucleoside comprising a 2'-substitution (e.g., a group other than hydrogen or hydroxyl) on the 2'-OH group of a ribosyl sugar moiety. A 2'-substituted nucleoside comprises at least one 2'-substituent other than H or OH on the 2' carbon of the nucleoside ribose.

[0054] '2'-MOE modified nucleoside' or '2'-MOE nucleoside' is a nucleoside containing a 2'-OCH2CH2OCH3(O-methoxyethyl) substitution at the 2'-OH group of the ribosyl sugar moiety.

[0055] '2'-NMA modified nucleoside' or '2'-NMA nucleoside' is a nucleoside containing a 2'-O-CH2-C(=O)-NH-CH3(ON-methylacetamide) substitution at the 2'-OH group of the ribosyl sugar moiety.

[0056] '2'-OMe modified nucleoside' or '2'-OMe nucleoside' is a nucleoside containing a 2'-OCH3 substitution at the 2'-OH group of a ribosyl sugar moiety.

[0057] '2'-F modified nucleoside' or '2'-F nucleoside' is a nucleoside containing a 2'-fluoro substitution instead of the 2'-OH group of the ribosyl sugar moiety.

[0058] A ‘cyclocyclic nucleoside’ (also called a cross-linked nucleoside) is a nucleoside containing a cyclocyclic sugar moiety. The ‘cyclocyclic sugar’ or ‘cyclocyclic sugar moiety’ is a modified sugar moiety containing two rings, wherein the second ring is formed through a bridge connecting two atoms within the first ring to form a cyclocyclic structure. In some embodiments, the first ring of the cyclocyclic sugar moiety is a furanosyl moiety, such as the ribosyl sugar moiety of the nucleoside.

[0059] A 'non-cyclic modified sugar moiety' is a modified sugar moiety that includes modifications such as substituents that do not form a bridge between two atoms of the sugar to form a second ring.

[0060] 'Restricted ethyl' or 'cEt' or 'cEt modified sugar moiety' or 'cEt sugar moiety' is a β-D ribosyl discyclic sugar moiety, wherein the second ring of the discyclic sugar is formed through a bridge connecting the 4'-carbon and 2'-carbon of the β-D ribosyl sugar moiety, where the bridge has the chemical formula 4'-CH(CH3)-O-2', where the methyl group of the bridge is in an S configuration. 'cEt modified nucleoside' or 'cEt nucleoside' is a nucleoside containing a cEt modified sugar moiety.

[0061] 'Lock nucleic acid', or 'LNA nucleoside', or 'LNA' is a bicyclic nucleoside having a 4'-CH2-O-2' bridge between the 4' and 2' furanosyl ring atoms.

[0062] A 'sugar substitute' is a moiety having a moiety other than the ribosyl moiety of the modified nucleoside. An oligonucleotide containing one or more suitable sugar substitutes retains the ability to hybridize to a complementary target nucleobase or nucleic acid sequence.

[0063] 'Internucleoside linkage' is a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, 'modified internucleoside linkage' refers to any internucleoside linkage other than phosphodiester internucleoside linkage.

[0064] 'Phosphorothioate nucleoside linkage' is a nucleoside linkage modified by replacing one of the non-crosslinked oxygen atoms of a phosphodiester nucleoside linkage with a sulfur atom.

[0065] '5-methylcytosine' includes cytosine modified by a methyl group attached to the 5 position. 5-methylcytosine is a modified nucleobase.

[0066] 'Abasic nucleosides' include nucleosides lacking a nucleobase.

[0067] "Chiral enrichment group" means multiple molecules having the same molecular formula, wherein the number or percentage of molecules within the group containing a specific stereochemical arrangement at a specific chiral center is greater than the number or percentage of molecules within the group expected to contain the same specific stereochemical arrangement at the same specific chiral center when the specific chiral center is stereorandom. A chiral enrichment group of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In some embodiments, the molecule is a modified oligonucleotide.

[0068] 'Stabilized phosphate group' refers to a 5'-chemical moiety that stabilizes the 5'-phosphate moiety of the 5'-terminal nucleoside of an oligonucleotide compared to the stability of the unmodified 5'-phosphate of the unmodified nucleoside under biological conditions. Stabilized phosphate groups include, but are not limited to, 5'-vinyl phosphonates and 5'-cyclopropyl phosphonates.

[0069] The SNCA ASO described herein may be synthesized using standard solid-phase or solution-phase synthesis techniques known in the art. In some embodiments, the SNCA ASO is synthesized using solid-phase phosphoramidite chemistry utilizing an automated synthesizer (U.S. Patent No. 6,773,885). Chemical synthesis of nucleic acids enables the generation of various forms of nucleic acids having modified bonds, chimeric compositions, and non-standard bases or modifiers attached at selected positions along the entire length of the nucleic acid.

[0070] As used herein, the term 'complementary' refers to the broad concept of complementary base pairing between two nucleic acids aligned at antisense positions relative to each other. Nucleic acids are considered complementary at those positions when nucleotide positions in two molecules are occupied by nucleotides that can normally form base pairs with each other. Thus, two nucleic acids are substantially complementary when at least about 50%, at least about 60%, or at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that normally form base pairs with each other [e.g., A:T (A:U in the case of RNA) and G:C nucleotide pairs].

[0071] With respect to two or more nucleotide sequences, the term 'complementary' percentage refers to two or more sequences or subsequences that are complementary or have a specified percentage of nucleotides, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, or have a specified region when compared and aligned to a maximum correspondence for a comparison range, or have a specified region as measured by using a sequence comparison algorithm or by manual alignment and visual inspection.

[0072] With respect to two or more nucleotide sequences, the term 'identical' or 'identity' percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of nucleotides, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, or have a specified region when compared and aligned to the maximum correspondence for a comparison range, or have an indicated region as measured by using a sequence comparison algorithm or by manual alignment and visual inspection.

[0073] It will be understood that chemical alterations are ignored when determining the percentage of complementarity or the percentage of identity, provided that the functional ability of the nucleobases forming Watson-Crick base pairs is maintained (e.g., 5-methylcytosine is considered identical to cytosine for the purpose of calculating the percentage of identity).

[0074] For the comparison of oligonucleotide sequences (e.g., to determine identity or complementarity), a single nucleotide sequence typically serves as a reference sequence to be compared with candidate sequences. Alignment can be performed using various methods available to a person skilled in the art, for example, by visual alignment, or by using publicly available software that employs algorithms known to achieve maximum alignment. Such programs include BLAST, ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR). The alignment parameters used to achieve maximum alignment can be determined by a person skilled in the art.

[0075] As used herein, ‘hybridize’ or ‘hybridization’ means the formation of pairs of complementary nucleotide sequences (e.g., between an antisense compound and its target nucleic acid, or between an antisense and sense strand). As used herein, ‘specifically hybridize’ means the ability of a reference nucleic acid to hybridize with one nucleic acid molecule with greater affinity than to hybridize with another nucleic acid molecule.

[0076] 'Expression' refers to the transcription and / or translation of endogenous genes, heterologous genes or nucleic acid segments, or transposable genes in a cell. For example, expression may refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of proteins.

[0077] The term 'gene' refers to a sequence of nucleic acids (e.g., DNA or RNA) containing a coding sequence necessary for the production of a polypeptide or precursor.

[0078] The phrase 'regulating the expression of a target gene or sequence' means a change (e.g., increase or decrease) in the expression of a target gene or sequence (e.g., through targeted degradation or translational inhibition). For example, it includes inhibiting, reducing, or decreasing the expression of a target gene or sequence. It also includes changes in alternative splicing that may result in changes in the absolute or relative amount of a specific splice variant.

