RNAi agent for inhibiting Ataxin-2 (ATXN2) expression, its composition, and method of use.

Abstract

RNAi agents, compositions containing RNAi agents, and methods for inhibiting the Ataxin-2 (ATXN2) gene are described herein. The ATXN2 RNAi agents and RNAi agent conjugates disclosed herein inhibit the expression of the ATXN2 gene. Pharmaceutical compositions containing one or more ATXN2 RNAi agents are also described, along with one or more additional therapeutic agents as may be required. In vivo delivery of the described ATXN2 RNAi agents to central nervous system (CNS) tissues provides inhibition of ATXN2 gene expression and reduction of ATXN2 activity, which may provide therapeutic benefits to subjects, including human subjects, for the treatment of various diseases, including spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 490,866, filed on 17 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy is named 30701-WO_SEQLIST.xml, was created on March 7, 2024, and is 5257kb in size.

[0003] Field of Invention This disclosure relates to RNA interference (RNAi) agents for inhibiting Ataxin-2 ("ATXN2") gene expression, such as double-stranded RNAi agents, compositions comprising ATXN2 RNAi agents, and methods of using the same. [Background technology]

[0004] background Spinocerebellar ataxia type 2 (SCA2) is a dominantly inherited neurodegenerative disorder caused primarily by CAG repeat elongation in exon 1 of the ATXN2 gene, affecting the cerebellum. CAG repeat elongation results in an elongated polyglutamine region and toxic gain-of-function in the ATXN2 protein (Scoles & Pulst, 2018). SCA2 is characterized by progressive ataxia, with a 10-year survival rate of 73% (Diallo et al., 2018). While the ATXN2 gene typically contains 22 or fewer CAG repeats, individuals with SCA2 have 33 or more CAG repeats in the ATXN2 gene. The elongated CAG repeats produce an elongated polyglutamine (poly-Q) region in the ATXN2 protein, which leads to toxic gain-of-function, particularly in neuronal populations essential for motor control and coordination, resulting in cellular dysfunction and cell death. The cerebellum, a brain region that plays a crucial role in motor coordination, is particularly affected in SCA2. Progressive degeneration of neurons in the cerebellum and brainstem leads to characteristic symptoms of SCA2, including ataxia, dysarthria, and oculomotor abnormalities. The exact mechanism by which the elongated ATXN2 protein triggers cell death is under investigation, but it may be involved in disrupting RNA metabolism.

[0005] Ataxin-2 is a protein encoded by the ATXN2 gene. Mutations in ATXN2 are known to cause SCA2 in humans. SCA2 is a progressive degenerative disease for which there is currently no available treatment, and it is often fatal. Patients with SCA2 may experience progressive cerebellar ataxia, slow saccadic eye movements, and other neurological symptoms such as neuropathy.

[0006] ATXN2 CAG elongation is also associated with parkinsonism and amyotrophic lateral sclerosis (ALS), which can be difficult to distinguish from idiopathic forms of these diseases. Due to the role of ATXN2 in SCA2 and other neurodegenerative disorders, therapeutic compounds that can inhibit ATXN2 expression in humans are needed. [Overview of the project] [Means for solving the problem]

[0007] overview Novel RNA interference (RNAi) agents (also called RNAi agents, RNAi triggers, or triggers), such as double-stranded RNAi agents, are needed to selectively and efficiently inhibit the expression of the ATXN2 gene, including for use as therapeutic or pharmaceutical products. Furthermore, there is a need for novel ATXN2-specific RNAi agent compositions for treating diseases or disorders associated with mutant ATXN2 expression, and / or disorders that can be at least partially mediated by reduced ATXN2 gene expression and / or ATXN2 protein expression.

[0008] The nucleotide sequences and chemical modifications of the ATXN2 RNAi agents disclosed herein, as well as their combinations with specific pharmacokinetic and pharmacodynamic (PK / PD) modulators suitable for selective and efficient delivery of the ATXN2 RNAi agents to relevant CNS cells in vivo, differ from those previously disclosed or known in the art. The ATXN2 RNAi agents disclosed herein provide highly potent and efficient inhibition of ATXN2 gene expression.

[0009] Generally, this disclosure features ATXN2 gene-specific RNAi agents, compositions comprising ATXN2 RNAi agents, and methods for inhibiting ATXN2 gene expression in vitro and / or in vivo using the ATXN2 RNAi agents and compositions comprising ATXN2 RNAi agents described herein. The ATXN2 RNAi agents described herein can selectively and efficiently reduce the expression of the ATXN2 gene, thereby reducing the expression of the ATXN2 protein.

[0010] The ATXN2 RNAi agents described may be used in methods for the therapeutic treatment (including preventative or prophylactic treatment) of a variety of central nervous system and neurodegenerative diseases, including SCA2 and ALS.

[0011] In one embodiment, the disclosure features an RNAi agent for inhibiting the expression of the ATXN2 gene, the RNAi agent comprising a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand). The sense strand and antisense strand may be partially, substantially, or completely complementary to each other. The length of the RNAi agent sense strand described herein may be 15 to 49 nucleotides. The length of the RNAi agent antisense strand described herein may each be 18 to 49 nucleotides. In some embodiments, the sense strand and antisense strand are independently 18 to 26 nucleotides long. The sense strand and antisense strand may be the same length or of different lengths. In some embodiments, the sense strand and antisense strand are independently 21 to 26 nucleotides long. In some embodiments, the sense strand and antisense strand are independently 21 to 24 nucleotides long. In some embodiments, both the sense strand and antisense strand are 21 nucleotides long. In some embodiments, the antisense strand is independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the sense strand is independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides long. The RNAi agents described herein, when delivered to ATXN2-expressing cells such as endothelial cells, neurons, microglia, and astrocytes, inhibit the expression of one or more ATXN2 gene variants in vivo and / or in vitro.

[0012] The ATXN2 RNAi agents disclosed herein target the human ATXN2 gene (see, for example, Sequence ID No. 1). In some embodiments, the ATXN2 RNAi agents disclosed herein target a portion of the ATXN2 gene having one of the sequences disclosed in Table 1.

[0013] In another embodiment, the disclosure features a composition comprising one or more disclosed ATXN2 RNAi agents capable of selectively and efficiently reducing the expression of the ATXN2 gene. Compositions comprising one or more ATXN2 RNAi agents as described herein can be administered to subjects such as humans or animals for the treatment (including prophylactic treatment or inhibition) of symptoms and diseases associated with ATXN2 mutant protein activity.

[0014] Tables 3, 4, 5, and 6 provide examples of ATXN2 RNAi agent sense strands and antisense strands that can be used in ATXN2 RNAi agents. Tables 7, 8, 9A, and 10 provide examples of ATXN2 RNAi agent double helical strands. Table 2 provides examples of 19-nucleotide core stretch sequences that may consist of, or may be included in, the sense strands and antisense strands of specific ATXN2 RNAi agents disclosed herein.

[0015] In another embodiment, the disclosure features a method for delivering ATXN2 RNAi agents in vivo to neurons, astrocytes, microglia, and endothelial cells in mammalian and other subjects. Compositions for use in such methods are also described herein. In some embodiments, a method for delivering ATXN2 RNAi agents in vivo to central nervous system cells (neurons, astrocytes, microglia, and endothelial cells) is disclosed herein. In some embodiments, the subjects are human subjects.

[0016] The methods disclosed herein include administration of one or more ATXN2 RNAi agents to a subject, such as a human or animal subject, by any suitable means known in the art. The pharmaceutical compositions disclosed herein containing one or more ATXN2 RNAi agents can be administered in several ways depending on whether local treatment or systemic treatment is desired. Administration can be, for example, intrathecal, intraventricular, intravenous, intraarterial, subcutaneous, intraperitoneal, subcutaneous (e.g., via an implanted device), and parenchymal administration, but is not limited thereto. In some embodiments, the pharmaceutical compositions described herein are administered by intrathecal injection or intraventricular injection.

[0017] In some embodiments, it is desirable for the ATXN2 RNAi agents described herein to inhibit the expression of the ATXN2 gene in central nervous system cells.

[0018] One or more ATXN2 RNAi agents can be delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. In some embodiments, the ATXN2 RNAi agent is delivered to cells or tissues by covalently linking the RNAi agent to a targeting moiety or lipid moiety.

[0019] The PK / PD modulator can be linked to the 3' or 5' end of the sense or antisense strand of the ATXN2 RNAi agent. In some embodiments, the PK / PD modulator is linked to the 3' or 5' end of the sense strand. In some embodiments, the PK / PD modulator is linked to the 5' end of the sense strand. In some embodiments, the PK / PD modulator is internally linked to nucleotides on the sense and / or antisense strand of the RNAi agent. In some embodiments, the PK / PD modulator is linked to the RNAi agent via a linker.

[0020] In another aspect, the present disclosure features a composition comprising one or more ATXN2 RNAi agents having the double-stranded structures disclosed in Tables 7, 8, 9A, and 10.

[0021] The use of an ATXN2 RNAi agent provides a method for the therapeutic (including prophylactic) treatment of a disease or disorder in which a decrease in ATXN2 protein activity may provide a therapeutic benefit. The ATXN2 RNAi agents disclosed herein can be used to treat various neurodegenerative diseases including SCA2 and ALS. Such methods of treatment include the administration of an ATXN2 RNAi agent to a human or animal having elevated or mutant ATXN2 protein or ATXN2 protein activity above desirable levels.

[0022] As used herein, the terms "oligonucleotide" and "polynucleotide" each independently mean a polymer of linked nucleosides that can be modified or unmodified.

[0023] As used herein, “RNAi agent” (also called “RNAi trigger”) means a chemical composition of a substance comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specific degradation or inhibition (e.g., degradation or inhibition under appropriate conditions) of the translation of a messenger RNA (mRNA) transcript of a target mRNA. As used herein, an RNAi agent may act by an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway. While RNAi agents are considered to act primarily by RNA interference mechanisms as used herein, the RNAi agents disclosed are not bound by any particular pathway or mechanism of action, and are not limited thereto. The RNAi agents disclosed herein consist of a sense strand and an antisense strand and include, but are not limited to, small (or short) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA (i.e., ATXN2 mRNA). The RNAi agent may contain one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0024] As used herein, the terms “silence,” “reduce,” “inhibit,” “downregulate,” or “knockdown,” when referring to the expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA, in a cell, cell population, tissue, organ, or subject on which the gene is transcribed, is reduced compared to a second cell, cell population, tissue, organ, or subject that has not been treated in the same manner, when the cell, cell population, tissue, organ, or subject has been treated with an RNAi agent as described herein.

[0025] As used herein, the terms “sequence” and “nucleotide sequence” mean a sequence or order of nucleic acid bases or nucleotides written as a sequence of letters using standard nomenclature.

[0026] As used herein, “base,” “nucleotide base,” or “nucleic acid base” refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, including the major purine bases adenine and guanine, as well as the major pyrimidine bases cytosine, thymine, and uracil. Nucleic acid bases may be further modified to include, but are not limited to, universal bases, hydrophobic bases, indiscriminate bases, size-expanded bases, and fluorinated bases. (See, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleic acid bases (including phosphoramidite compounds containing modified nucleic acid bases) is known in the art.

[0027] Where used herein, unless otherwise specified, the term “complementary” means that when used to describe a first nucleic acid base or nucleotide sequence (e.g., RNAi agent sense strand or target mRNA) with respect to a second nucleic acid base or nucleotide sequence (e.g., RNAi agent antisense strand or single-stranded antisense oligonucleotide), the oligonucleotide or polynucleotide containing the first nucleotide sequence hybridizes with the oligonucleotide containing the second nucleotide sequence (under mammalian physiological conditions (or other appropriate in vivo or in vitro conditions) and base-pair hydrogen bonding). This refers to the ability to form a double-stranded or double-helical structure under specific standard conditions. Those skilled in the art will be able to select the set of conditions best suited for hybridization testing. Complementary sequences include natural or modified nucleotides or nucleotide mimeographs, and include Watson-Crick base pairs or non-Watson-Crick base pairs, as long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification. For example, a and Af as defined herein are complementary to U (or T) and identical to A for the purpose of determining identity or complementarity.

[0028] As used herein, “perfectly complementary” or “fully complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, all (100%) of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may consist of all or part of the first or second nucleotide sequence.

[0029] As used herein, “partially complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, at least 70% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0030] As used herein, “substantially complementary” means that in a hybridized pair of nucleic acid bases or nucleotide sequence molecules, at least 85% of, but not all, of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0031] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used in reference to the matching of nucleic acid bases or nucleotides between the sense strand and antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of ATXN2 mRNA.

[0032] As used herein, the terms “substantially identical” or “substantially identical” applied to nucleic acid sequences mean that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or more sequence identity, e.g., at least 90%, at least 95%, or at least 99%, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window. The percentage is calculated by determining the number of positions where the same type of nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The inventions disclosed herein encompass nucleotide sequences that are substantially identical to those disclosed herein.

[0033] As used herein, the terms “to treat,” “treatment,” etc., mean a method or step taken to provide relief or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, “to treat” and “treatment” may include prevention, control, or prophylactic treatment of one or more symptoms of a disease in a subject, and / or inhibition or reduction of their number, severity, and / or frequency.

[0034] As used herein, the phrase “introducing into cells” when referring to RNAi agents means functionally delivering the RNAi agent to cells. The phrase “functional delivery” means delivering the RNAi agent to cells in a manner that enables the RNAi agent to have its expected biological activity, such as sequence-specific inhibition of gene expression.

[0035] Unless otherwise specified, the symbols used in this specification [ka] The use of means that any one or more bases in accordance with the scope of the invention described herein may be linked thereto.

[0036] As used herein, the term “isomer” refers to compounds that have the same molecular formula but differ in the nature or arrangement of the bonds between their atoms or in the spatial arrangement of those atoms. Isomers that differ in the spatial arrangement of their atoms are called “stereoisomers.” Stereoisomers that are not mirror images of each other are called “diastereomers,” and stereoisomers that are mirror images that cannot be superimposed are called “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a “chiral center.”

[0037] Where used herein, unless specifically identified as having a particular conformation in its structure, each structure disclosed herein is intended to represent all such possible isomers, including optically pure and racemic forms, for each structure in which a chiral center is present and thus results in an enantiomer, diastereomer, or other stereoisomer configuration. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0038] As used in the claims of this specification, the phrase "consisting of" excludes any element, step, or component not specified in the claims. As used in the claims of this specification, the phrase "consisting essentially of" limits the claims to specific materials or steps and any basic and novel features(s) of the claimed invention that do not substantially affect them.

[0039] Those skilled in the art will readily understand and recognize that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is placed. Therefore, when used herein, the structures disclosed herein assume that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of their protonation state based on the environment (e.g., pH), as will be readily understood by those skilled in the art. Accordingly, compounds described herein that have an unstable proton or basic atom should also be understood to represent a salt form of the corresponding compound. The compounds described herein may be in the form of free acids, free bases, or salts. A pharmaceutically acceptable salt of a compound described herein should be understood to be within the scope of the invention.

[0040] As used herein, the terms “linked” or “conjugated” refer to a connection between two compounds or molecules, meaning that the two compounds or molecules are joined by a covalent bond. Unless otherwise specified herein, the terms “linked” and “conjugated” may refer to a connection between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0041] As used herein, the term “including” is used herein to mean the phrase “including but not limited to” and is interchangeable with it. The term “or” is used herein to mean the term “and / or” and is interchangeable with it unless the context clearly indicates otherwise.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing the present invention, but suitable methods and materials are listed below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope of the invention.

[0043] Other objects, features, embodiments, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims. [Modes for carrying out the invention]

[0044] Detailed explanation RNAi agents RNAi agents (referred to herein as ATXN2 RNAi agents or ATXN2 RNAi triggers) for inhibiting the expression of the ATXN2 (or ATXN2) gene are described herein. Each ATXN2 RNAi agent disclosed herein comprises a sense strand and an antisense strand. The sense strand and antisense strand may each be 15 to 49 nucleotides long. The antisense strand may be 18 to 30 nucleotides long. The antisense strand may be the same length as or different in length. In some embodiments, the sense strand and antisense strand are each independently 18 to 27 nucleotides long. In some embodiments, both the sense strand and antisense strand are each 21 to 26 nucleotides long. In some embodiments, the sense strand and antisense strand are each 21 to 24 nucleotides long. In some embodiments, the sense strand and antisense strand are each independently 19 to 21 nucleotides long. In some embodiments, the sense strand is about 19 nucleotides long, while the antisense strand is about 21 nucleotides long. In some embodiments, the sense strand is approximately 21 nucleotides long, while the antisense strand is approximately 23 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides long. In some embodiments, the RNAi agent sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides long. In some embodiments, the RNAi agent antisense strand is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the double-stranded RNAi agent has a double-strand length of approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0045] Tables 2, 3, 4, 5, 6, and 10 provide examples of nucleotide sequences used to form ATXN2 RNAi agents. Tables 7, 8, 9A, and 10 show examples of RNAi agent double helices, including the sense and antisense strand sequences in Tables 2, 3, 4, 5, and 6.

[0046] In some embodiments, the region of complete, substantial, or partial complementarity between the sense strand and the antisense strand is 16–26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides long and occurs at or near the 5' end of the antisense strand (e.g., this region may be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not fully, substantially, or partially complementary).

[0047] The sense strand of the ATXN2 RNAi agent described herein comprises at least 15 consecutive nucleotides having at least 85% identity with the core stretch sequence (hereinafter also referred to herein as the “core stretch” or “core sequence”) of the same number of nucleotides in the ATXN2 mRNA. In some embodiments, the sense strand core stretch sequence is 100% (completely) complementary or at least about 85% (substantially) complementary to the core stretch sequence in the antisense strand, and therefore the sense strand core stretch sequence is typically completely identical or at least about 85% identical to a nucleotide sequence of the same length (sometimes referred to as the target sequence) present in the ATXN2 mRNA target. In some embodiments, this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this sense strand core stretch is 17 nucleotides long. In some embodiments, this sense strand core stretch is 19 nucleotides long.

[0048] The antisense strand of the ATXN2 RNAi agent described herein comprises at least 16 consecutive nucleotides having at least 85% complementarity to the same number of nucleotides in the ATXN2 mRNA core stretch and the same number of nucleotides in the corresponding sense strand core stretch. In some embodiments, the antisense strand core stretch is 100% (completely) complementary or at least about 85% (substantially) complementary to a nucleotide sequence of the same length present in the ATXN2 mRNA target (e.g., the target sequence). In some embodiments, this antisense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this antisense strand core stretch is 19 nucleotides long. In some embodiments, this antisense strand core stretch is 17 nucleotides long. The sense strand core stretch sequence may be the same length as the corresponding antisense core sequence or may be of a different length.

[0049] ATXN2 RNAi agents consist of a sense strand and an antisense strand that anneal to form a double helix. The sense strand and antisense strand of an ATXN2 RNAi agent may be partially, substantially, or completely complementary to each other. Within the complementary double helix region, the sense strand core stretch sequence is at least 85% or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% or 100% complementary to the corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of an ATXN2 RNAi agent have regions of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that are at least 85% or 100% base-paired).

[0050] In some embodiments, the antisense strand of the ATXN2 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 or Table 3 by only 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the ATXN2 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10 by only 0, 1, 2, or 3 nucleotides.

[0051] In some embodiments, the sense strand and / or antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3' end, 5' end, or both the 3' and 5' ends of the core stretch sequence. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding sequence in the ATXN2 mRNA. The additional nucleotides of the sense strand, if present, may or may not be identical to the corresponding sequence in the ATXN2 mRNA. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding additional nucleotides of the sense strand.

[0052] As used herein, the extension includes 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' ends of the sense strand core stretch sequence and / or antisense strand core stretch sequence. The extension nucleotides on the sense strand may or may not be complementary to any nucleotide in the corresponding antisense strand, either the core stretch sequence nucleotide or the extension nucleotide. Conversely, the extension nucleotides on the antisense strand may or may not be complementary to any nucleotide in the corresponding sense strand (either the core stretch nucleotide or the extension nucleotide). In some embodiments, both the sense and antisense strands of the RNAi agent include 3' and 5' extensions. In some embodiments, one or more 3' extension nucleotides on one strand base-pair with one or more 5' extension nucleotides on the other strand. In other embodiments, one or more 3' extension nucleotides on one strand do not base-pair with one or more 5' extension nucleotides on the other strand. In some embodiments, the ATXN2 RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, the extension nucleotide(s) are unpaired and form an overhang. As used herein, “overhang” refers to one or more unpaired nucleotides located at the terminal end of either the sense strand or the antisense strand that do not form part of the hybridized or duplexed portion of the RNAi agent disclosed herein.

[0053] In some embodiments, the ATXN2 RNAi agent comprises an antisense chain having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the ATXN2 RNAi agent comprises an antisense chain having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more antisense chain extension nucleotides comprise nucleotides complementary to the corresponding ATXN2 mRNA sequence. In some embodiments, one or more antisense chain extension nucleotides comprise nucleotides that are not complementary to the corresponding ATXN2 mRNA sequence.

[0054] In some embodiments, the ATXN2 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more sense strand extension nucleotides include adenosine, uracil, or thymidine nucleotides, AT dinucleotides, or nucleotides corresponding to or identical to nucleotides in the ATXN2 mRNA sequence. In some embodiments, the 3' sense strand extension comprises, but is not limited to, one of the following sequences: T, UT, TT, UU, UUT, TTT, or TTTT (each listed from 5' to 3').

[0055] The sense strand may have a 3' extension and / or a 5' extension. In some embodiments, the ATXN2 RNAi agent includes a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more sense strand extension nucleotides include nucleotides that correspond to or are identical to nucleotides in the ATXN2 mRNA sequence.

[0056] Tables 2, 3, 4, 5, 6, and 10 provide examples of sequences used in the formation of ATXN2 RNAi agents. In some embodiments, the ATXN2 RNAi agent antisense strand includes one of the sequences in Tables 2, 3, or 10. In certain embodiments, the ATXN2 RNAi agent antisense strand includes or consists of one of the modified sequences in Table 3. In some embodiments, the ATXN2 RNAi agent antisense strand includes the nucleotide sequences (5' end → 3' end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 of any of the sequences in Table 2 or Table 3. In some embodiments, the ATXN2 RNAi agent sense strand includes one of the sequences in Tables 2, 4, 5, or 6. In some embodiments, the ATXN2 RNAi agent sense strand contains a sequence of nucleotides 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 (5' end → 3' end) of any of the sequences in Table 2, 4, 5, or 6. In certain embodiments, the ATXN2 RNAi agent sense strand contains or consists of one of the modified sequences in Table 4, 5, 6, or 10.

[0057] In some embodiments, the sense and antisense strands of the RNAi agent described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agent described herein contain different numbers of nucleotides. In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a blunt end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form a blunt end. In some embodiments, both ends of the RNAi agent form a blunt end. In some embodiments, neither end of the RNAi agent is a blunt end. As used herein, “blunt end” refers to the end of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands are complementary (form a complementary base pair).

[0058] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a frayed end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form a frayed end. In some embodiments, both ends of the RNAi agent form a frayed end. In some embodiments, neither end of the RNAi agent is a frayed end. As used herein, a frayed end refers to the end of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands form a pair (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of a double-stranded RNAi agent form an overhang. Unpaired nucleotides may be present on the sense strand or antisense strand, resulting in either a 3' overhang or a 5' overhang. In some embodiments, the RNAi agent includes a blunt end and a frayed end, a blunt end and a 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends. Typically, if present, the overhang is located at the 3' terminal end of the sense strand, the antisense strand, or both the sense and antisense strands.

[0059] The ATXN2 RNAi agents disclosed herein may also consist of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand of the ATXN2 RNAi agent are modified nucleotides. The ATXN2 RNAi agents disclosed herein may further consist of one or more modified nucleoside bonds, for example, one or more phosphorothioate bonds. In some embodiments, the ATXN2 RNAi agent contains one or more modified nucleotides and one or more modified nucleoside bonds. In some embodiments, 2'-modified nucleotides are combined with modified nucleoside bonds.

[0060] In some embodiments, the ATXN2 RNAi agent is prepared or provided as a salt, a mixed salt, or a free acid. In some embodiments, the ATXN2 RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, the ATXN2 RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms well known in the art are within the scope of the invention disclosed herein.

[0061] Modified nucleotides Modified nucleotides, when used in various oligonucleotide constructs, can protect the activity of compounds in cells while simultaneously increasing the serum stability of these compounds, and can minimize the potential for activating interferon activity in humans when oligonucleotide constructs are administered.

[0062] In some embodiments, the ATXN2 RNAi agent comprises one or more modified nucleotides. As used herein, “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxylnucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides may include, but are not limited to, deoxyribonucleotides, nucleotide mimes, debasalized nucleotides, 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleic acid bases, cross-linked nucleotides, peptide nucleic acids (PNAs), 2',3'-seconucleotide mimes (unlocked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy(2'-nucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholinonucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. Examples of 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a five-membered sugar ring) include, but are not limited to, 2'-O-methylnucleotide (also known herein or in the art as 2'-methoxynucleotide), 2'-fluoronucleotide (also known herein or in the art as 2'-deoxy-2'-fluoronucleotide), 2'-deoxynucleotide, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotide (also known herein or in the art as 2'-MOE nucleotide), 2'-aminonucleotide, and 2'-alkylnucleotide. Not all positions in a given compound need to be uniformly modified. Conversely, more than one modification can be incorporated into a single ATXN2 RNAi agent, or even into its single nucleotide. The sense and antisense strands of an ATXN2 RNAi agent can be synthesized and / or modified by methods known in the art.Modifications in one nucleotide are independent of modifications in other nucleotides.

[0063] Modified nucleic acid bases include synthetic and natural nucleic acid bases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl or 6-n-butyl) derivatives, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl or 2-n-butyl) and other adenine and guanine alkyl derivatives, and 2-methyl Examples include ouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azouracil, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0064] In some embodiments, the 5' and / or 3' ends of the antisense strand may contain a debasic residue (Ab), which may also be called a “debasic site” or “debasic nucleotide.” A debasic residue (Ab) is a nucleotide or nucleoside lacking a nucleic acid base at the 1' position of the sugar moiety. (See, for example, U.S. Patent No. 5,998,203). In some embodiments, the debasic residue may be located within a nucleotide sequence. In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may contain one or more additional debasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab is added to the 3' end of the sense strand. In some embodiments, the debasic (deoxyribose) residue may be replaced with a ribitol (debasic ribose) residue.

[0065] In some embodiments, all or substantially all of the nucleotides in an RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand, which are ribonucleotides (i.e., unmodified). As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand, which is an unmodified ribonucleotide. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the antisense strand, which is an unmodified ribonucleotide. In some embodiments, one or more nucleotides in an RNAi agent are unmodified ribonucleotides. The chemical structures for specific modified nucleotides are shown in Table 11 herein.