[0079] The terms ‘object’, ‘entity’, and ‘patient’ used interchangeably herein refer to mammals including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0080] The terms 'treatment,' 'treating,' etc. are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. 'Treating' or 'treatment' may refer to any indication of success in the treatment or improvement of an impairment, disease, or condition, including any objective or subjective parameter, such as decline, remission, improvement in patient survival, increase in survival time or survival rate, reduction or impairment of symptoms, making the disease or condition more tolerable to the patient, slowing the rate of regression or decline, or improvement in the patient's physical or mental well-being. Additionally, 'treating' or 'treatment' may refer to the regulation of target gene expression, such as gene knockdown or gene knockout. For example, the expression of a target gene or sequence is suppressed or reduced by, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to expression in a control group. Treatment or improvement of symptoms may be based on objective or subjective parameters. Treatment effects may be compared to untreated individuals or a pool of individuals, or to the same patient at different times before or during treatment.

[0081] As used herein, “improvement” refers to the use of an effective amount of SNCA ASO and a conjugate containing it to improve at least one symptom (e.g., compared to the same symptom when not treated). In some embodiments, improvement is a reduction in the severity or frequency of the symptom, or a delay in the onset or a reduction in the rate of progression of the severity or frequency of the symptom.

[0082] The term 'prevent' or 'prevention' refers to the use of an effective amount of SNCA ASO and a conjugate containing it to reduce or eliminate the occurrence or recurrence of a disease or disease symptoms in a subject, for example, a subject prone to the occurrence or recurrence of the disease.

[0083] The term 'pharmaceuticalally acceptable excipient' refers to a non-active pharmaceutical ingredient that is biologically or pharmaceutically compatible for use in humans or animals, such as but not limited to buffers, carriers, or preservatives.

[0084] The 'therapeutic dose' or 'therapeutic effective dose' of medication used in this institution is an amount of medication that treats, alleviates, weakens, or reduces the severity of disease symptoms in the subject. The 'therapeutic dose' or 'therapeutic effective dose' of medication may improve patient survival, increase survival time or rate, reduce symptoms, make impairment, disease, or condition more tolerable, slow the rate of degeneration or decline, or improve the patient's physical or mental well-being.

[0085] The term "administer" refers to a method of delivering a drug, compound, or composition to a site intended for biological action. Such methods include, but are not limited to, local delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the protein described herein is administered intravenously.

[0086] The terms 'control group' or 'control value' refer to a reference value or standard value. An appropriate control group may be determined by a person skilled in the art. In some cases, the control value may be determined as a relative value to a baseline within the same subject or experiment, for example, measured prior to treatment with the SNCA ASO or its conjugate or composition described herein. SNCAGene expression levels may be control values ​​for measuring SNCA levels after treatment in the same subjects. In other examples, control values ​​may be determined as relative values ​​to the mean of a control group (e.g., healthy control group or disease control group) or a population of control subjects (e.g., healthy control group or disease control group, e.g., a population of 10, 20, 50, 100, 200, 500, 1000 control subjects or more), e.g., at baseline or after treatment of the subjects SNCA Gene expression levels can be compared to healthy control values.

[0087] 3. Oligonucleotides targeting SNCA

[0088] human being SNCA SNCA antisense oligonucleotides (SNCA ASOs) complementary to genes are disclosed herein. The SNCA ASOs described herein are human SNCAIt is at least 90% complementary to the gene. In some embodiments, the SNCA ASO described herein is at least 90% complementary to SEQ ID NO. 49. In some embodiments, the SNCA ASO described herein comprises or is composed of a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO. 49. In some embodiments, the SNCA ASO described herein comprises or is composed of a nucleic acid sequence of length 10 to 25 nucleotides that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO. 49. In some embodiments, the SNCA ASO described herein comprises or is composed of a nucleic acid sequence of length 10 to 25 nucleotides that is at least 90% or at least 95% complementary to SEQ ID NO. 49. In some embodiments, the SNCA ASO described herein comprises or is composed of a nucleic acid sequence of length 10 to 25 nucleotides that is 100% complementary to SEQ ID NO. 49.

[0089] ASO is in the range of about 10 to 30 base pairs (bp) in length, but may be longer or shorter. For example, in some embodiments, ASO is about 10 to about 60 nucleotide lengths (i.e., about 10 to about 60 linked nucleoside lengths), or about 10 to about 50 nucleotide lengths, or about 10 to about 40 nucleotide lengths. In some embodiments, the ASO is about 10 to 30 nucleotide lengths, or about 12 to 30 nucleotide lengths, or about 14 to about 30 nucleotide lengths, or about 15 to about 30 nucleotide lengths, or about 16 to about 30 nucleotide lengths, or about 17 to about 30 nucleotide lengths, or about 18 to about 30 nucleotide lengths, or about 18 to about 28 nucleotide lengths, or about 18 to 26 nucleotide lengths, or about 18 to about 24 nucleotide lengths, or about 15 to about 25 nucleotide lengths, or about 16 to about 20 nucleotide lengths. In some embodiments, ASO is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide lengths.

[0090] Although the length of the SNCA ASO may vary, in some embodiments the SNCA ASO is about 10 to about 60 nucleotide lengths, or about 10 to about 30 nucleotide lengths, or about 18 to about 30 nucleotide lengths, or about 15 to about 25 nucleotide lengths, or about 16 to about 20 nucleotide lengths. In some embodiments the ASO is about 16 to about 20 nucleotide lengths. In some embodiments the ASO is 16, 17, 18, 19, or 20 nucleotide lengths. In some embodiments the ASO is 16 nucleotide lengths. In some embodiments the ASO is 17 nucleotide lengths. In some embodiments the ASO is 18 nucleotide lengths. In some embodiments the ASO is 19 nucleotide lengths. In some embodiments, ASO is 20 nucleotides long.

[0091] In some embodiments, the SNCA ASO disclosed herein comprises or is composed of a nucleic acid sequence comprising or consisting of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleobases of a sequence selected from any one of SEQ ID NOs 1 to 15 and 18 to 23, and at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO disclosed herein comprises a nucleic acid sequence selected from any one of SEQ ID NOs 1 to 15 and 18 to 23, and further comprises at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO disclosed herein comprises a nucleic acid sequence selected from any one of SEQ ID NOs 1–15 and 18–23, and further comprises at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO disclosed herein comprises SNCA Targeting an exon of a gene, comprising or composed of any one of SEQ ID NOs. 1 and 6–8, and further comprising at least one modified nucleoside linkage and / or at least one modified sugar. In one embodiment, the SNCA ASO disclosed herein is SNCA Targeting an intron of a gene, comprising or composed of a nucleic acid sequence selected from any one of SEQ ID NOs. 2 and 10–12, and further comprising at least one modified nucleoside linkage and / or at least one modified sugar. In one embodiment, the SNCA ASO disclosed herein is SNCATargeting an intron of a gene, comprising or composed of a nucleic acid sequence selected from any one of SEQ ID NOs. 3 and 13–15, and further comprising at least one modified nucleoside linkage and / or at least one modified sugar. In one embodiment, the SNCA ASO disclosed herein is SNCA Targeting an intron of a gene, comprising or composed of a nucleic acid sequence selected from any one of SEQ ID NOs. 4 and 18–20, and further comprising at least one modified nucleoside linkage and / or at least one modified sugar. In one embodiment, the SNCA ASO disclosed herein is SNCA Targeting an intron of a gene, comprising or composed of a nucleic acid sequence selected from any one of SEQ ID NOs. 5 and 21–23, and further comprising at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO disclosed herein comprises 15 to 25 linked nucleosides. In some embodiments, the SNCA ASO disclosed herein comprises 16 to 20 linked nucleosides. In some embodiments, the SNCA ASO disclosed herein comprises 17 to 19 linked nucleosides. In some embodiments, the SNCA ASO described herein (e.g., 15 to 25 nucleotide lengths or 17 to 19 nucleotides) is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO. 1.

[0092] In some embodiments, the SNCA ASO disclosed herein has a length of 16 to 20 nucleotides and comprises any one of the sequences SEQ ID NOs 24 to 38 and 41 to 46, wherein the SNCA ASO further comprises at least one modified nucleoside linkage and / or at least one modified sugar. In some embodiments, the SNCA ASO disclosed herein comprises a gapmer ASO having a gap segment comprising any one of the sequences SEQ ID NOs 24 to 38 and 41 to 46. In some embodiments, the SNCA ASO disclosed herein comprises a gapmer ASO having a gap segment composed of any one of the sequences SEQ ID NOs 24 to 38 and 41 to 46. In some embodiments, SNCA ASO comprises a sequence having 100% identity with respect to any of SEQ ID NOs 24–38 and 41–46 and at least 85%, at least 90%, at least 95%, or 100% identity with respect to any of SEQ ID NOs 1–15 and 18–23.

[0093]

[0094]

[0095]

[0096]

[0097] Exemplary oligonucleotide modification

[0098] In some embodiments, the SNCA ASOs described herein may comprise at least one nucleic acid modification selected from the group consisting of, e.g., a modified nucleoside linkage, a modified nucleobase, a modified sugar, and combinations thereof (e.g., at least one modified nucleoside linkage and / or at least one modified sugar). Such modifications may be used to alter pharmacokinetics (improved nuclease resistance resulting in a longer half-life), pharmacodynamics (excellent affinity for target RNA), or endocytotic uptake. However, many modifications render cleavage by RNA degradase H, which is the intended mechanism of action for many ASOs, impossible. Thus, a specific RNA degradase H ASO may be designed as a chimera in which different bases are a mixture of different chemistry, or as a gapmer in which some modifications are located in the 'wing' rather than the central base.

[0099] Accordingly, the SNCA ASO described herein may comprise one or more nucleic acid modifications. In some embodiments, the SNCA ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 40 or more modifications. In some embodiments, the SNCA ASO described herein comprises one or more nucleotide modifications (e.g., for a nucleobase or sugar moiety). In some embodiments, 25% or more of the nucleotides present in the SNCA ASO are modified. In some embodiments, more than 50% of the nucleotides present in the SNCA ASO are modified. In some embodiments, more than 75% of the nucleotides present in the SNCA ASO are modified. In some embodiments, 100% of the nucleotides present in the SNCA ASO are modified.

[0100] Modified sugar moiety, nucleoside / nucleotide

[0101] In some embodiments, the SNCA ASO comprises one or more nucleobase modifications. In some embodiments, the SNCA ASO comprises one or more modifications to the sugar moiety (e.g., furanosyl including substitutions at the 2'-position, 3'-position, 4'-position, and / or 5'-position). In some embodiments, the substituted sugar moiety comprises a bicyclic sugar moiety. In some embodiments, the bicyclic sugar moiety comprises a chemical bridge between the 4' and 2' positions of the sugar, wherein the chemical bridge is each independently selected from 4'-CH(R)-O-2' and 4'-(CH2)2-O-2', where R is each independently selected from H, C1-C6 alkyl, and C1-C6 alkoxy. In one embodiment, the bicyclic sugar moiety comprises a chemical bridge between the 4' and 2' positions of the sugar, wherein the chemical bridge is each 4'-CH(R)-O-2', where R is each independently H.

[0102] Modified nucleosides / nucleotides are 2'- O Methyl (2'OMe) residue, 2' O- Includes, but is not limited to, methoxyethyl (MOE) residues, restricted nucleic acid residues (e.g., S-cEt, R-cEt, S-cMOE and R-cMOE), peptide nucleic acid (PNA) residues, locked nucleic acid (LNA) residues and 5-methylcytidine residues (methylated cytosine residues) (see also [Scoles, et al., Neurol Genet Apr 2019, 5 (2) e323]). In some embodiments, SNCA ASO includes one or more 2'-MOE residues. In some embodiments, SNCA ASO includes one or more OMe residues or F residues (e.g., 2'-F or 2'OMe). In some embodiments, the SNCA ASO comprises one or more restricted (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE) and / or LNA residues. The nucleic acid is considered a 'lock' when it has a methylene bridge link formed between the 2'-oxygen and 4'-carbon of a ribose sugar molecule. In some embodiments, the SNCA ASO is a morpholino (i.e., contains a specific modification in the sugar moiety).

[0103] In some embodiments, the SNCA ASO described herein comprises one or more LNA residues. In some embodiments, the SNCA ASO described herein comprises one or more 5-methyl cytidine residues. In some embodiments, the SNCA ASO described herein comprises one or more LNA residues and one or more 5-methyl cytidine residues. In some embodiments, all cytosines in the SNCA ASO are 5-methyl cytidine.

[0104] Linking between modified nucleosides

[0105] In some embodiments, the SNCA ASO comprises one or more modifications to the nucleoside backbone [i.e., natural phosphodiester (PO) bonds are modified]. In some embodiments, these modifications are made, for example, to reduce nuclease activity. Accordingly, in some embodiments, the SNCA ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more modified nucleoside linkages. In some embodiments, at least 25% of the nucleoside linkages are modified. In some embodiments, at least 50% of the nucleoside linkages are modified. In some embodiments, more than 75% of the nucleoside linkages are altered. In some embodiments, 100% of the nucleoside linkages present in SNCA ASO are altered.

[0106] Skeletal modifications are known in the art and include, but are not limited to, phosphorothioate (PS) bonds, chiral phosphorothioate bonds, phosphorodiamidate bonds, phosphorodithioate bonds, aminoalkylphosphorister bonds, phosphorister bonds, thiophosphate bonds, phosphonate bonds, methylphosphonate bonds, alkylphosphonate bonds, 3' alkylene phosphonate bonds, chiral phosphonate bonds, 3'-aminophosphoramidate bonds, aminoalkylphosphoramidate bonds, phosphinate bonds, thionoalkylphosphonate bonds, thionophosphoramidate bonds, thionoalkyl-phosphorister bonds, borano-phosphate bonds, morpholino bonds, and peptide nucleic acid (PNA) bonds. For example, in some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) internucleoside linkages in the SNCA ASO are replaced by phosphorothioate (PS) linkages. In some embodiments, the SNCA ASO comprises a mixture of modified linkages and non-modified linkages. The modification in one internucleoside linkage may be independent of the modification in another internucleoside linkage. In some embodiments, all internucleoside linkages in the SNCA ASO are modified linkages. In some embodiments, all nucleoside linkages in SNCA ASO are PS bonds. In some embodiments, all nucleoside linkages in SNCA ASO are phosphorothioates.In some other embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) internucleoside linkages in the SNCA ASO are replaced by phosphorodiamidate linkages. In some embodiments, the SNCA ASO is a phosphorodiamidate morpholino (PMO).

[0107] In some embodiments, the linkage between nucleosides is stereorandom with respect to the chiral centers (Rp and Sp). In some other embodiments, the stereoconfiguration of Rp and Sp in SNCA ASO is optimized at a specific stereoconfiguration.