[0066] Inter-modified nucleoside bonding In some embodiments, one or more nucleotides of the ATXN2 RNAi agent are linked by non-standard bindings or skeletons (i.e., modified nucleoside bindings or modified skeletons). Modified nucleoside bonds or skeletons include, but are not limited to, phosphorothioate groups (represented herein by lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl phosphonates, thionoalkylphosphotriesters, morpholino bonds, boranophosphates having the usual 3'-5' bond, 2'-5' bonded analogs of boranophosphates, or boranophosphates with reverse polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified nucleoside bond or skeleton lacks a phosphorus atom. Examples of modified nucleoside bonds lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl sugar bonds, mixed heteroatoms and alkyl or cycloalkyl sugar bonds, or one or more short-chain heteroatoms or heterocyclic sugar bonds. In some embodiments, examples of modified nucleoside skeletons include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons with a mixture of N, O, S and CH2 components.

[0067] In some embodiments, the sense strand of the ATXN2 RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, the antisense strand of the ATXN2 RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds.

[0068] In some embodiments, the ATXN2 RNAi agent sense strand contains at least two phosphorothioate nucleoside bonds. In some embodiments, the phosphorothioate nucleoside bonds are located between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate nucleoside bond is located at the 5' end of the sense strand nucleotide sequence, and another phosphorothioate bond is located at the 3' end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate nucleoside bonds are located at the 5' end of the sense strand, and another phosphorothioate bond is located at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate nucleoside bonds between nucleotides, but contains one, two, or three phosphorothioate bonds between both the 5' and 3' terminal nucleotides and the inverted debasal residue terminal caps, if present. In some embodiments, the targeting ligand is ligated to the sense strand via phosphorothioate bonds.

[0069] In some embodiments, the ATXN2 RNAi agent antisense strand contains four phosphorothioate nucleoside bonds. In some embodiments, the four phosphorothioate nucleoside bonds are located between nucleotides at positions 1–3 from the 5' end of the antisense strand and between nucleotides at positions 19–21, 20–22, 21–23, 22–24, 23–25, or 24–26 from the 5' end. In some embodiments, three phosphorothioate nucleoside bonds are located between positions 1–4 from the 5' end of the antisense strand, and a fourth phosphorothioate nucleoside bond is located between positions 20–21 from the 5' end of the antisense strand. In some embodiments, the ATXN2 RNAi agent contains at least three or four phosphorothioate nucleoside bonds on the antisense strand.

[0070] Capping residue or portion In some embodiments, the sense strand may include one or more capping residues or portions, which are sometimes referred to in the art as “caps,” “terminal caps,” or “capping residues.” As used herein, “capping residues” are non-nucleotide compounds or other portions that can be incorporated into one or more terminals of the nucleotide sequence of an RNAi agent disclosed herein. In some examples, capping residues can provide the RNAi agent with certain beneficial properties, such as protection against exonuclease degradation. In some embodiments, inverted debase residues (invAb) (also referred to in the art as “inverted debase sites”) are added as capping residues (see Table 11). (See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are commonly known in the art, and include, for example, inverted debase residues and terminal C3H7 (propyl), C6H 13 (Hexyl) or C 12 H 25The capping residue includes a carbon chain such as a (dodecyl) group. In some embodiments, the capping residue is located at either the 5' or 3' terminus of the sense chain, or at both the 5' and 3' terminus. In some embodiments, the 5' and / or 3' terminus of the sense chain may contain one or more inverted debasic deoxyribose moieties as capping residues.

[0071] In some embodiments, one or more inverted debase residues (invAbs) are added to the 3' end of the sense strand. In some embodiments, one or more inverted debase residues (invAbs) are added to the 5' end of the sense strand. In some embodiments, one or more inverted debase residues or sites are inserted between the targeting ligand and the nucleotide sequence of the RNAi agent's sense strand. In some embodiments, including one or more inverted debase residues or sites at or near the terminal end of the RNAi agent's sense strand can enhance the activity or other desired properties of the RNAi agent.

[0072] In some embodiments, one or more inverted debasing residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted debasing residues may be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted debasing residues may be linked via phosphate, phosphorothioate (e.g., indicated herein as (invAb)) or other nucleoside bonds. In some embodiments, including one or more inverted debasing residues at or near the terminal end of one or more of the sense strands of the RNAi agent may enhance the activity or other desired properties of the RNAi agent. In some embodiments, the inverted debasing (deoxyribose) residue may be replaced with an inverted ribitol (debased ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence or the 3' end of the antisense strand sequence may contain an inverted debasing residue. The chemical structures of the inverted debased deoxyribose residues are shown in Table 11 below.

[0073] ATXN2 RNAi agent The ATXN2 RNAi agents disclosed herein are designed to target specific locations on the ATXN2 gene (e.g., Sequence ID No. 1 (NM_001310123.1)). As defined herein, an antisense strand sequence is designed to target the ATXN2 gene at a given location on the gene when the 5' terminal nucleic acid base of the antisense strand aligns with a location 21 nucleotides downstream (towards the 3' end) from the location on the gene when base-pairing with the gene. For example, as shown in Tables 1 and 2 herein, an antisense strand sequence designed to target the ATXN2 gene at position 304 requires that the 5' terminal nucleic acid base of the antisense strand aligns with position 324 on the ATXN2 gene when base-pairing with the gene.

[0074] Where provided herein, ATXN2 RNAi agents do not require the nucleic acid base at position 1 (5'→3') of the antisense strand to be complementary to the gene, provided that there is at least 85% complementarity between the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity). For example, in the case of an ATXN2 RNAi agent disclosed herein designed to target position 304 of the ATXN2 gene, the 5' terminal nucleic acid base of the antisense strand of the ATXN2 RNAi agent must be aligned with position 324 of the gene, but the 5' terminal nucleic acid base of the antisense strand may, but is not necessarily, be complementary to position 324 of the ATXN2 gene, provided that there is at least 85% complementarity between the antisense strand and the gene transcript across a core stretch sequence of at least 16 consecutive nucleotides (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity). In particular, as illustrated by the various examples disclosed herein, the specific gene binding site of the antisense strand of an ATXN2 RNAi agent (e.g., whether the ATXN2 RNAi agent is designed to target the ATXN2 gene at position 127, position 130, position 136, or some other position) is a crucial factor for the level of inhibition achieved by the ATXN2 RNAi agent. (See, for example, Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), Figure 1 (2015)).

[0075] In some embodiments, the ATXN2 RNAi agents disclosed herein target the ATXN2 gene at or near the location of the ATXN2 sequence shown in Table 1. In some embodiments, the antisense strand of the ATXN2 RNAi agents disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to the 19-mer sequence of the target ATXN2 disclosed in Table 1. [Table 1-1] [Table 1-2] Homo sapiens Ataxin-2 (ATXN2), GenBank NM_001310123.1 (SEQ ID NO: 1), gene transcript (3755 base pairs): [ka] [ka] [ka]

[0076] In some embodiments, the ATXN2 RNAi agent includes an antisense strand, and position 19 (5'→3') of the antisense strand can form a base pair with position 1 of the 19-mer target sequence disclosed in Table 1.

[0077] In some embodiments, the ATXN2 agent includes an antisense strand, and position 2 (5'→3') of the antisense strand can base-pair with position 18 of the 19-mer target sequence disclosed in Table 1. In some embodiments, the ATXN2 agent includes an antisense strand, and positions 2-18 (5'→3') of the antisense strand can base-pair with each of the complementary bases located at positions 18-2 of the 19-mer target sequence disclosed in Table 1.

[0078] In the case of the RNAi agents disclosed herein, the nucleotide at position 1 (5' end → 3' end) of the antisense strand may be either perfectly complementary to the ATXN2 gene or non-complementary to the ATXN2 gene. In some embodiments, the nucleotide at position 1 (5' end → 3' end) of the antisense strand is U, A, or dT. In some embodiments, the nucleotide at position 1 (5' end → 3' end) of the antisense strand forms an A:U or U:A base pair with the sense strand.

[0079] In some embodiments, the ATXN2 RNAi antisense strand includes a sequence of 2-18 or 2-19 nucleotides (5' end to 3' end) from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the ATXN2 RNAi sense strand includes a sequence of 1-17, 1-18 or 2-18 nucleotides (5' end to 3' end) from any of the sense strand sequences in Table 2, Table 4, Table 5 or Table 6.

[0080] In some embodiments, the ATXN2 RNAi agent comprises (i) an antisense strand containing a sequence of 2-18 or 2-19 nucleotides (5' end to 3' end) of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand containing a sequence of 1-17 or 1-18 nucleotides (5' end to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5 or Table 6.

[0081] In some embodiments, the ATXN2 RNAi agent contains the core 19-mer nucleotide sequence shown in Table 2 below. Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 [Table 2-18]

[0082] The sense and antisense strands of an ATXN2 RNAi agent containing or consisting of the nucleotide sequences in Table 2 may be modified or unmodified nucleotides. In some embodiments, an ATXN2 RNAi agent having sense and antisense strand sequences containing or consisting of any of the nucleotide sequences in Table 2 is entirely or substantially entirely modified nucleotides.

[0083] In some embodiments, the antisense strand of the ATXN2 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 by only 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the ATXN2 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2 by only 0, 1, 2, or 3 nucleotides.

[0084] When used herein, each N listed in the sequences disclosed in Table 2 may be independently selected from any and all nucleic acid bases (including those found in both modified and unmodified nucleotides). In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are complementary to the N nucleotide at the corresponding position in the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are not complementary to the N nucleotide at the corresponding position in the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are the same as the N nucleotide at the corresponding position in the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are different from the N nucleotide at the corresponding position in the other strand.

[0085] The sense and antisense strands of specific modified ATXN2 RNAi agents are provided in Tables 3, 4, 5, 6, and 10. The antisense strands of specific modified ATXN2 RNAi agents, as well as their underlying unmodified nucleic acid sequences, are provided in Table 3. The sense strands of specific modified ATXN2 RNAi agents, as well as their underlying unmodified nucleic acid sequences, are provided in Tables 4, 5, and 6. In the formation of the ATXN2 RNAi agent, each nucleotide in each of the underlying sequences listed in Tables 3, 4, 5, and 6, and in Table 2 above, may be a modified nucleotide.

[0086] The ATXN2 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing the sequences listed in Table 2, Table 4, Table 5, or Table 6 can be hybridized to any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity across a sequence of 16, 17, 18, 19, 20, or 21 consecutive nucleotides.

[0087] In some embodiments, the ATXN2 RNAi agent antisense strand comprises a nucleotide sequence from either Table 2 or Table 3.

[0088] In some embodiments, the ATXN2 RNAi agent comprises or consists of a double helix having the nucleic acid base sequences of the sense strand and antisense strand of any of the sequences in Table 2, Table 3, Table 4, Table 5, Table 6, or Table 10.

[0089] Examples of antisense strands containing modified nucleotides are provided in Table 3. Examples of sense strands containing modified nucleotides are provided in Tables 4, 5, and 6.

[0090] When used in Tables 3, 4, 5, 6, and 10, the following notation is used to indicate modified nucleotides, targeting groups, and linking groups. A = adenosine-3'-phosphate C = cytidine-3'-phosphate G = Guanosine-3'-phosphate U = uridine-3'-phosphate I = Inosine-3'-phosphate a=2'-O-methyladenosine-3'-phosphate as=2'-O-methyladenosine-3'-phosphorothioate c=2'-O-methylcytidine-3'-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i=2'-O-methylinosine-3'-phosphate is=2'-O-methylinosine-3'-phosphorothioate t=2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us=2'-O-methyluridine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate dT = 2'-deoxythymidine-3'-phosphate A UNA = 2',3'-seco-adenosine-3'-phosphate A UNA s = 2',3'-seco-adenosine-3'-phosphorothioate C UNA = 2',3'-seco-cytidine-3'-phosphate C UNA s = 2',3'-seco-cytidine-3'-phosphorothioate G UNA = 2',3'-seco-guanosine-3'-phosphate G UNA s = 2',3'-seco-guanosine-3'-phosphorothioate U UNA = 2',3'-seco-uridine-3'-phosphate U UNA s = 2',3'-seco-uridine-3'-phosphorothioate a_2N = Refer to Table 11 a_2Ns = Refer to Table 11 (invAb) = Inverted abasic deoxyribonucleotide-5'- phosphate, refer to Table 11<​​​​​​​​​​​​​​​​cPrpus = 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphorothioate (see Table 11) (Alk-SS-C6)=See Table 11 (C6-SS-Alk)=See Table 11 (C6-SS-C6)=See Table 11 (6-SS-6) = See Table 11. (C6-SS-Alk-Me)=See Table 11 (NH2-C6) = See Table 11. -C6- = See Table 11 -C6s- = See Table 11 -L6-C6-=See Table 11 -L6-C6s-=See Table 11 LP183s = See Table 11. LP183rs = See Table 11 cC16 = See Table 11. aC16 = See Table 11. gC16 = See Table 11. uC16 = See Table 11 ALNA = See Table 11. c16s = See Table 11. C22s = See Table 11. HO-C16s = See Table 11. LP293 = See Table 11 LP310 = See Table 11

[0091] As will be readily apparent to those skilled in the art, unless otherwise indicated by the sequence (e.g., by a phosphorothioate bond "s"), nucleotide monomers in oligonucleotides are linked to each other by a 5'-3'-phosphodiester bond. As will be clearly apparent to those skilled in the art, the inclusion of a phosphorothioate bond, as shown in the modified nucleotide sequences disclosed herein, replaces the phosphodiester bond typically present in oligonucleotides. Furthermore, those skilled in the art will readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence typically has a hydroxyl (-OH) group at the 3' position of each given monomer in ex vivo, instead of a phosphate moiety. Furthermore, in the embodiments disclosed herein, observing each chain 5'→3', the inverted debasing residue is inserted such that the 3' position of the deoxyribose is linked to the 3' end of the preceding monomer on each chain (see, for example, Table 11). Furthermore, as will be readily understood and recognized by those skilled in the art, the phosphorothioate chemical structures shown herein typically represent an anion on a sulfur atom, but the inventions disclosed herein encompass all phosphorothioate tautomers (e.g., when the sulfur atom has a double bond and the anion is on an oxygen atom). Unless otherwise expressly indicated herein, such understanding of those skilled in the art is used when describing the ATXN2 RNAi agents and compositions of ATXN2 RNAi agents disclosed herein.