[0108] Antisense oligonucleotide motif

[0109] In some embodiments, nucleic acid modification using SNCA ASO is included in the pattern. In some embodiments, the SNCA ASO is a gapmer. The modification pattern of a gapmer SNCA ASO is generally 5'-X a -Y a -Z a It has the chemical formula -3', and X a and Z a is the gap region Y a It is a surrounding flanking region. In some embodiments, Y a The region is a continuous elongation of nucleotides (i.e., linked nucleosides) capable of mobilizing RNA degrading enzymes such as RNA degrading enzyme H, for example, a region of at least six DNA nucleotides. In some embodiments, Y a The region is at least 8 DNA nucleotides. In some embodiments, Y a The region is about 9 to about 15 DNA nucleotides. In some embodiments, Y aThe region is about 11 to about 13 DNA nucleotides. In some embodiments, Y a The region is 10, 11, 12, or 13 DNA nucleotides. In some embodiments, the capmer binds to the target nucleic acid, a RNA degradase is mobilized at that site, and subsequently, the target nucleic acid can be cleaved. In some embodiments, Y a The area is X a Area and Z a A modified nucleotide, e.g., X, which is adjacent to both the 5' and 3' ends by the region. a and Z a Each of comprises 1 to 6 modified nucleotides. In some embodiments, Y a The area is X a Area and Z a It is adjacent to both 5' and 3' by the region, but X a and Z a comprises a modified nucleotide having a modified sugar. In some embodiments, X a and Z a In each nucleotide, the modified nucleotide having a sugar modification. The modified nucleotide may be, but is not limited to, a 2-MOE modified nucleotide, a bicyclic nucleotide, an LNA nucleotide, or a cET modified nucleotide. In some embodiments, the modified nucleotide is present in the 5' and 3' regions of the SNCA ASO, whereas specific modified nucleotides and / or modified bonds may or may not be present in the central region of the molecule. In some embodiments, the modified nucleotide is present in the 5' and 3' regions of the SNCA ASO, and specific modified nucleotides are not present in the central region of the molecule (e.g., LNA residues are not present in the central region, but the central region may contain modified bonds such as PS bonds). In some embodiments, X a and Z aEach is independently about 3 to 6 nucleotide long. In some embodiments, X a and Z a Each is independently 3, 4, or 5 nucleotide lengths. In some embodiments, X a and Z a Each comprises three modified nucleotides (e.g., containing a modified sugar). In some embodiments, the three modified nucleotides are X a and Z a They are arranged in series in each of them.

[0110] In some embodiments, SNCA ASO is a gapmer comprising LNA and PS modifications. For example, in some embodiments, SNCA ASO is of formula 5'-X a -Y a -Z a It is a gapmer with a modification pattern of -3', and X a and Z a is the gap region Y a The surrounding lateral region, but X a and Z a Each contains three LNA-modified nucleotides (e.g., three consecutive LNA-modified nucleotides), and the gap region Y a It includes PS bonds. In some embodiments, all internucleotide linkages in the SNCA ASO include PS bonds. In some embodiments, the SNCA ASO contains a mixture of PS bonds and other modified internucleotide linkages. In some embodiments, the SNCA ASO further includes one or more 5-methyl cytidine residues. In some embodiments, Y is a gap region. a It does not contain LNA residues.

[0111] In some embodiments, the SNCA ASO comprises a 5' wing segment having 1 to 6 nucleosides in the 5' to 3' direction, wherein each nucleoside of the 5' wing segment comprises a modified sugar; a gap segment having 8 to 15 nucleosides, wherein each nucleoside of the gap segment comprises a deoxynucleoside; and a 3' wing segment having 1 to 6 nucleosides, wherein each nucleoside of the 3' wing segment comprises a modified sugar. In some embodiments, all nucleotide-to-nucleotide linkages in the SNCA ASO comprise PS bonds. In some embodiments, the SNCA ASO comprises a mixture of PS bonds and other modified nucleotide-to-nucleotide linkages. In some embodiments, all nucleosides within the 5' wing segment and the 3' wing segment comprise 2-MOE modified nucleotides, cyclocyclic nucleotides, LNA nucleotides, or cET modified nucleotides.

[0112] In some embodiments, the SNCA ASO comprises a 5' wing segment having three nucleosides in the 5' to 3' direction, wherein each nucleoside of the 5' wing segment comprises a modified sugar; a gap segment having 11 to 13 nucleosides, wherein each nucleoside of the gap segment is a deoxynucleoside; and a 3' wing segment having three nucleosides, wherein each nucleoside of the 3' wing segment comprises a modified sugar. In some embodiments, all internucleotide connections in the SNCA ASO comprise PS bonds. In some embodiments, all nucleosides within the 5' wing segment and the 3' wing segment comprise 2-MOE modified nucleotides, cyclocyclic nucleotides, LNA nucleotides, or cET modified nucleotides.

[0113] In some embodiments Table 7An SNCA ASO comprising or composed of a modified nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to the modified sequence as shown in [the reference]. In some embodiments, the SNCA ASO Table 7 It includes a modified nucleic acid sequence as shown in. In some embodiments, SNCA ASO is Table 7 It consists of a modified nucleic acid sequence as shown in [figure]. The SNCA ASO in Table 7 is a capmer having a wing segment containing an LNA-modified nucleoside. Similar SNCA ASO capmers can be prepared using a wing segment having other ribose-modified nucleosides, including but not limited to 2'-MOE nucleotides and / or cEt nucleosides.

[0114] In some embodiments, a modified oligonucleotide according to any one of the following chemical structures is provided herein.

[0115] Sequence No. 1: (Refer to Table 7 for symbols)

[0116] +[%C]*+A*+[%C]*dA*dT*dT*dG*dG*dA*dA*dC*dT*dG*dA*dG*dC*+A*+[%C]*+T

[0117]

[0118] Sequence No. 2: (Refer to Table 7 for symbols)

[0119] +G*+T*+T*dA*dA*dA*dT*dC*dT*dA*dG*dT*dT*dG*dT*+[%C]*+[%C]*+A

[0120]

[0121] Sequence No. 3: (Refer to Table 7 for symbols)

[0122] +T*+[%C]*+T*dC*dT*dA*dT*dA*dT*dA*dA*dC*dA*dT*dC*+A*+[%C]*+T

[0123]

[0124] Sequence No. 4: (Refer to Table 7 for symbols)

[0125] +A*+A*+[%C]*dT*dG*dC*dT*dT*dA*dG*dT*dG*dA*dT*dT*+[%C]*+[%C]*+A

[0126] , or

[0127] Sequence No. 5: (Refer to Table 7 for symbols)

[0128] +G*+G*+T*dA*dA*dC*dT*dT*dA*dG*dG*dA*dC*dA*dA*+G*+G*+T

[0129] , or inflammation of the tooth.

[0130] ASO terminal clipping

[0131] In some embodiments, the SNCA ASO targeted to the Asyn nucleic acid may be shortened or cleaved. In some embodiments, the SNCA ASO may be shortened or cleaved by nucleic acid endosclerotic activity. For example, a single subunit (e.g., a nucleotide or part thereof) may be deleted at the 5' end (5' cleavage) or alternatively at the 3' end (3' cleavage). The shortened or cleaved antisense compound targeted to the Asyn nucleic acid may have two or more subunits deleted from the 5' end of the SNCA ASO, or alternatively, two or more subunits deleted from the 3' end thereof.

[0132] II. SNCA ASO Conjugate

[0133] Any described SNCA ASO may be connected to a targeter or a delivery vehicle. The SNCA ASO may be connected directly or indirectly to a targeter or a delivery vehicle. The SNCA ASO may be connected to a targeter or a delivery vehicle at the 5' end of the ASO, or to the 3' end of the ASO or to any nucleotide within the ASO. The targeter or delivery vehicle may be attached (connected) to the sugar moiety, base moiety, nucleoside linkage, or end (5' or 3') of the SNCA ASO. Any targeter or delivery vehicle suitable for facilitating or increasing the delivery of the SNCA ASO to target cells or tissues of a subject known in the art may be conjugated to the SNCA ASO. The targeter may be, but is not limited to, a molecule that specifically binds to the transferrin receptor (TfR) or a molecule expressed on the luminal surface of the blood-brain barrier. The molecule may be an anti-TfR antibody or its TfR-binding fragment or an Fc polypeptide modified to bind to TfR, but is not limited thereto. The delivery vehicle may be a molecule, brain shuttle, protein, liposome, lipoplex, or lipid nanoparticle, but is not limited thereto.

[0134] In some embodiments, the SNCA ASO is connected to a targeting device or a delivery vehicle through a linker. Any linker known in the art suitable for conjugating the SNCA ASO to another molecule may be used.

[0135] III. How to Use

[0136] SNCA ASO can be used for various purposes, including therapeutic indications.

[0137] In some embodiments, at the target SNCA A method for reducing gene expression is described, comprising the step of administering an effective amount of the SNCA ASO, conjugate, or composition described herein to a subject. In some embodiments, in the subject's cells SNCA An SNCA ASO, a conjugate, or a composition thereof is provided for use in reducing gene expression. When the SNCA ASO described in some embodiments is administered to a cell or target, in the cell or target SNCA The expression of the gene is reduced. Expression may be reduced by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% compared to expression in a control group (e.g., cells or subjects not administered the SNCA ASO, conjugate, or composition described herein) or compared to the expression level of SNCA in cells or subjects prior to administration of SNCA ASO.

[0138] Some embodiments are SNCA A method for reducing expression in a subject requiring reduction of mRNA expression is provided, comprising the step of administering the SNCA ASO, conjugate, or composition described herein to the subject.

[0139] Some embodiments also provide a method for reducing expression in a subject where reduction of ASyn expression is required, comprising the step of administering the SNCA ASO, conjugate, or composition described herein to the subject.

[0140] In some embodiments, the SNCA ASO described herein may be used to reduce SNCA expression in brain cells. The brain cells may be neurons, oligodendrocytes, deep brain cells, hippocampal cells, or cells of the entorhinal cortex, but are not limited thereto. Accordingly, some embodiments provide a method for generating neurons or oligodendrocytes with reduced ASyn expression, comprising the step of delivering the SNCA ASO, conjugate, or composition described herein to the neurons or oligodendrocytes, wherein the SNCA ASO SNCA Genes (e.g., endogenous SNCAA method for reducing the expression level of a gene) is provided. Accordingly, some embodiments provide a method for modifying a neuron or oligodendrocyte to reduce ASyn expression, comprising the step of delivering the SNCA ASO, conjugate, or composition described herein to the neuron or oligodendrocyte, wherein the SNCA ASO is SNCA Genes (e.g., endogenous SNCA It also provides a method to reduce the expression level of a gene. In some embodiments, SNCA ASO SNCA Binds to transcripts and is intracellular SNCA It reduces the expression level of transcripts. In some embodiments, SNCA ASO is SNCA It mobilizes RNA degrading enzyme H, which binds to the transcript and degrades the transcript.

[0141] In the decrease of SNCA expression SNCA Reducing the transcription of transcripts or mRNA, SNCA Transcript or mRNA knockdown SNCA This includes reducing gene expression or reducing the level of SNCA protein.

[0142] In some embodiments, the SNCA ASO described herein may be used to reduce SNCA expression in the spinal cord or spinal cord cells.

[0143] In some embodiments, the SNCA ASO described herein is delivered to target brain cells such as neurons, glial cells, oligodendrocytes, deep brain cells, hippocampal cells, or cells of the entorhinal cortex.

[0144] Some embodiments are methods for delivering an SNCA ASO to the CNS of a subject requiring SNCA ASO delivery, comprising the step of administering the SNCA ASO, conjugate, or composition described herein to the subject, wherein the SNCA ASO SNCA Genes (e.g., endogenous SNCA Provides a method to reduce the expression level of genes.

[0145] In some embodiments, SNCA ASO is the same as when SNCA ASO is not administered. SNCA Endogenous compared to transcript levels SNCA Gene expression levels or SNCA Reduces the transferor level by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some embodiments, endogenous SNCA Gene or SNCA The level of the transcript is reduced by at least about 50% compared to the expression level before administration of SNCA ASO. In some embodiments, endogenous SNCA Gene or SNCA The level of the transcript is reduced by at least about 70% compared to the expression level before administration of SNCA ASO.

[0146] The SNCA ASO and conjugates and compositions containing SNCA ASO described herein may also be used to treat, prevent, or improve diseases, disorders, and conditions associated with aSyn. Accordingly, methods for treating, preventing, or improving a target requiring treatment, prevention, or improvement of diseases, disorders, and conditions associated with aSyn are provided herein in some embodiments. In some embodiments, diseases, disorders, and conditions associated with aSyn are aSyn-associated neurodegenerative disorders (i.e., synucleinopathy). In some embodiments, synucleinopathy includes, but is not limited to, Parkinson's disease, dementia with Lewy bodies (DLB), multiple system atrophy [MSA], PAF, and RBD.

[0147] Accordingly, some embodiments provide a method for treating a subject requiring treatment for aSyn-associated neurodegenerative disease, comprising the step of administering to the subject the SNCA ASO or a conjugate or composition containing the SNCA ASO described herein.

[0148] In some embodiments, the aSyn-associated neurodegenerative disease is Parkinson's disease. Accordingly, some embodiments provide a method for treating Parkinson's disease comprising the step of administering the SNCA ASO or a conjugate or composition containing the SNCA ASO described herein to a subject requiring treatment. The subject has been diagnosed with Parkinson's disease, has been diagnosed with one or more symptoms of Parkinson's disease, or is at risk of developing Parkinson's disease or one or more symptoms associated with Parkinson's disease.

[0149] In some embodiments, the subject is a human subject.

[0150] The SNCA ASO or conjugates or compositions containing SNCA ASO described herein may be administered to a subject at a therapeutically effective amount or dose. However, the dosage may vary depending on several factors, including the selected route of administration, the formulation of the composition, patient response, severity of the condition, the subject's body weight, and the prescribing physician's judgment. The dosage may be increased or decreased over time as required by the individual patient.

[0151] In some embodiments, the SNCA ASO or the conjugate or composition containing the SNCA ASO described herein is administered parenterally. In some embodiments, the SNCA ASO, conjugate or composition is administered intravenously. Intravenous administration may be by infusion over a period of, for example, about 5 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, the SNCA ASO, conjugate or composition is administered as an intravenous bolus. A combination of infusion and bolus administration may also be used.

[0152] In some parenteral embodiments, the SNCA ASO, or a conjugate or composition containing the SNCA ASO, is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the SNCA ASO, conjugate, or composition is administered intradermally or intramuscularly. In some embodiments, the SNCA ASO, conjugate, or composition is administered intrathecally, such as intraventricular administration into the lateral ventricle.

[0153] In other embodiments, the SNCA ASO, or a conjugate or composition containing SNCA ASO described herein, may be administered orally, by pulmonary administration, by nasal administration, by ocular administration, or by topical administration. Pulmonary administration may also be performed, for example, by an inhaler or nebulizer, or by formulation with an aerosolizing agent.

[0154] IV. Pharmaceutical Composition and Kit

[0155] In some embodiments, pharmaceutical compositions and kits comprising the SNCA ASO or conjugate described herein are provided.

[0156] Pharmaceutical composition

[0157] Guidelines for preparing formulations for use as described herein can be found in numerous textbooks on pharmaceutical manufacturing and formulations known to a person skilled in the art.

[0158] In some embodiments, the pharmaceutical composition comprises the SNCA ASO or a conjugate thereof described herein and further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0159] As used herein, the term "pharmaceuticalally acceptable carrier" comprises any solvent, dispersion medium, or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the activator. Various pharmaceutically acceptable excipients are well known. In some embodiments, the carrier is suitable for intravenous, intrathecal, intraventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds that act to stabilize the composition or increase or decrease the absorption of the SNCA ASO or conjugate. Physiologically acceptable compounds may include, for example, carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, compositions that reduce the removal rate or hydrolysis of the activator, excipients, or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also known in the art.

[0160] The pharmaceutical compositions described herein may be prepared in a manner known to a person skilled in the art, for example, by conventional mixing, dissolving, granulating, dragé preparation, emulsifying, encapsulating, capturing, or freeze-drying processes. The following methods and excipients are merely exemplary and are not intended to be limiting.

[0161] For oral administration, the SNCA ASO or conjugate described herein may be formulated by combining it with one or more pharmaceutically acceptable carriers known in the art. In some embodiments, the pharmaceutically acceptable carrier may be used to formulate the compound (e.g., SNCA ASO or conjugate) as a tablet, pill, draze, capsule, emulsion, lipophilic and hydrophilic suspension, liquid, gel, syrup, slurry, suspension, etc., for oral intake by a patient to be treated. Oral pharmaceutical formulations may be obtained by mixing the SNCA ASO or conjugate with a solid excipient after adding a suitable adjuvant to obtain a tablet or draze core if necessary, optionally grinding the resulting mixture, and processing the granulated mixture. Suitable excipients include, for example, fillers such as sugars comprising lactose, sucrose, mannitol, or sorbitol; Cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone. If desired, a disintegrant, for example, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, for example, sodium alginate, may be added.