[0092] Specific examples of targeting and linking groups used with the ATXN2 RNAi agents disclosed herein are included in the chemical structures provided in Table 11 below. Each sense strand and / or antisense strand may have any of the targeting or linking groups listed herein, as well as other targeting or linking groups conjugated to the 5' and / or 3' ends of the sequence. [Table 3-1] [Table 3-2] Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 6-1 Table 6-2 Table 6-3 [Table 6-4] [Table 6-5]

[0093] The ATXN2 RNAi agents disclosed herein are formed by annealing an antisense strand with a sense strand. A sense strand containing the sequences listed in Table 2, Table 4, Table 5, or Table 6 can be hybridized to any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity across a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0094] As shown in Table 5 above, certain exemplary ATXN2 RNAi agent nucleotide sequences are shown to further include reactive linking groups at one or both of the 5' and 3' terminal ends of the sense strand. For example, many of the ATXN2 RNAi agent sense strand sequences shown in Table 5 above have a (NH2-C6) linking group at the 5' end of the nucleotide sequence. Other linking groups, such as a (6-SS-6) linking group or a (C6-SS-C6) linking group, may also be present, or in certain embodiments. Such reactive linking groups are positioned to facilitate the linking of targeting ligands, targeting groups, and / or PK / PD modulators to the ATXN2 RNAi agents disclosed herein. Linking or conjugation reactions are well known in the art and provide the formation of a covalent bond between two molecules or reactants. Suitable conjugation reactions for use within the scope of the present invention as described herein include, but are not limited to, amide coupling reactions, Michael addition reactions, hydrazone formation reactions, inverse-demand Diels-Alder cycloaddition reactions, oxim ligation, and copper(I)-catalyzed or strain-promoted azide-alkyne cycloaddition reactions.

[0095] In some embodiments, the targeting ligand may be synthesized as an activated ester, such as a tetrafluorophenyl (TFP) ester, which can be replaced by a reactive amino group (e.g., NH2-C6) to conjugate the targeting ligand to the ATXN2 RNAi agent disclosed herein. In some embodiments, the targeting ligand may be synthesized as an azide that can be conjugated to a propargyl or DBCO group, for example, via a copper(I) catalyzed or strain-promoting azide-alkyne cycloaddition reaction.

[0096] Furthermore, certain nucleotide sequences can be synthesized using a dT nucleotide at the 3' terminal end of the sense strand, followed by a (3'→5') linker (e.g., C6-SS-C6). In some embodiments, the linker can facilitate binding to additional components, such as lipids or one or more targeting ligands. As described herein, the disulfide bond of C6-SS-C6 can be first reduced to remove dT from the molecule, and then facilitate conjugation of the desired component. Thus, the terminal dT nucleotide is not part of the fully conjugated construct.

[0097] In some embodiments, the antisense strand of the ATXN2 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 3 or Table 10 by only 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the ATXN2 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 4, Table 5, Table 6, or Table 10 by only 0, 1, 2, or 3 nucleotides.

[0098] In some embodiments, the ATXN2 RNAi agent antisense strand includes a nucleotide sequence from either Table 2 or Table 3. In some embodiments, the ATXN2 RNAi agent antisense strand includes a nucleotide sequence (5' end → 3' end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 from either Table 2, Table 3, or Table 10. In certain embodiments, the ATXN2 RNAi agent antisense strand includes or consists of one of the modified sequences from either Table 3 or Table 10.

[0099] In some embodiments, the ATXN2 RNAi agent sense strand includes a nucleotide sequence of any of the sequences in Table 2 or Table 4. In some embodiments, the ATXN2 RNAi agent sense strand includes a nucleotide sequence (5' end → 3' end) of any of the sequences in Table 2, Table 4, Table 5, Table 6 or Table 10 (5' end → 3' end) 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23, 1-24, 2-24, 3-24, or 4-24. In certain embodiments, the ATXN2 RNAi agent sense strand comprises or consists of one of the modified sequences shown in Table 3 or Table 10.

[0100] In the case of the RNAi agents disclosed herein, the nucleotide at position 1 (5' end → 3' end) of the antisense strand may be either perfectly complementary to the ATXN2 gene or non-complementary to the ATXN2 gene. In some embodiments, the nucleotide at position 1 (5' end → 3' end) of the antisense strand is U, A, or dT (or a modified version of U, A, or dT). In some embodiments, the nucleotide at position 1 (5' end → 3' end) of the antisense strand forms an A:U or U:A base pair with the sense strand.

[0101] In some embodiments, the ATXN2 RNAi antisense strand includes a sequence of 2-18 or 2-19 nucleotides (5' end to 3' end) from any of the antisense strand sequences in Table 2, Table 3, or Table 10. In some embodiments, the ATXN2 RNAi sense strand includes a sequence of 1-17 or 1-18 nucleotides (5' end to 3' end) from any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0102] In some embodiments, the ATXN2 RNAi agent comprises (i) an antisense strand containing a sequence of 2-18 or 2-19 nucleotides (5' end to 3' end) of any of the antisense strand sequences in Table 2, Table 3, or Table 10, and (ii) a sense strand containing a sequence of 1-17 or 1-18 nucleotides (5' end to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0103] A sense strand containing the sequences listed in Table 2 or Table 4 can be hybridized to any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity across a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, the ATXN2 RNAi agent comprises a sense strand consisting of a modified sequence from any of the modified sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of a modified sequence from any of the modified sequences in Table 3 or Table 10. Specific representative sequence pairings are exemplified by the double-stranded ID numbers shown in Tables 7, 8, and 9A.

[0104] In some embodiments, the ATXN2 RNAi agent comprises, consists of, or essentially consists of a double helix represented by one of the double helix ID numbers presented herein. In some embodiments, the ATXN2 RNAi agent comprises one of the double helix ID numbers presented herein. In some embodiments, the ATXN2 RNAi agent comprises the sense and antisense nucleotide sequences of one of the double helix ID numbers presented herein. In some embodiments, the ATXN2 RNAi agent comprises the sense and antisense nucleotide sequences of one of the double helix ID numbers presented herein, along with a targeting group, a linking group, and / or other non-nucleotide groups, the targeting group, linking group, and / or other non-nucleotide groups being covalently linked (i.e., conjugated) to the sense or antisense strand. In some embodiments, the ATXN2 RNAi agent comprises the modified nucleotide sequences of the sense and antisense strands of one of the double helix ID numbers presented herein. In some embodiments, the ATXN2 RNAi agent comprises a sense-strand and antisense-strand modified nucleotide sequence of any of the dual-strand ID numbers presented herein, and a targeting group, a linking group, and / or other non-nucleotide groups, the targeting group, the linking group, and / or other non-nucleotide groups being covalently linked to the sense-strand or antisense-strand.

[0105] In some embodiments, the ATXN2 RNAi agent comprises an antisense strand and a sense strand having one of the nucleotide sequences of the antisense / sense strand duplexes in Tables 2, 7, 8, 9A, or 10, and includes a targeting group. In some embodiments, the ATXN2 RNAi agent comprises an antisense strand and a sense strand having one of the nucleotide sequences of the antisense / sense strand duplexes in Tables 2, 7, 8, 9A, or 10, and includes one or more lipid moieties.

[0106] In some embodiments, the ATXN2 RNAi agent comprises an antisense strand and a sense strand having one of the nucleotide sequences of the antisense / sense strand duplexes in Tables 2, 7, 8, 9A, or 10, and includes a lipid moiety.

[0107] In some embodiments, the ATXN2 RNAi agent comprises an antisense strand and a sense strand having one of the modified nucleotide sequences of the antisense / sense strand double helix shown in Tables 7, 8, 9A, and 10.

[0108] In some embodiments, the ATXN2 RNAi agent comprises an antisense strand and a sense strand having one of the modified nucleotide sequences of the antisense / sense strand double helix shown in Tables 7, 8, 9A, and 10, and includes a lipid moiety.

[0109] In some embodiments, the ATXN2 RNAi agent comprises, consists of, or essentially consists of, one of the double helixes shown in Tables 7, 8, 9A, and 10.

[0110] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0111] [Table 8-1] [Table 8-2]

[0112] [Table 9A-1] [Table 9A-2]

[0113] [Table 10-1] [Table 10-2]

[0114] In some embodiments, the ATXN2 RNAi agent is prepared or provided as a salt, a mixed salt, or a free acid. In some embodiments, the ATXN2 RNAi agent is prepared or provided as a pharmaceutically acceptable salt. In some embodiments, the ATXN2 RNAi agent is prepared or provided as a pharmaceutically acceptable sodium salt or potassium salt. When delivered to cells expressing the ATXN2 gene, the RNAi agents described herein inhibit or knock down the expression of one or more ATXN2 genes in vivo and / or in vitro.

[0115] Targeting group, linking group, lipid moiety, and delivery vehicle In some embodiments, the ATXN2 RNAi agent includes or is conjugated to one or more non-nucleotide groups, including, but not limited to, targeting groups, linking groups, pharmacokinetic / pharmacodynamic (PK / PD) modulators, delivery polymers, or delivery vehicles. The non-nucleotide groups can enhance the targeting, delivery, or binding of the RNAi agent. The non-nucleotide groups can be covalently linked to the 3' and / or 5' ends of either the sense strand and / or antisense strand. In some embodiments, the ATXN2 RNAi agent includes a non-nucleotide group linked to the 3' and / or 5' ends of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the ATXN2 RNAi agent sense strand. The non-nucleotide groups can be linked directly or indirectly to the RNAi agent via linker / linking groups. In some embodiments, the non-nucleotide groups are linked to the RNAi agent via unstable, cleavable, or reversible bonds or linkers.

[0116] In some embodiments, non-nucleotide groups enhance the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which they are bound, thereby improving the cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, non-nucleotide groups enhance the endocytosis of the RNAi agent.

[0117] Targeting groups or moieties enhance the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent to which they are bound, thereby improving the cell-specific (and possibly organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi agent. Targeting groups can be monovalent, divalent, trivalent, or tetravalent, or may have a higher valency relative to the target they are directed to. Typical targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity for cell surface molecules. In some embodiments, targeting groups are linked to the RNAi agent using a linker, such as a PEG linker, or, in some examples, one, two, or three debased and / or ribitol (debased ribose) residues that can act as a linker.

[0118] With or without a linker, the targeting group can be attached to the 5' or 3' end of either the sense chain and / or antisense chain disclosed in Tables 2, 3, 4, 5, 6, and 10. The linker can be attached to the 5' or 3' end of either the sense chain and / or antisense chain disclosed in Tables 2, 3, 4, 5, 6, and 10, with or without a targeting group.

[0119] The ATXN2 RNAi agents described herein can be synthesized to have reactive groups such as amino groups (also referred to herein as amines) at their 5' and / or 3' ends. The reactive groups can then be used to attach the targeting moiety using methods typical in the art.

[0120] For example, in some embodiments, the ATXN2 RNAi agents disclosed herein are synthesized to have an NH2-C6 group at the 5' end of the sense strand of the RNAi agent. The terminal amino group can then be reacted to form a conjugate with a group, for example, one containing a lipid moiety. In some embodiments, the ATXN2 RNAi agents disclosed herein are synthesized to have one or more alkyne groups at the 5' end of the sense strand of the RNAi agent.

[0121] In some embodiments, the targeting group is linked to the ATXN2 RNAi agent without the use of an additional linker. In some embodiments, the targeting group is designed to have a readily present linker to facilitate binding to the ATXN2 RNAi agent. In some embodiments, if two or more RNAi agents are included in the composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linker. In some embodiments, if two or more RNAi agents are included in the composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0122] In some embodiments, the linking group is conjugated to the RNAi agent. The linking group facilitates the covalent bonding of the agent to a targeting group, pharmacokinetic modulator, delivery polymer, or delivery vehicle. The linking group can be linked to the 3' and / or 5' ends of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' end of the RNAi agent sense strand. Examples of linking groups, but not limited to, include reactive groups such as C6-SS-C6, 6-SS-6, primary amines (e.g., NH2-C6), and alkynes, alkyl groups, debased residues / nucleotides, amino acids, trialukine functionalized groups, ribitols, and / or PEG groups. Examples of specific linking groups are provided in Table 11.

[0123] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (e.g., a targeting group, a pharmacokinetic modulator, or a delivery polymer) or segment of interest via one or more covalent bonds. Unstable linkages include unstable bonds. Linkages may optionally include spacers that increase the distance between the two joined atoms. Spacers may further add flexibility and / or length to the linkage. Examples of spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkyl groups, each of which may include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the preceding list is not intended to limit the scope of the foregoing description. In some embodiments, the ATXN2 RNAi agent is conjugated to a polyethylene glycol (PEG) moiety or a hydrophobic group having 12 or more carbon atoms, such as cholesterol or a palmitoyl group.

[0124] In some embodiments, the ATXN2 RNAi agent is conjugated to one or more lipid moieties. The lipid moieties can enhance the pharmacodynamic or pharmacokinetic properties of the RNAi agent. In some embodiments, the lipid moieties may be conjugated to a linker at the 3' or 5' end of the sense or antisense strand of the RNAi agent described herein. In some embodiments, the lipid moieties may be conjugated to both the 3' or 5' end of either the sense or antisense strand of the RNAi agent described herein.

[0125] In some embodiments, the lipid moiety can be conjugated to an ATXN2 RNAi agent by reacting it with an ATXN2 RNAi agent containing an amine-containing linker, for example (NH2-C6) (see Table 11). In some embodiments, the amine-containing linker may be located at the 5' end of the sense or antisense strand of the ATXN2 RNAi agent. In some embodiments, the amine-containing linker may be located at the 3' end of the sense or antisense strand of the RNAi agent.

[0126] In some embodiments, an RNAi agent containing an amine-containing linker, such as one (NH2-C6) or more (NH2-C6), may be reacted with a lipid containing an activated ester moiety. Examples of lipids having an activated ester moiety include LP183-p, LP293-p, and LP310-p, as shown in Table 11 below.

[0127] In some embodiments, the ATXN2 RNAi agent may be conjugated to a lipid moiety using phosphoramidite synthesis. The synthesis of oligonucleotides using phosphoramidites is well known in the art. In some embodiments, the lipid moiety may be conjugated to the 5' end of the sense or antisense strand of the ATXN2 RNAi agent using phosphoramidite. In some embodiments, the lipid moiety may be conjugated to the 3' end of the sense or antisense strand of the ATXN2 RNAi agent using phosphoramidite. In some embodiments, phosphoramidites selected from HO-C16-p, C16-p, or C22-p may be used to conjugate the lipid moiety to the ATXN2 RNAi agent, as shown in Table 11 below.

[0128] In some embodiments, the ATXN2 RNAi agent may contain a lipid moiety on an internal nucleotide (i.e., not on the 3' or 5' terminal nucleotide). In some embodiments, the internal nucleotide may be ligated to the 2' position of ribose. In some embodiments, the ATXN2 RNAi agent may contain aC16, uC16, cC16, or gC16, as shown in Table 11 below.

[0129] Any of the ATXN2 RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 10, whether modified or unmodified, may contain 3' and / or 5' targeting groups, linking groups, and / or lipid moieties. Any ATXN2 RNAi agent sequence listed in Tables 3, 4, 5, 6, and 10, or any other ATXN2 RNAi agent sequence described herein in a different form, that contains 3' or 5' targeting groups, linking groups, and / or lipid moieties may, alternatively, not contain 3' or 5' targeting groups, linking groups, or lipid moieties, or may contain different 3' or 5' targeting groups, linking groups, or lipid moieties (including, but not limited to, those shown in Table 11). Any of the ATXN2 RNAi agent duplexes listed in Tables 7, 8, 9A, and 10, whether modified or unmodified, may further contain targeting or linking groups, including but not limited to those shown in Table 11, and these targeting or linking groups may be attached to the 3' or 5' end of either the sense strand or the antisense strand of the ATXN2 RNAi agent duplex.

[0130] Examples of specific modified nucleotides, capping moieties, lipid moieties, and linking groups are provided in Table 11. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0131] Alternatively, other linking groups known in the art may be used. In many cases, linking groups are commercially available or, alternatively, incorporated into commercially available nucleotide phosphoramidites. (See, for example, International Patent Application Publication WO2019 / 161213, which is incorporated in its entirety herein by reference).

[0132] In some embodiments, the ATXN2 RNAi agent is delivered without being conjugated to a targeting ligand or pharmacokinetic / pharmacodynamic (PK / PD) modulator (referred to as "naked" or "naked RNAi agent").

[0133] In some embodiments, the ATXN2 RNAi agent is conjugated to a targeting group, a linking group, a PK modulator, and / or another non-nucleotide group to facilitate delivery of the ATXN2 RNAi agent to selected cells or tissues, e.g., CNS cells in vivo. In some embodiments, the ATXN2 RNAi agent is conjugated to a lipid moiety.

[0134] In some embodiments, a delivery vehicle may be used to deliver RNAi agents to cells or tissues. The delivery vehicle is a compound that improves the delivery of RNAi agents to cells or tissues. The delivery vehicle may include, but is not limited to, polymers such as amphiphilic polymers, membrane-active polymers, peptides, melittin peptides, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines.

[0135] In some embodiments, RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art for nucleic acid delivery. RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to, cholesteryl and cholesteryl derivatives), encapsulated in nanoparticles, liposomes, micelles, or conjugated in polymers or DPCs (see, for example, International Publication Nos. 2000 / 053722, 2008 / 022309, 2011 / 104169, and 2012 / 083185, 2013 / 032829, and 2013 / 158141, each of which is incorporated herein by reference) (by iontophoresis or by incorporation into other delivery vehicles or systems available in the art, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or protein vectors). In some embodiments, RNAi agents can be conjugated to antibodies having affinity for CNS cells. In some embodiments, the RNAi agent can be linked to a targeting ligand that has affinity for CNS cells or receptors present on CNS cells.

[0136] Pharmaceutical compositions and formulations The ATXN2 RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “pharmaceuticals”). In some embodiments, the pharmaceutical compositions comprise at least one ATXN2 RNAi agent. These pharmaceutical compositions are particularly useful in inhibiting the expression of ATXN2 mRNA in target cells, cell populations, tissues, or organisms. The pharmaceutical compositions can be used to treat subjects with diseases, disorders, or conditions that would benefit from a reduction in the level of target mRNA or inhibition of the expression of a target gene. The pharmaceutical compositions can be used to treat subjects at risk of developing diseases or disorders that would benefit from a reduction in the level of target mRNA or inhibition of the expression of a target gene. In one embodiment, the method comprises administering an ATXN2 RNAi agent linked to a targeting ligand or lipid moiety as described herein to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable additives (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition comprising the ATXN2 RNAi agent to form a pharmaceutical formulation or pharmaceutical suitable for in vivo delivery to subjects, including humans.

[0137] Pharmaceutical compositions and methods comprising ATXN2 RNAi agents disclosed herein include administering a therapeutically effective amount of the ATXN2 RNAi agent described herein to a subject, thereby reducing the level of target mRNA in cells, cell populations, tissues, organs, or subjects, including inhibiting the expression of ATXN2 mRNA in the subject. In some embodiments, the subject has been previously identified or diagnosed with a disease or disorder that can be at least partially mediated by a reduction in ATXN2 expression. In some embodiments, the subject has been previously diagnosed with one or more neurodegenerative diseases, such as SCA2 and ALS. In some embodiments, the neurodegenerative disease is SCA2.

[0138] In some embodiments, the subjects have been previously diagnosed with a neurodegenerative disease.

[0139] Embodiments of this disclosure include pharmaceutical compositions for delivering ATXN2 RNAi agents to CNS cells in vivo. Such pharmaceutical compositions may, for example, include ATXN2 RNAi agents conjugated to a lipid moiety.

[0140] In some embodiments, the described pharmaceutical compositions comprising an ATXN2 RNAi agent are used to treat or manage clinical findings in subjects who would benefit from inhibition of ATXN2 expression. In some embodiments, one or more therapeutically or prophylactically effective doses of the pharmaceutical composition are administered to subjects requiring such treatment. In some embodiments, administration of any of the disclosed ATXN2 RNAi agents can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0141] In some embodiments, the described ATXN2 RNAi agent is combined with one or more additional (i.e., second, third, etc.) therapeutic agents as needed. The second therapeutic agent may be another ATXN2 RNAi agent (e.g., an ATXN2 RNAi agent targeting a different sequence within the ATXN2 gene). In some embodiments, the second therapeutic agent may be an RNAi agent targeting the ATXN2 gene. The additional therapeutic agents may also be small molecule drugs, antibodies, antibody fragments, and / or aptamers. The ATXN2 RNAi agent can be combined with one or more excipients, with or without one or more additional therapeutic agents, to form a pharmaceutical composition.

[0142] The described pharmaceutical compositions comprising an ATXN2 RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder and who would benefit from a reduction or inhibition of ATXN2 mRNA expression. In some embodiments, the subject is administered one or more pharmaceutical compositions comprising a therapeutically effective dose of the ATXN2 RNAi agent to thereby treat the symptom. In other embodiments, the subject is administered one or more preventively effective doses of the ATXN2 RNAi agent to thereby prevent or inhibit at least one symptom.

[0143] In some embodiments, one or more of the ATXN2 RNAi agents described are administered to a mammal in a pharmaceutically acceptable carrier or diluent. In some embodiments, the mammal is a human.

[0144] The route of administration is the route through which the ATXN2 RNAi agent comes into contact with the body. In general, methods for administering drugs, oligonucleotides, and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The ATXN2 RNAi agents disclosed herein can be administered via any suitable route in the form of preparations appropriately adjusted for a specific route. Thus, in some embodiments, the pharmaceutical compositions described herein are administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. In some embodiments, the pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraventricularly, intra-articularly, intraocularly, or intraperitoneally, or locally.

[0145] Pharmaceutical compositions comprising the ATXN2 RNAi agents described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. In general, any suitable method (in vitro or in vivo) recognized in the art for delivering nucleic acid molecules can be adapted for use with the compositions described herein. For example, delivery may be by local administration (e.g., direct injection, implantation, or local administration), systemic administration, or parenteral routes including subcutaneous, intravenous, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intraventricular, intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or local (including buccal and sublingual) administration. In some embodiments, the compositions are administered by inhalation, intranasal administration, oropharyngeal inhalation administration, or intratracheal administration. For example, in some embodiments, it is desirable that the ATXN2 RNAi agents described herein inhibit the expression of the ATXN2 gene in the CNS.

[0146] In some embodiments, the pharmaceutical compositions described herein include one or more pharmaceutically acceptable additives. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0147] When used herein, a pharmaceutical composition or drug comprises a pharmacologically effective amount of at least one described therapeutic compound and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API (Active Pharmaceutical Ingredient), therapeutic product, e.g., ATXN2 RNAi agent) that is intentionally included in the drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dose. An excipient may act to a) assist in the processing of the drug delivery system during manufacturing, b) protect, assist or enhance the stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0148] Examples of additives include, but are not limited to, absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, dyes, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, flow enhancers, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0149] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, New Jersey, USA), or phosphate-buffered saline (PBS). These should be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of injectable compositions can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0150] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing, if necessary, one or a combination of the components listed above, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders and disperses for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying, thereby obtaining powders and disperses of the active ingredient and any additional desired components from their pre-sterile filtered solution.

[0151] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the drug, which may be in the form of a microcrystalline form, such as an aqueous microcrystalline suspension. Liposome formulations or biodegradable polymer systems can also be used to provide drugs for both intra-articular and ocular administration.

[0152] The active compounds can be prepared using carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are obvious to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0153] ATXN2 RNAi agents can be formulated into dosage unit formulations for ease of administration and uniformity of dosage. A dosage unit formulation refers to a physically distinct unit suitable as a unit dose for the subject being treated, and each unit contains a predetermined amount of the active compound calculated to associate with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the dosage unit formulations in this disclosure are determined and directly depend on the inherent characteristics of the active compound and the therapeutic effect achieved, as well as the limitations inherent in the technology for formulating such active compounds for the treatment of an individual.

[0154] A pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to, antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramines, etc.). It is also conceivable that cells, tissues, or isolated organs expressing or containing an RNAi agent as defined herein may be used as a “pharmaceutical composition.” As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of RNAi agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0155] In some embodiments, the methods disclosed herein further include the step of administering a second therapeutic agent or treatment in addition to administering the RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another ATXN2 RNAi agent (e.g., an ATXN2 RNAi agent targeting a different sequence within the ATXN2 target). In other embodiments, the second therapeutic agent may be a small molecule drug, an antibody, an antibody fragment, and / or an aptamer.

[0156] In some embodiments, compositions comprising a combination or cocktail of at least two ATXN2 RNAi agents having different sequences are described herein. In some embodiments, two or more ATXN2 RNAi agents are each separately and independently linked to a lipid.

[0157] Compositions for delivering ATXN2 RNAi agents to central nervous system cells are described herein. Furthermore, compositions for delivering ATXN2 RNAi agents to cells, including neurons, astrocytes, microglia, and endothelial cells, in vivo are generally described herein.

[0158] Generally, the effective dose of the ATXN2 RNAi agents disclosed herein ranges from about 0.0001 to about 20 mg / kg body weight, for example, from about 0.001 to about 5 mg / kg body weight. In some embodiments, the effective dose of the ATXN2 RNAi agent ranges from about 0.01 mg / kg to about 3.0 mg / kg body weight per dose. In some embodiments, the effective dose of the ATXN2 RNAi agent ranges from about 0.03 mg / kg to about 2.0 mg / kg body weight per dose. In some embodiments, the effective dose of the ATXN2 RNAi agent ranges from about 0.01 to about 1.0 mg / kg dose per body weight. In some embodiments, the effective dose of the ATXN2 RNAi agent ranges from about 0.50 to about 1.0 mg / kg dose per body weight. The dose administered may also depend on variables such as the patient's overall health, the relative biological efficacy of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. Furthermore, it should be understood that the initial dose administered may be increased beyond the above upper limit to rapidly achieve the desired blood or tissue level, or the initial dose may be smaller than the optimal dose. In some embodiments, the dose is administered daily. In some embodiments, the dose is administered weekly. In further embodiments, the dose is administered every two weeks, every three weeks, every month, or every quarter (i.e., every three months).