[0162] As disclosed above, the SNCA ASO or conjugate described herein may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the SNCA ASO or conjugate may be formulated into a formulation by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the SNCA ASO or conjugate may be formulated in an aqueous solution, preferably in a physiologically suitable buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. The injectable formulation may be provided in a unit dose form, for example, in an ampoule or multi-dose container with an added preservative. The composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain a formulation agent, such as a suspending agent, stabilizer, and / or dispersant.

[0163] For injection administration, the SNCA ASO or conjugate described herein may be formulated by combining it with one or more pharmaceutically acceptable carriers known in the art. In some embodiments, the pharmaceutically acceptable carrier may be used to formulate the compound (e.g., SNCA ASO or conjugate) as an emulsion, lipophilic and hydrophilic suspension, liquid, suspension, etc., for injection to a patient to be treated. In some embodiments, the SNCA ASO or SNCA ASO conjugate is formulated for intravenous injection, intrathecal injection, intra-arterial injection, or intraventricular injection.

[0164] For intranasal administration, the SNCA ASO or conjugate described herein may be formulated by combining it with one or more pharmaceutically acceptable carriers known in the art as suitable for intranasal administration of drugs (Reference [Shah P et al. "Intranasal delivery: An attractive route for the administration of nucleic acid based therapeutics for CNS disorders" Front Pharmacol. 2022 Aug 30;13:974666.]).

[0165] In general, pharmaceutical compositions intended for in vivo administration are sterilized. Sterilization can be achieved by methods known in the art, for example, heat sterilization, steam sterilization, sterile filtration, or light irradiation.

[0166] The dosage and target drug concentration of the pharmaceutical compositions described herein may vary depending on the specific intended use.

[0167] Kit

[0168] In some embodiments, a kit comprising the SNCA ASO or conjugate described herein is provided. In some embodiments, the kit is intended for use in reducing SNCA expression.

[0169] The described SNCA ASO (including the SNCA ASO conjugate) and the pharmaceutical composition comprising the SNCA ASO disclosed herein may be packaged or contained in a kit, container, pack, or dispenser. The SNCA ASO and the pharmaceutical composition comprising said SNCA ASO may be packaged in a pre-filled syringe or vial. Any of the SNCA ASO identified herein or the pharmaceutical composition containing the SNCA ASO may be formulated or packaged in a single-dose or multiple-dose format. Any of the SNCA ASO identified herein or the pharmaceutical composition containing the SNCA ASO may be formulated for repeated administration.

[0170] In some embodiments, the kit further comprises instructional material (e.g., instructions for using the kit to administer a composition across the blood-brain barrier) containing instructions (i.e., protocols) for the method described herein. Instructional material generally comprises, but is not limited to, written or printed material. Any medium capable of storing such instructions and delivering them to an end user is considered herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such media may include an address to an internet site providing such instructional material.

[0171] Examples

[0172] The present invention will be described in more detail through specific embodiments. The following embodiments are provided for illustrative purposes only and are not intended to limit the invention in any way. A person skilled in the art will readily recognize various non-critical parameters, which may be varied or modified to yield essentially the same results. While efforts are made to ensure accuracy with respect to the numbers used (e.g., quantities, temperatures, etc.), some experimental error and deviation may exist. Unless otherwise indicated, the practice of the present invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the art. These techniques are sufficiently described in the literature.

[0173] Example 1: SNCA In vitro screening of ASO for genes

[0174] The selection of an ASO sequence specific to a given target sequence is based on the analysis of the selected target sequence and the evaluation of multiple factors, including in vitro and in vivo efficacy and hepatotoxicity profiles.

[0175] Initial SNCA ASO screening

[0176] SNCA pre-mRNA(Ensembl ENST00000394991)(Sequence No. 48) and SNCA 309 SNCA ASO sequences that are 100% complementary to mRNA (refseq NM_000345) (sequence number 49) were initially identified based on computational predictions of target binding, specificity, efficacy, and safety.

[0177] In human HeLa and EFO-21 cells SNCA The knockdown efficacy of specific ASOs was tested. Without using transfection reagents. SNCA Specific ASO or control oligonucleotides were treated at a concentration of 5 μM. After 3 days of treatment, cells were lysed. SNCAHPRT1 mRNA expression was analyzed using QuantiGene Singleplex assay (ThermoFisher). SNCA expression values ​​were normalized to HPRT1 values, and the degree of knockdown was measured by comparison with mock-treated cells. Twenty-two ASOs were selected to investigate TLR9 activation ability and concentration-response relationships (IC50 measurement).

[0178] Investigation into TLR9 activation ability

[0179] The potential for TLR9-dependent inflammation induction was tested on 22 ASOs. Accordingly, in reporter cell lines (HEK-Blue-hTLR9 cells, Invivogen) SNCA Specific ASO, positive control, and negative control were treated at a concentration of 5 μM without transfection reagent. After 20 hours, QUANTI-Blue TM A solution (Invivogen) was added to the cells. Optical density was measured after 2 hours to confirm the TLR9 activating ability of the tested ASO.

[0180] In vitro IC50 measurement

[0181] 22 selected SNCA The concentration-response relationship for specific ASOs in EFO-21 cells was investigated, and half maximal inhibitory concentration (IC50) values ​​were measured. Cells were treated at concentrations of 5000 nM, 1667 nM, 556 nM, 185 nM, 62 nM, 21 nM, and 7 nM without the addition of transfection reagents. SNCA Specific ASO was treated. After 3 days of treatment, the cells were lysed. SNCA HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (Thermo Fisher). SNCA expression values ​​were normalized to HPRT1 values, and the degree of knockdown was measured by comparison with mock-treated cells. The results are below. Table 5 It was shown in.

[0182]

[0183] Based on comprehensive consideration of in vitro efficacy evaluation and the absence of significant hepatotoxicity in mice, six sequences—ASO_S2, ASO_S3, ASO_S4, ASO_S5, ASO_S6, and ASO_S7—were selected to conduct additional in vivo safety and efficacy analyses.

[0184] Example 2: In vivo knockdown screening

[0185] The six ASOs selected above (based on in vitro efficacy evaluation and the absence of significant hepatotoxicity in mice) and the control ASO_1 were diluted in sterile saline, and hSNCA (mSnca - / - 50 μg (ASO_S1, ASO_S2, ASO_S3, ASO_S4, ASO_S5, ASO_S7) or 25 μg (ASO_S6) due to acute neurotoxicity was administered to mice via ICV injection. All ASOs were administered at a final volume of 10 μL. Two weeks after administration, tissues were collected, and after high-throughput RNA isolation according to the protocol described below SNCA SNCA expression in the brain and spinal cord was measured via qPCR of and Gapdh. In the cortex and spinal cord SNCA Knockdowns are shown in Figures 1a and 1b, respectively. Gfap and Itgax expression were measured in the brain in the same manner to evaluate glial activation for ASO deposition (Figure 1c). ASO_S2, ASO_S3, ASO_S4, ASO_S5, and ASO_S6 were selected for further investigation. ASO_S7 was excluded due to low efficacy along with induced glial activation.

[0186] To confirm consistent dosage, the concentrations of ASO_S2, ASO_3, ASO_S4, and ASO_S5 in the brain were also measured as described below (Fig. 3). The data in Fig. 1 show that when ASO_S2, ASO_S3, ASO_S4, ASO_S5, and ASO_S6 are delivered to the brains of mice at high concentrations, they significantly reduce hSNCA.