[0159] For the treatment of a disease, or for the formation of a pharmaceutical or composition for the treatment of a disease, the pharmaceutical compositions described herein, comprising an ATXN2 RNAi agent, may be combined with excipients or with a second therapeutic agent or treatment (including, but not limited to, a second or other RNAi agent, small molecule drug, antibody, antibody fragment, peptide, and / or aptamer).

[0160] When the ATXN2 RNAi agents described are added to pharmaceutically acceptable excipients or adjuvants, they may be packaged in kits, containers, packs, or dispensers.

[0161] Methods for treating and inhibiting ATXN2 expression The ATXN2 RNAi agents disclosed herein can be used to treat subjects (e.g., humans or other mammals) who have a disease or disorder and who would benefit from the administration of RNAi agents. In some embodiments, the RNAi agents disclosed herein can be used to treat subjects (e.g., humans) who would benefit from reduced and / or inhibition of ATXN2 mRNA expression and / or reduced ATXN2 protein levels.

[0162] In some embodiments, the RNAi agents disclosed herein can be used to treat subjects (e.g., humans) who have a disease or disorder and who benefit from a reduction in mutant ATXN2 protein, including but not limited to spinocerebellar ataxia type 2 and ALS. Treatment of subjects may include therapeutic and / or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more ATXN2 RNAi agents described herein. The subject may be a human, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. Administration of the pharmaceutical compositions described herein may be to humans or animals.

[0163] Mutant ATXN2 activity is known to promote neurodegenerative disorders. In some embodiments, the described ATXN2 RNAi agents are used to treat at least one symptom that is at least partially mediated by a decrease in mutant ATXN2 levels in a subject. The subject is administered a therapeutically effective dose of any one or more of the described ATXN2 RNAi agents. In some embodiments, the subject is treated by administering a prophylactically effective dose of any one or more of the described RNAi agents, thereby preventing or inhibiting at least one symptom.

[0164] In certain embodiments, the Disclosure provides a method to a patient in need of treatment for a disease, disorder, condition, or pathological state at least partially mediated by ATXN2 gene expression, the method comprising administering one of the ATXN2 RNAi agents described herein to the patient.

[0165] In some embodiments, the ATXN2 RNAi agent is used to treat or manage a clinical finding or pathological condition in a subject, where the clinical finding or pathological condition is at least partially mediated by a decrease in ATXN2 expression. The subject is administered a therapeutically effective amount of one or more of the ATXN2 RNAi agent or ATXN2 RNAi agent-containing composition described herein. In some embodiments, the method includes administering a composition containing the ATXN2 RNAi agent described herein to the subject to be treated.

[0166] In further embodiments, the disclosure features methods (including prophylactic or preventative measures) for treating diseases or conditions that can be addressed by reducing mutant ATXN2 levels, the methods comprising administering to subjects in need an ATXN2 RNAi agent comprising an antisense strand containing one of the sequences in Table 2, Table 3, or Table 10. Compositions for use in such methods are also described herein.

[0167] The ATXN2 RNAi agents and / or compositions described herein, including ATXN2 RNAi agents, can be used in methods for the therapeutic treatment of diseases or conditions caused by enhancement or elevation of mutant ATXN2 levels. Such methods include administering the ATXN2 RNAi agents described herein to a subject, for example, a human or animal subject.

[0168] In another embodiment, the present disclosure provides a method for treating (including prophylactic treatment) a pathological condition (such as a state or disease) at least partially mediated by ATXN2 expression, wherein the method comprises administering a therapeutically effective amount of an RNAi agent to a target, comprising an antisense strand containing any of the sequences in Table 2, Table 3, or Table 10.

[0169] In some embodiments, methods for inhibiting the expression of the ATXN2 gene are disclosed herein, which include administering an RNAi agent to cells comprising an antisense strand containing one of the sequences in Table 2, Table 3, or Table 10.

[0170] In some embodiments, methods for treating (including prophylactic treatment) pathological conditions at least partially mediated by ATXN2 expression are disclosed herein, which include administering a therapeutically effective amount of an RNAi agent to a target, comprising a sense strand containing any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0171] In some embodiments, methods for inhibiting the expression of the ATXN2 gene are disclosed herein, which include administering an RNAi agent to cells comprising a sense strand containing one of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0172] In some embodiments, methods for treating (including prophylactic treatment) pathological conditions at least partially mediated by ATXN2 expression are disclosed herein, the methods comprising administering a therapeutically effective amount of an RNAi agent to a target comprising a sense strand containing a sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand containing a sequence of any of the sequences in Table 3 or Table 10.

[0173] In some embodiments, methods for inhibiting the expression of the ATXN2 gene are disclosed herein, where the method comprises administering to a cell an RNAi agent comprising a sense strand comprising any of the sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand comprising any of the sequences in Table 3 or Table 10.

[0174] In some embodiments, methods for inhibiting the expression of the ATXN2 gene are disclosed herein, where the method comprises administering to a subject an ATXN2 RNAi agent comprising a sense strand consisting of any of the nucleic acid sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of any of the nucleic acid sequences in Table 3 or Table 10. In other embodiments, methods for inhibiting the expression of the ATXN2 gene are disclosed herein, where the method comprises administering to a subject an ATXN2 RNAi agent comprising a sense strand consisting of any of the modified sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of any of the modified sequences in Table 3 or Table 10.

[0175] In some embodiments, methods for inhibiting the expression of the ATXN2 gene in a cell are disclosed herein, where the method comprises administering one or more ATXN2 RNAi agents comprising one of the duplex structures described in Tables 7, 8, 9A and 10.

[0176] In some embodiments, the gene expression level and / or mRNA level of the ATXN2 gene in specific CNS cells of a subject to whom the described ATXN2 RNAi agent is administered is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower compared to the subject before the administration of the ATXN2 RNAi agent or a subject not receiving the ATXN2 RNAi agent. In some embodiments, the ATXN2 mRNA or ATXN2 protein level in specific CNS cells of a subject to whom the described ATXN2 RNAi agent is administered is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower compared to the subject before the administration of the ATXN2 RNAi agent or a subject not receiving the ATXN2 RNAi agent. The gene expression level, protein level, and / or mRNA level in a subject can be decreased in the cells, cell populations, and / or tissues of the subject. In some embodiments, the ATXN2 mRNA level in specific CNS cells of a subject administered the described ATXN2 RNAi agent is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% lower compared to the subject before the administration of the ATXN2 RNAi agent or a subject not receiving the ATXN2 RNAi agent.

[0177] The decrease in gene expression, mRNA, and protein levels can be evaluated by any method known in the art. The decrease or reduction in ATXN2 mRNA and / or ATXN2 protein levels are collectively referred to herein as a decrease, reduction, or inhibition of ATXN2 expression. The examples described herein illustrate known methods for evaluating the inhibition of ATXN2 expression and ATXN2 gene expression.

[0178] Cells, tissues, organs and non-human organisms Cells, tissues, organs, and non-human organisms comprising at least one of the ATXN2 RNAi agents described herein are intended. Cells, tissues, organs, or non-human organisms are created by delivering the RNAi agent to the cells, tissues, organs, or non-human organisms.

[0179] Additional exemplary embodiments Herein, certain additional exemplary embodiments of the disclosed technology are provided. These embodiments are illustrative only and do not limit the scope of this disclosure or the claims appended herein.

[0180] Embodiment 1. An RNAi agent for inhibiting the expression of the Ataxin-2 (ATXN2) gene, An antisense strand containing at least 17 consecutive nucleotides that differ by only 0 or 1 nucleotide from any one of the sequences provided in Table 2 or Table 3, An RNAi agent comprising a sense strand having a nucleotide sequence at least partially complementary to the antisense strand.

[0181] Embodiment 2. The RNAi agent according to Embodiment 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence provided in Table 2 or Table 3.

[0182] Embodiment 3. The RNAi agent according to Embodiment 1 or Embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs by only 0 or 1 nucleotide from any one of the sequences provided in Table 2 or Table 4, and the sense strand has a region of at least 85% complementarity with respect to the antisense strand across the 17 consecutive nucleotides.

[0183] Embodiment 4. The RNAi agent according to any one of Embodiments 1 to 3, wherein at least one nucleotide of the ATXN2 RNAi agent is a modified nucleotide or comprises a modified nucleoside bond.

[0184] Embodiment 5. The RNAi agent according to any one of Embodiments 1 to 4, wherein all or substantially all of the nucleotides are modified nucleotides.

[0185] Embodiment 6. An RNAi agent according to any one of Embodiments 4 to 5, wherein the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methylnucleotide, inverted 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methylnucleotide.

[0186] Embodiment 7. The RNAi agent according to Embodiment 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

[0187] Embodiment 8. The RNAi agent according to any one of Embodiments 1 to 7, wherein the antisense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 3.

[0188] Embodiment 9. The RNAi agent according to any one of Embodiments 1 to 8, wherein the sense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 4.

[0189] Embodiment 10. The RNAi agent according to Embodiment 1, wherein the antisense strand comprises one nucleotide sequence of any of the modified sequences provided in Table 3, and the sense strand comprises one nucleotide sequence of any of the modified sequences provided in Table 4.

[0190] Embodiment 11. The RNAi agent according to any one of Embodiments 1 to 10, wherein the sense strand has a length of 18 to 30 nucleotides and the antisense strand has a length of 18 to 30 nucleotides.

[0191] Embodiment 12. The RNAi agent according to Embodiment 11, wherein the sense strand and the antisense strand each have a length of 18 to 27 nucleotides.

[0192] Embodiment 13. The RNAi agent according to Embodiment 12, wherein the sense strand and the antisense strand each have a length of 18 to 24 nucleotides.

[0193] Embodiment 14. The RNAi agent according to Embodiment 13, wherein the sense strand and the antisense strand each have a length of 21 nucleotides.

[0194] [[ID=�6]]Embodiment l5. The RNAi agent according to Embodiment 14, wherein the RNAi agent has two blunt ends.

[0195] Embodiment 16. The RNAi agent according to any one of Embodiments 1 to 15, wherein the sense strand contains one or two terminal caps.

[0196] Embodiment 17. The RNAi agent according to any one of Embodiments 1 to 16, wherein the sense strand contains one or two inverted abasic residues.

[0197] Embodiment 18. The RNAi agent according to Embodiment 1, comprising a sense strand and an antisense strand that form a double strand having the structure of any one of the double strands in Table 7, Table 8, Table 9A or Table 10.

[0198] Embodiment 19. The RNAi agent according to Embodiment 18, wherein all or substantially all of the nucleotides are modified nucleotides.

[0199] Embodiment 20. The RNAi agent according to Embodiment 19, wherein all or substantially all of the nucleotides are modified nucleotides.

[0200] Embodiment 21. The RNAi agent according to any one of Embodiments 19 to 20, wherein the sense strand further comprises an inverted debase residue at the 3' terminal end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[0201] Embodiment 22. The RNAi agent according to any one of Embodiments 1 to 21, wherein the RNAi agent is linked to a lipid portion.

[0202] Embodiment 23. The lipid portion is as follows: [ka] [ka] Selected from the group consisting of, in the formula, [ka] This is the RNAi agent according to Embodiment 22, which indicates a linkage point with the RNAi agent.

[0203] Embodiment 24. The RNAi agent according to Embodiment 22 or Embodiment 23, wherein the lipid portion is conjugated to the sense strand.

[0204] Embodiment 25. The RNAi agent according to Embodiment 24, wherein the lipid portion is conjugated to the 5' terminal end of the sense strand.

[0205] Embodiment 26. A composition comprising an RNAi agent according to any one of Embodiments 1 to 25, wherein the composition further comprises a pharmaceutically acceptable additive.

[0206] Embodiment 27. The composition according to Embodiment 26, further comprising a second RNAi agent capable of inhibiting the expression of the Ataxin-2 gene.

[0207] Embodiment 28. The composition according to any one of Embodiments 26 to 27, further comprising one or more additional therapeutic agents.

[0208] Embodiment 29. The composition according to any one of Embodiments 26 to 28, wherein the RNAi agent is a sodium salt.

[0209] Embodiment 30. The composition according to any one of Embodiments 26 to 29, wherein the pharmaceutically acceptable additive is water for injection.

[0210] Embodiment 31. The composition according to any one of Embodiments 26 to 29, wherein the pharmaceutically acceptable additive is buffered saline.

[0211] Embodiment 32. A method for inhibiting the expression of the ATXN2 gene in cells, comprising introducing an effective amount of an RNAi agent according to any one of Embodiments 1 to 25 or a composition according to any one of Embodiments 26 to 31 into the cells.

[0212] Embodiment 33. The method according to Embodiment 32, wherein the cells are located within the target.

[0213] Embodiment 34. The method according to Embodiment 33, wherein the subject is a human subject.

[0214] Embodiment 35. The method according to any one of Embodiments 32 to 34, wherein the Ataxin-2 (ATXN2) gene expression is inhibited by at least about 30% after administration of the RNAi agent.

[0215] Embodiment 36. A method for treating one or more symptoms or diseases related to enhancement or elevation of membrane ATXN2 activity levels, comprising administering a therapeutically effective amount of any one of Embodiments 26 to 31 to a human subject in need thereof.

[0216] Embodiment 37. The method according to Embodiment 36, wherein the disease is a neurodegenerative disease.

[0217] Embodiment 38. The method according to Embodiment 37, wherein the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2).

[0218] Embodiment 39. The method according to Embodiment 37, wherein the disease is ALS.

[0219] Embodiment 40. The method according to any one of Embodiments 32 to 39, wherein the RNAi agent is administered in a dose of approximately 0.01 mg per kg of body weight of the subject to approximately 5.0 mg per kg.

[0220] Embodiment 41. The method according to any one of Embodiments 32 to 40, wherein the RNAi agent is administered in a dose of approximately 0.03 mg to approximately 2.0 mg per kg of body weight of the subject.

[0221] Embodiment 42. The method according to any one of Embodiments 32 to 41, wherein the RNAi agent is administered in a dose of 2 or more.

[0222] Embodiment 43. Use of an RNAi agent according to any one of Embodiments 1 to 25 for the treatment of a disease, disorder, or condition that is at least partially mediated by mutant ATXN2 activity and / or ATXN2 gene expression.

[0223] Embodiment 44. Use of any one of Embodiments 26 to 31 for the treatment of a disease, disorder or condition that is at least partially mediated by ataxin-2 (ATXN2) activity and / or ataxin-2 (ATXN2) gene expression.

[0224] Embodiment 45. Use of the composition according to any one of Embodiments 26 to 31 for the manufacture of a pharmaceutical product for the treatment of a disease, disorder or condition at least partially mediated by Ataxin-2 (ATXN2) gene expression.

[0225] Embodiment 46. The use according to any one of Embodiments 43 to 45, wherein the disease is a neurodegenerative disease.

[0226] Embodiment 47. A method for producing an RNAi agent according to any one of Embodiments 1 to 25, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

[0227] Embodiment 48. The method according to Embodiment 47, wherein the sense chain includes a lipid portion.

[0228] Embodiment 49. The method according to Embodiment 48, comprising conjugating the lipid portion to the sense chain. [Examples]

[0229] Example 1. Synthesis of ATXN2 RNAi agent.

[0230] The ATXN2 RNAi agent double helix disclosed herein was synthesized according to the following:

[0231] A. Synthesis. The sense and antisense strands of the ATXN2 RNAi agent were synthesized according to solid-phase phosphoramidite techniques used for oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was performed on solid supports made of controlled-pore glass (CPG, 500 Å or 600 Å, available from Prime Synthesis (Aston, Pennsylvania, USA)). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, Wisconsin, USA). Specifically, the 2'-O-methylphosphoramidite used contained: (5'-O-dimethoxytrityl-N 6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-deoxy-2'-fluoro-phosphoramidite had the same protecting group as 2'-O-methylRNAamidite. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). The reverse debase (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from ChemGenes (Wilmington, Massachusetts, USA). The following UNA phosphoramidites were used: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxy Trityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxy-trityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA aminolink phosphoramidite was also purchased commercially (ThermoFisher). Linker L6 was purchased from BroadPharm (catalog no. BP-20907) as propargyl-PEG5-NHS and coupled to the NH2-C6 group from the aminolink phosphoramidite using standard coupling conditions to form -L6-C6-.Linker Alk-cyHex was similarly commercially purchased from Lumiprobe (alkyne phosphoramidite, 5' terminus) as a propargyl-containing phosphoramidite compound to form the linker-Alk-cyHex-. In each case, a phosphorothioate bond was introduced as specified using the conditions described herein. Cyclopropylphosphonate phosphoramidites were synthesized according to International Patent Application Publication WO2017 / 214112 (see also Altenhofer et al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).

[0232] Tri-alkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me), and 60 seconds (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc. (Reminster, Massachusetts, USA)) in anhydrous acetonitrile was used.

[0233] Alternatively, the tri-alkyne moiety was introduced after synthesis (see Section E below). For this pathway, the sense strand was functionalized with 5' and / or 3' terminal nucleotides containing a primary amine. TFA aminolink phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 min (RNA), 90 sec (2'O-Me), and 60 sec (2'F). To introduce the phosphorothioate bond, a 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc. (Reminster, Massachusetts, USA)) in anhydrous acetonitrile was used.

[0234] B. Cleavage and deprotection of support-bound oligomers. After completion of solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine and 28-31% ammonium hydroxide solution (Aldrich) in water at 30°C for 1.5 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0235] C. Purification. Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% ​​acetonitrile, and Buffer B was the same as Buffer A with 1.5 M sodium chloride added. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then run by size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 Fine and a running buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile or filtered water. Alternatively, the pooled fractions were desalted and replaced with appropriate buffers or solvent systems by tangential flow filtration.

[0236] D. Annealing. RNAi agents were formed by mixing complementary strands with equimolar RNA solutions (sense and antisense) in 1×PBS (phosphate-buffered saline, 1×, Corning, Cellgro). Several RNAi agents were lyophilized and stored at -15 to -25°C. The double-strand concentration was determined by measuring the solution absorbance in 1×PBS using a UV-Vis spectrometer. The double-strand concentration was then determined by multiplying the solution absorbance at 260 nm by a conversion factor (0.050 mg / (mL·cm)) and a dilution factor.

[0237] E. Lipid synthesis

[0238] If the lipids described herein are not included in Example 1E, it is assumed that the compounds are commercially available.

[0239] Synthesis of LP183 phosphoramidite [ka]

[0240] To a solution of compound 2 (2.00 g) in DCM, TEA (2.27 mL) was added, followed by the dropwise addition of compound 1 (4.931 g) at room temperature. The mixture was then stirred at room temperature for 2 hours. The mixture was then filtered. The white solid was dried overnight. The product was a white solid with a yield of 4.267 g, 74%. LC-MS: Calculated [M+H] 356.35, measured 356.63. [ka]

[0241] Compound 3 (0.61 g) and then Compound 2 (5.37 g) were added dropwise to a mixture of Compound 1 (2.54 g) in 120 mL of DCM at room temperature. The mixture was then stirred overnight at room temperature. 5 mL of TEA was added, followed by Celite. After removing the solvent under vacuum, the residue was loaded onto a 40 g column by dry method. The product was purified using hexane (2% TEA) to hexane (2% TEA) with 50% SiO(2% TEA) as the gradient. The product was a white waxy solid with a yield of 3.462 g, 87%. LC-MS: Calculated [M+H] 556.46, measured 556.64.

[0242] LP293-p synthesis [ka]

[0243] Compound 2 (48.9 mg) was added under ambient conditions to a solution of Compound 1 (73 mg), NEt3 (0.112 mL), and COMU (126 mg) in DMF. The reaction was stirred until complete conversion was observed by LC-MS. The reaction was stirred for 30 minutes until the conversion could not be clearly observed by LC-MS, and instead the mixture changed from bright yellow (before addition of Compound 2) to honey orange, indicating that all materials were mostly dissolved. The reaction mixture was then washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® via a DCM liquid load onto a 12-g column using a hexane gradient to 100% siRNA, and the product was eluted with 30% B. The product was concentrated under vacuum to obtain a white solid residue, which was confirmed by 1H NMR in CDCl3.

[0244] LP-310p synthesis [ka]

[0245] To a solution of 1 in DCM, DIPEA (0.057 mL), COMU (0.077 g), and 2 (0.0300 g) were added at room temperature. After stirring at room temperature for 2 hours, the reaction mixture was quenched with 0.1 N HCl. The organic layer was washed with brine. After removing the solvent, the residue was loaded onto a 4 g column. Purification was performed using 50% hexane in hexane-SiO as a gradient. The product was a white solid, 46 mg, 44%. LC-MS: Calculated [M+H] 422.36, measured 422.61. [ka]

[0246] A solution of 1 (0.046 g) in 4N HCl / dioxane (2 mL) was stirred overnight at room temperature. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. The residue was then dissolved in DCM at room temperature, and then COMU (0.0700 g), DIPEA (0.038 mL), and 2 (0.036 g) were added at room temperature. After stirring at room temperature for 2 hours, the solvent was removed under vacuum. The residue was loaded onto a 4 g column. Purification was performed using 50% hexane in hexane-SiO as a gradient. The product was a white solid, 21 mg, 38%. LC-MS: Calculated [M+H] 514.29, measured 514.61.

[0247] Synthesis of HO-C16 phosphoramidite [ka]

[0248] 1,16-Hexadecanediol and N,N-diisopropylethylamine (0.100 g) were dissolved in 2 mL of THF. 4,4'-Dimethoxytrityl chloride (2.2 g, 6.6 mmol) was slowly added as a solid. After 2 hours, the reaction mixture was concentrated by rotational evaporation, and the product was purified by column chromatography (25% ethyl acetate / 75% hexane).

[0249] DMT-OC 16-OH (0.200 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.227 mL), and bisdiisopropylammonium tetrazolide (0.0611 g) were dissolved in anhydrous DCM at room temperature. The reaction mixture was sealed and stirred overnight. The conversion was determined by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture and concentrated under vacuum until a white powder remained. To prevent hydrolysis from silica gel, the mixture was loaded dry onto a silica column (12 g) using an ELISA / hexane (1% triethylamine) solvent system. [1] Products 31 PNMR, 1 The characteristics were evaluated by HNMR and LC-MS.

[0250] Synthesis of C16 phosphoramidites [ka]

[0251] Cetyl alcohol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.88 mL), and bisdiisopropylammonium tetrazolide (0.778 g) were dissolved in DCM solution at room temperature. The reaction mixture was sealed and stirred overnight. The conversion was determined by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture and concentrated under vacuum until a white powder remained. To prevent hydrolysis from silica gel, the mixture was loaded dry onto a silica column (12 g) using an siRNA / hexane (1% triethylamine) solvent system. The desired product was not retained on the column and emerged immediately after loading. The isolated product was then analyzed by LC-MS. 1 HNMR and 31 Characterization was performed by PNMR. Final yield: 856.5 mg (93.8%).

[0252] Synthesis of C22 phosphoramidites [ka]

[0253] Docosanol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.1 mL), and bisdiisopropylammonium tetrazolide (0.577 g) were dissolved in DCM solution at room temperature. The reaction mixture was sealed and stirred overnight. The conversion was determined by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture and concentrated under vacuum until a white powder remained. To prevent hydrolysis from silica gel, the mixture was loaded dry onto a silica column (12 g) pre-treated with 3 mL of triethylamine using an siRNA / hexane (1% triethylamine) solvent system. The isolated product was then analyzed by LC-MS. 1 HNMR and 31 Characterization was performed by PNMR. Final yield: 2.1085g (118.8%).

[0254] Synthesis of phosphoramidites for aC16 internal nucleotides [ka]

[0255] Compound 2: Sodium hydride (60% dispersion in mineral oil, 4.17 g, 104 mmol) was added in two portions (15 minutes apart) to a cold solution of adenosine 1 (12.5 g, 46.77 mmol) in anhydrous DMF (250 mL). The cooling bath was removed, and the reaction mixture was stirred at room temperature for 1.5 hours, after which 1-bromohexadecane (18 g, 59 mmol) was added. After stirring at room temperature for 16 hours, ethanol (5 mL) was added, and the mixture was stirred for 15 minutes. The DMF was removed using a rotary evaporator, and residual DMF was removed by evaporating toluene twice. The product was isolated using a 220 g SiO2 column with a solid load of 40 g silica gel followed by CombiFlash®. Eluent: DCM (A) - 20% MeOH (B) in DCM, B = 0-20%, for 15 minutes, then at 20%, for 5 minutes. The product was dried overnight in vacuum. Yield 2.726g. Calculated value: MW 491.68. Measured value: MS (ES, positive): 492.46[M+H]+.

[0256] Compound 3: A solution of Compound 2 (2.7 g, 5.49 mmol) in anhydrous pyridine (40 mL) was cooled on an ice bath, and TMS-Cl (2.25 mL, 17.73 mmol) was added. The reaction mixture was stirred on an ice bath for 30 minutes, benzoyl chloride (1.5 mL, 12.9 mmol) was added, the cooling bath was removed after 30 minutes, and stirring was continued overnight. After 16 hours, the reaction mixture was cooled on an ice bath, water (7.5 mL) was added, and stirring was continued for another 30 minutes. Concentrated NH4OH (7.5 mL) was added, and all volatile substances were removed using a rotary evaporator. To remove the side bis-acylated adduct, the crude substance was dissolved in MeOH (125 mL) and treated with NH4OH (13 mL) for 25 minutes. The solvent was removed using a rotary evaporator, and toluene was evaporated once. CombiFlash® purification was performed using a solid load of silica gel (18g) on ​​an 80g column with eluent: 20% MeOH in DCM-DCM, 0-20%, 50 minutes. Yield: 2.66g. Calculated value: MW 595.79. Measured value: MS (ES, positive): 596.53[M+H]+.