[0187] Example 3: In vivo hepatotoxicity screening

[0188] The hepatic safety of each ASO was evaluated by measuring serum ALT, AST, and LDH levels in mice. ASO_S2, ASO_S3, ASO_S4, ASO_S5, and ASO_S6 were administered to wild-type mice by subcutaneous injection at a dose of 20 mpk daily for 5 consecutive days. No significant hepatotoxicity was observed in mice with any of the ASOs.

[0189] Example 4: In vivo ED50 screening

[0190] To evaluate the efficacy of ASO, ASO_S1 control and ASO_S2, S ASO_3, ASO_S4, ASO_S5, and ASO_S6 were diluted in sterile saline and hSNCA(mSNCA - / -) 10 μg (ASO_S1, ASO_S2, ASO_S3, ASO_S4, ASO_S5, ASO_S6) and 25 μg (ASO_S1, ASO_S2, ASO_S3, ASO_S4, ASO_S5) were administered to mice via ICV injection. All ASOs were administered at a final volume of 10 μL. Two weeks after administration, tissues were collected, and after large-scale RNA isolation according to the protocol described below... SNCASNCA expression in the brain and spinal cord was measured via qPCR of Gapdh. SNCA knockdown in the cortex and spinal cord is shown in Figures 2a and 2b, respectively. ED50 values ​​were calculated using Graphpad Prism software with data from all doses (10 μg, 25 μg, and 50 μg where available) (Figures 2a and 2b). The data in Figure 2 show that ASO_S2, ASO_S3, ASO_S4, ASO_S5, and ASO_S6 ASOs exhibit very potent effects in the hSNCA mouse CNS, with ED50 values ​​as low as approximately 11 μg.

[0191]

[0192] Example 6: Measurement of rat nephrotoxicity in vivo

[0193] All ASOs (i.e., ASO_S2, ASO_S3, ASO_S4, ASO_S5, and ASO_S6) were well tolerated in Wista Hanover rats in a 2-week repeated-dose study (two subcutaneous injections of ASO 40 mg / kg on days 1 and 8), and there were no changes in clinical signs or body weight throughout the treatment period. In all treatment groups, serum blood urea nitrogen (BUN) and creatinine levels (indicators of renal function) were within the normal historical range and similar to those of the vehicle control group. At day 15, the change in the urinary KIM-1:creatinine ratio (an index of renal impairment) was considered below the critical threshold and was regarded as the degree of change typically observed in rats under similar study conditions. In conclusion, no evidence of renal dysfunction or histopathological abnormalities was found in mice administered ASOs.

[0194] In vivo method

[0195] Tissue homogenization for RNA measurement. For RNA analysis, a 10-fold volume of Qiazol reagent was added to weighed frozen tissue samples for treatment. The samples were homogenized in 2 mL Eppendorf tubes using 5 mm tungsten carbide beads and shaken using a Qiagen TissueLyzer II (Product No. / ID: 85300) (twice at 27 Hz for 3 minutes). After lysis, the samples were left to stand at room temperature for 5 minutes, after which chloroform was added. The samples were vortexed and left to stand at room temperature for 3 minutes, then centrifuged at 12,000 µg at 4 ℃ for 15 minutes. The aqueous phase was then separated. Subsequently, isopropanol was added, the samples were vortexed and left to stand at room temperature for 10 minutes, and then centrifuged at 12,000 µg at 4 ℃ for 10 minutes to isolate RNA. The generated precipitate was resuspended in 75% ethanol, vortexed, and centrifuged at 7500 lb for 5 minutes at 4°C. The final precipitate was resuspended in water.

[0196] qPCR analysis. qRT-PCR was performed on RNA extracted from tissue lysates to evaluate target mRNA levels. Target mRNA levels were assessed using Taqman probes (hSNCA, mGapdh) and the Express One-Step kit. For each sample hSNCA mRNA levels were normalized relative to the housekeeping gene Gapdh. The glial cell activation markers Gfap and Itgax were measured using the same approach. qRT-PCR was performed using the QuantStudio 6 Flex system (Applied Biosystems), and the mean CT value was calculated for each probe through technical replicate measurements. Next, delta-delta CT values ​​were calculated for the group not treated with ASO and graphed as relative expression levels.

[0197] Tissue homogenization for drug concentration measurement. For biochemical analysis, frozen tissue samples weighed were treated by adding 10 times the volume of cold 1% NP40+PBS homogenization buffer containing cOmplete Protease Inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitors. The samples were homogenized in 1.5 mL Eppendorf tubes using 3 mm tungsten carbide beads and shaken using a Qiagen TissueLyzer II (Product No. / ID: 85300) (twice for 3 minutes at 27 Hz).

[0198] Total ASO Analysis.The total ASO (conjugated and free forms) in mouse plasma and tissue homogenates was quantified using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, a custom biotinylated and digoxigenin-conjugated antisense probe [synthesized by Integrated DNA Technologies (Coralville, Iowa)] at a working concentration was combined with a prepared test sample (pre-diluted where appropriate) and a relevant standard in TE buffer (10 mM Tris-HCl containing 1 mM EDTA). The prepared sample in TE buffer was added in a 1:1 mixture to 1x SSC buffer (Sigma-Aldrich, St. Louis, Missouri) containing a working concentration of recombinant protease K enzyme (ThermoFisher, Waltham, Massachusetts). Subsequently, the hybridization / enzyme mixture was decomposed, denatured, and annealed, and cooled in a thermal cycler. After settling the hybrid product, the sample was added to the wells of an MSD GOLD 96-well streptavidin-coated microquantification plate (Meso Scale Discovery, Rockville, Maryland) and set for approximately 30 minutes. After the settling and plate washing steps, a secondary ruthenylated (sulfo-tagged) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, Colorado) at the working concentration in the assay diluent was added to the plate and set for approximately 30 minutes. After plate washing, 1x MSD Read Buffer T (Meso Scale Discovery, Rockville, Maryland) was added to generate an electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All analytical reaction steps were performed at ambient temperature while shaking on a plate shaker (where appropriate), and all test samples were pre-diluted at an analytical MRD of 1:20 before analysis on the analytical plate.Subsequently, the sample ECLU signals generated from the analysis were processed into concentrations by inversely calculating the analysis calibration (CS) curve. To use in calculating the concentrations for unknown / test samples, the analysis CS curve was fitted to a weighted 4-parameter nonlinear logistic regression.

[0199] Intraventricular bolus (ICV) surgery. The subsequent procedure described in the literature included by reference in this text [DeVos, J Vis Exp (2013)] is briefly summarized as follows: The surgical site was disinfected with 70% ethanol for surgical preparation. The mice were anesthetized with 4% isoflurane. Hair was removed from the shoulder area to the area between the eyes before positioning the mice on the stereotactic fixation surface. An incision was made from the base of the neck to the area between the eyes while maintaining the isoflurane level at 2%. After rinsing with hydrogen peroxide, the needle was slowly inserted into the skull at a speed of 1 mm / s. After waiting 2–3 minutes for the brain area around the needle to seal, a 10 μL dose of ASO was administered at a rate of 1 μL / s. With a cotton swab placed against the skull at the base of the needle, the needle was raised at a speed of 1 mm / s. To limit drug leakage, the cotton swab was held at the injection site for 1 minute. After administering the ICV bolus, the incision was sutured and treated with antibiotic ointment. The mice were transferred to a heated recovery pad to observe for complete recovery. After surgery, the mice were monitored daily to check for pain, discomfort, or infection.

[0200]

Claims

Claim 1 An SNCA antisense oligonucleotide (SNCA ASO) comprising a nucleic acid sequence comprising at least nine consecutive nucleobases of a sequence selected from any one of SEQ ID NOs 1 to 15 and 18 to 23, at least one modified nucleoside linkage, and / or at least one modified sugar. Claim 2 In claim 1, the SNCA ASO is an SNCA ASO composed of 15 to 25 linked nucleosides. Claim 3 In claim 1, the SNCA ASO is an SNCA ASO having a length of 16 to 20 nucleotides. Claim 4 In claim 1, the SNCA ASO is an SNCA ASO of length 16 nucleotides. Claim 5 In claim 1, the SNCA ASO comprises any one of the sequences 24 to 38 and 41 to 46. Claim 6 In any one of claims 1 to 5, the nucleic acid sequence is at least 90% complementary to SEQ ID NO. 49, SNCA ASO. Claim 7 SNCA ASO according to any one of claims 1 to 6, wherein the linkage between at least one modified nucleoside is a linkage between phosphorothioate nucleosides. Claim 8 In any one of claims 1 to 7, the at least one modified sugar is a bicyclic sugar, SNCA ASO. Claim 9 SNCA ASO comprising a nucleic acid sequence described in any one of SEQ ID NOs 1–15, 18–38 and 41–46, comprising at least one modified nucleoside linkage and / or at least one modified sugar. Claim 10 In claim 9, the at least one modified nucleoside linkage is a phosphorothioate nucleoside linkage, SNCA ASO. Claim 11 In claim 9 or 10, the at least one modified sugar is a bicyclic sugar, SNCA ASO. Claim 12 In claim 11, the dicyclic sugar comprises a chemical bridge between the 4' and 2' positions of the sugar, wherein the chemical bridge is independently selected from 4'-CH(R)-O-2' and 4'-(CH2)2-O-2', where R is independently selected from H, C1-C6 alkyl and C1-C6 alkoxy, SNCA ASO. Claim 13 In paragraph 12, the bicyclic sugars each comprise a chemical bridge between the 4' and 2' positions of the said sugar, wherein the chemical bridge is each 4'-CH(R)-O-2', where R is independently H, SNCA ASO. Claim 14 SNCA ASO comprising a modified nucleobase in any one of paragraphs 9 through 13. Claim 15 In paragraph 14, the modified nucleobase is 5-methylcytosine, SNCA ASO. Claim 16 SNCA ASO according to any one of claims 9 to 15, comprising: a 5' wing segment having 1 to 6 nucleosides in the 5' to 3' direction, wherein each nucleoside of the 5' wing segment comprises a modified sugar; a gap segment having 8 to 15 nucleosides, wherein each nucleoside of the gap segment comprises a deoxynucleoside; and a 3' wing segment having 1 to 6 nucleosides, wherein each nucleoside of the 3' wing segment comprises a modified sugar. Claim 17 SNCA ASO according to any one of claims 9 to 16, comprising: a 5' wing segment having three nucleosides in the 5' to 3' direction, wherein each nucleoside of the 5' wing segment comprises a modified sugar; a gap segment having 11 to 13 nucleosides, wherein each nucleoside of the gap segment comprises a deoxynucleoside; and a 3' wing segment having three nucleosides, wherein each nucleoside of the 3' wing segment comprises a modified sugar. Claim 18 SNCA ASO according to the following chemical structure, or a salt thereof: . Claim 19 SNCA ASO according to the following chemical structure, or a salt thereof: . Claim 20 SNCA ASO according to the following chemical structure, or a salt thereof: . Claim 21 SNCA ASO according to the following chemical structure, or a salt thereof: . Claim 22 SNCA ASO according to the following chemical structure, or a salt thereof: . Claim 23 In any one of claims 1 to 22, the SNCA ASO is an SNCA ASO conjugated to a targeting ligand or a delivery vehicle. Claim 24 In paragraph 23, the targeting ligand is an SNCA ASO that specifically binds to a transferrin receptor (TfR) or a molecule expressed on the lumen surface of the blood-brain barrier. Claim 25 A pharmaceutical composition comprising an SNCA ASO of any one of claims 1 to 24 and a pharmaceutically acceptable carrier or diluent. Claim 26 A method for generating neurons, glial cells, or oligodendrocytes with reduced alpha-synuclein expression, comprising the step of delivering an SNCA ASO of any one of claims 1 to 24 to said neurons, said glial cells, or said oligodendrocytes, wherein said SNCA ASO is endogenous SNCA A method for reducing the expression level of a gene. Claim 27 A method for modifying a neuron, a glial cell, or an oligodendrocyte to reduce alpha-synuclein expression, comprising the step of delivering an SNCA ASO according to any one of claims 1 to 24 to the neuron, the glial cell, or the oligodendrocyte, wherein the SNCA ASO is endogenous SNCA A method for reducing the expression level of a gene. Claim 28 A method for modifying a neuron, a glial cell, or an oligodendrocyte to reduce alpha-synuclein expression, comprising the step of delivering an SNCA ASO according to any one of claims 1 to 24 to the neuron, the glial cell, or the oligodendrocyte, wherein the SNCA ASO is in the cell SNCA A method for specifically reducing the expression level of a transcript. Claim 29 In paragraph 28, the above SNCA ASO is SNCA A method for mobilizing RNA degrading enzyme H, which binds to a transcript and degrades the transcript. Claim 30 A method for reducing alpha-synuclein expression in spinal cord cells of a subject, comprising the step of administering an SNCA ASO of any one of claims 1 to 24 or a pharmaceutical composition of claim 25 by intrathecal administration. Claim 31 A method for reducing alpha-synuclein expression in a subject, comprising the step of administering to the subject an SNCA ASO of any one of claims 1 to 23 or a pharmaceutical composition of claim 25. Claim 32 In paragraph 31, a method in which alpha-synuclein expression is reduced in the CNS of the subject. Claim 33 In paragraph 31, the method wherein the SNCA ASO is administered to the subject by intrathecal administration. Claim 34 In any one of paragraphs 26 to 33, the SNCA ASO is endogenous compared to the level where the SNCA ASO was not administered SNCA Gene expression levels or SNCA A method for reducing the level of a transcribed material by at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Claim 35 In Clause 34, the above endogenous SNCA Gene expression or the above SNCA A method in which the level of the transcribed material is reduced by at least about 50%. Claim 36 In Clause 34, the above endogenous SNCA Gene expression or the above SNCA A method in which the level of the transcribed material is reduced by at least about 70%. Claim 37 A method according to any one of claims 31 to 36, wherein the SNCA ASO is administered intravenously to the subject. Claim 38 A method for treating a human subject requiring treatment for an alpha-synuclein-associated neurodegenerative disorder, comprising the step of administering to the human subject an SNCA ASO of any one of claims 1 to 24 or a pharmaceutical composition of claim 25. Claim 39 In paragraph 38, the above alpha-synuclein-associated neurodegenerative syndrome is Parkinson's disease, method. Claim 40 In paragraph 38, the above alpha-synuclein-associated neurodegenerative syndrome is multiple system atrophy, method. Claim 41 A method for treating Parkinson's disease comprising the step of administering the pharmaceutical composition of claim 25 to a human subject requiring treatment. Claim 42 A method for treating multiple system atrophy comprising the step of administering the pharmaceutical composition of claim 25 to a human subject requiring treatment. Claim 43 SNCA A method for reducing messenger ribonucleic acid (mRNA) expression in a human subject requiring reduction of mRNA expression, comprising the step of administering to the human subject an SNCA ASO of any one of claims 1 to 24 or a pharmaceutical composition of claim 25. Claim 44 In claim 25, as a pharmaceutical composition for use in delivering said SNCA ASO to the CNS of a human subject requiring SNCA ASO delivery, said SNCA ASO is endogenous SNCA A pharmaceutical composition that reduces gene expression levels. Claim 45 A pharmaceutical composition for use in treating human subjects requiring treatment for alpha-synuclein-associated neurodegenerative disorders, according to claim 25. Claim 46 In claim 45, the above alpha-synuclein-associated neurodegenerative disorder is Parkinson's disease, a pharmaceutical composition. Claim 47 A pharmaceutical composition according to claim 45, wherein the alpha-synuclein-associated neurodegenerative disorder is multiple system atrophy. Claim 48 In paragraph 25, SNCA A pharmaceutical composition for use in reducing expression in human subjects requiring reduction of mRNA expression.

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