[0257] Compound 4: Compound 3 (1.55 g, 2.60 mmol) was dried by sequentially evaporating toluene and pyridine anhydride using a rotating dry evaporator. This was dissolved in pyridine anhydride (10 mL), DMAP (12 mg, 0.1 mmol) was added, followed by dimethoxytrityl chloride (965 mg, 2.9 mmol). The reaction mixture was stirred at room temperature for 16 hours. All volatile substances were removed using a rotating evaporator, and residual pyridine was removed by evaporation of toluene. The residue was partitioned between DCM and aqueous NaHCO3. The organic phase was separated, the aqueous phase was extracted with DCM, and the combined organic phase was dried (Na2SO4) and concentrated. The product was isolated by CombiFlash® using a 40 g column, eluent: hexane(A)-ethyl acetate(B) + 1% Et3N, B=20-60%, 40 minutes. Yield 1.845 g. Calculated MW: 898.16. Actual value: MS (ES, positive): 899.65 [M+H]+.

[0258] Compound 5: Compound 4 (1.845 g, 2.052 mmol) was dried by two evaporations of toluene. This was dissolved in anhydrous DCM (30 mL), and diisopropylammonium tetrazolide (176 mg, 1.03 mmol) and dried molecular sieves (100 mg) were added, and the mixture was stirred for 30 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (804 mg, 2.67 mmol) was added, and the mixture was stirred for 16 hours. The reaction mixture was diluted 3-fold with anhydrous DCM, filtered, and stirred with 150 mL of cold NaHCO3 solution for 5 minutes. The organic phase was separated, the aqueous phase was extracted with DCM, and the combined organic phase was washed with NaHCO3 and dried (Na2SO4). The product was isolated by CombiFlash® using a 40g column, eluent: hexane(A)-ethyl acetate(B) + 1% Et3N, B=15-60%, for 30 minutes. Yield: 1.386g. Calculated value: MW 1098.38. Measured value: MS (ES, positive): 1099.17[M+H]+.

[0259] Synthesis of phosphoramidites for gC16 internal nucleotides [ka]

[0260] A solution of compound 2:N2-isobutyrylguanosine 1 (5 g, 14.15 mmol) in anhydrous DMF (120 mL) was added to a cold flask containing sodium hydride (60% dispersion in mineral oil, 1.3 g, 32.55 mmol). The cooling bath was removed, and the reaction mixture was stirred at room temperature for 3 hours, after which 1-bromohexadecane (5.61 g, 18.4 mmol) was added. The reaction mixture was stirred at 50 °C for 72 hours, after which EtOH (2 mL) was added. DMF was removed using a rotary evaporator, and residual DMF was removed by evaporating toluene twice. The product was isolated using a CombiFlash® following a solid load of 30 g silica gel using a 120 g column. Separation system: A = DCM: Depositphotos (1:1); B = DCM: Depositphotos:MeOH (9:9:2). B = 0-100%, 50 min. Retention fraction B = 32-70%. Yield: 2.45g. Calculated value: MW 577.77. Measured value: MS (ES, positive): 579.39[M+H]+.

[0261] Compound 3: Compound 2 (2.129 g, 3.69 mmol) was dried by two evaporations of toluene, followed by one evaporation of anhydrous pyridine. This was dissolved in anhydrous pyridine (40 mL), and dimethoxytrityl chloride (1.375 g, 4.06 mmol) and DMAP (18 mg, 0.148 mmol) were added, and the mixture was stirred overnight. All volatile substances were removed using a rotary evaporator, followed by two evaporations of toluene. The residue was collected in DCM (150 mL) and stirred with NaHCO3 (50 mL) for 3 minutes. The organic phase was separated, the aqueous phase was extracted with DCM, and the combined organic phase was washed with NaHCO3 and dried (Na2SO4). The product was isolated by CombiFlash® using a 40 g column, eluent: hexane(A)-ethyl acetate(B) + 1% Et3N, B=20-90%, 40 minutes. Retained fraction B = 58-70%. Yield 1.606g. Calculated value: MW 880.14. Measured value: MS (ES, positive): 881.34[M+H]+.

[0262] Compound 4: Compound 3 (1.4 g, 1.59 mmol) was dried by two evaporations of toluene. This was dissolved in anhydrous DCM (30 mL), and diisopropylammonium tetrazolide (136 mg, 0.8 mmol) and dried molecular sieve (100 mg) were added, and the mixture was stirred for 30 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (767 mg, 2.54 mmol) was added, and the mixture was stirred for 16 hours. Et3N (0.3 mL) was added, followed by silica gel (6 g). The mixture was concentrated under vacuum and solid-loaded using CombiFlash®. The product was isolated using a 40 g column, eluent: hexane(A)-ethyl acetate(B) + 1% Et3N, B=20-90%, 35 minutes. Yield: 836 mg. Calculated value: MW1080.36. Actual value: MS (ES, positive): 1081.38 [M+H]+.

[0263] Synthesis of phosphoramidites for uC16 internal nucleotides [ka]

[0264] Compound 2: Dried 2,2'-cyclouridine 1 (18 g, 79.6 mmol) and DMAP (486 mg, 3.98 mmol) were suspended in anhydrous pyridine (120 mL) and treated with tert-butyl(chloro)diphenylsilane (25.6 g, 93.14 mmol). The reaction mixture was stirred at room temperature for 48 hours. Pyridine was removed by rotary evaporator, and residual pyridine was co-evaporated with toluene. The residue was dissolved in ethyl acetate (200 mL), washed with 10% H3PO4 (75 mL), 5% NaCl, and brine, and then dried over Na2SO4. Compound 2 was isolated after CombiFlash® purification using two 120 g SiO2 columns, eluent DCM (A) - 20% MeOH in DCM (B), B = 0-60%. Yield 14 g. Calculated MW: 464.59. Actual value: MS (ES, positive): 465.71 [M+H]+.

[0265] Compound 3: Hexadecanol (49.6 g, 204.4 mmol) was dried overnight in vacuum. It was dissolved in dried diglyme (38 mL) while heating and cooled to room temperature. AlMe3 (2 M solution in heptane, 31.25 mL, 62.5 mmol) was slowly added under a flow of N2, and the reaction mixture was heated at 110°C until methane evolution stopped (30 minutes). It was cooled to room temperature, dried uridine derivative 2 (13.2 g, 28.39 mmol) was added as a solid, and dried diglyme (30 mL) was added. The reaction mixture was heated at 140°C for 18 hours. It was partitioned into 10% H3PO4 (300 mL) and RINKAN (200 mL). The aqueous layer was extracted with RINKAN, and the combined organic phase was washed twice with 5% NaCl and brine, and dried over Na2SO4. After filtration and concentration, the crude solid was dried by evaporation of toluene and maintained in vacuum overnight. Crude product 3 (115.8 g) was used directly in the next step.

[0266] Compound 4: Crude compound 3 was dissolved in THF (130 mL), triethylamine trishydrofluoride (18 mL, 114 mmol) was added, and the reaction mixture was stirred for 3 days. The product was partitioned into SiO2 (300 mL) and 5% NaCl aqueous solution (250 mL). The organic phase was separated, the aqueous phase was extracted with SiO2, the combined organic phase was washed with brine, dried (Na2SO4), concentrated and dried. Product 4 was isolated after CombiFlash® purification on two 220 g SiO2 columns with a solid load of 60 g silica gel. Eluent: DCM (A), 10% MeOH in DCM (B), B = 0-60% after 60 mins. The product was dried by two evaporations of toluene. Yield 5.63 g. Calculated MW: 468.64. Actual values: MS (ES, positive): 469.52 [M+H]+; 491.52 [M+Na]+.

[0267] Compound 5: Compound 4 (5.63 g, 12 mmol), 4,4'-dimethoxytrityl chloride (5.2 g, 15.36 mmol), and DMAP (293 mg, 2.4 mmol) were dissolved in anhydrous pyridine (40 mL), Et3N (2.17 mL, 15.36 mmol) was added, and the mixture was stirred for 16 hours. The reaction mixture was quenched with MeOH (0.6 mL), stirred for 15 minutes, pyridine was removed under vacuum, and the residue was partitioned into HCl (230 mL) and 5% NaCl aqueous solution (230 mL). The organic phase was separated, the aqueous phase was extracted with HCl, the combined organic phase was washed with brine, and dried over Na2SO4. Purification was performed using CombiFlash® with a 120 g SiO2 column, eluent: hexane (A)-HCl (B), B = 10-50%, for 60 minutes. Yield: 8.271 g. Calculated value: MW 771.01. Measured value: MS (ES, positive): 772.35 [M+H]+.

[0268] Compound 6: Compound 5 (2.33 g, 3.024 mmol) was dried by two evaporations of anhydrous ACN and placed under high vacuum for 2 hours. The dried compound 5 was dissolved in anhydrous DCM (40 mL) and stirred with diisopropylammonium tetrazolide (704 mg, 4.11 mmol) and molecular sieves for 20 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (1.404 g, 4.66 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted to 100 mL with anhydrous DCM, the solid was filtered, and shaken with cold NaHCO3 (100 mL) for 5 minutes. The organic layer was separated, washed with NaHCO3, dried (Na2SO4), and the product was purified by CombiFlash® using a 40 g SiO2 column. Eluent: Hexane(A)-1% Et3N-containing siRNA(B), B=15-50%. Yield 2g. Calculated value: MW 971.23. Measured value: MS (ES, positive): 972.17[M+H]+.

[0269] Synthesis of phosphoramidites for cC16 internal nucleotides [ka]

[0270] Compound 2: The 5'-O-DMT-protected uracil derivative 1 (11.687 g, 15.15 mmol) described in the preparation of cC16 was dissolved in anhydrous pyridine (100 mL) and cooled in an ice bath. TMS-Cl (7.8 mL, 61 mmol) was added, the cooling bath was removed, the reaction mixture was stirred at room temperature for 30 minutes, and then cooled again in an ice bath.

[0271] In a separate flask, 1,2,4-triazole (35 g, 50.7 mmol) was suspended in anhydrous CAN (200 mL), cooled in an ice bath, and POCl3 (11.2 mL, 120 mmol) was slowly added over 10 minutes, followed by stirring at 0°C for 5 minutes. Et3N (84 mL, 595 mmol) was slowly added over 30 minutes, followed by stirring at 0°C for 30 minutes.

[0272] The cooled contents of the first flask, containing the silylated uracil derivative, were rapidly added in one go to the mixture with 1,2,4-triazole, and the mixture was stirred for 10 minutes, after which the cooling bath was removed. Stirring was continued at room temperature for 5 hours. The reaction mixture was concentrated to 1 / 3 of its volume in a rotary evaporator, diluted with ethyl acetate (600 mL), and washed with 5% NaCl (2 × 400 mL). The aqueous phase was back-extracted with ethyl acetate (200 mL). The combined ethyl acetate layers were washed with brine and dried (Na₂SO₄). The ethyl acetate solution was filtered, concentrated, and dried under vacuum to obtain crude derivative 2 (17.46 g).

[0273] Compound 3: Crude compound 4 was dissolved in 220 mL of dry dioxane in a 1 L thick-walled RB flask, and 50 mL of concentrated ammonium hydroxide solution was added. The flask was sealed with a rubber septum, and the reaction mixture was stirred at room temperature for 40 hours. All volatile substances were removed using a rotary evaporator. Toluene was evaporated twice, and the residue was dried. The product was isolated using CombiFlash® following a solid load of 35 g of silica gel using a 220 g SiO2 column. Eluent: DCM (A) - 10% MeOH (B) in DCM, B = 0-45%, 60 min. Yield 7.81. Calculated value: MW 770.02. Measured value: MS (ES, positive): 770.31 [M+H]+, 1541.24 [2M+H]+.

[0274] Compound 4: Compound 3 (6.94 g, 9 mmol) was dried by evaporation of anhydrous DMF and dissolved in anhydrous DMF (60 mL). Acetic anhydride (1.75 mL, 18 mmol) was added and the mixture was stirred at room temperature for 16 hours. NaHCO3 solution (250 mL) was added and the product was extracted with DCM (2 × 200 mL). This was washed with brine (50 mL), dried to (Na2SO4), concentrated, and dried by two consecutive evaporations of toluene. CombiFlash® purification was performed using a 120 g SiO2 column, eluent: DCM (A), 5% MeOH in DCM (B), B = 0-40%, 60 minutes. Yield 6.55 g. Calculated value: MW 812.06. Measured value: MS (ES, positive): 813.36 [M + H]+.

[0275] Compound 5: Compound 4 (6.55 g, 8.07 mmol) was dried by two evaporations of toluene. This was dissolved in anhydrous DCM (166 mL) and stirred with diisopropylammonium tetrazolide (2.188 g, 12.78 mmol) and molecular sieve (500 mg) for 20 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (4.376 g, 14.52 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted to 300 ml with anhydrous DCM, the solid was filtered, and shaken with cold NaHCO3 (150 mL) for 5 minutes. The organic layer was separated, washed with NaHCO3, dried (Na2SO4), and the product was purified by CombiFlash® using a 120 g SiO2 column. Eluent: Hexane(A)-siRNA(B), B=10-50%, 60 minutes. Yield 6.29g. Calculated value: MW 1012.28. Measured value: MS (ES, positive): 1013.89[M+H]+.

[0276] Example 2. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were subcutaneously injected with either physiological saline or a 3 mg / kg (mpk) compound formulated in physiological saline, in an injection volume of 200 μL / 20 g animal body weight, according to Table 12 below: [Table 12]

[0277] Four mice (n=4) were administered the drug to each group. The mice received a subcutaneous injection on day 1. On day 8, the mice were euthanized, and 50 mg of liver was collected from each animal. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 13 below: [Table 13]

[0278] As shown in Table 13, all treatment groups showed improved knockdown compared to the saline-administered group. AD10271 showed the greatest knockdown in the liver (approximately 76% reduction compared to ATXN2).

[0279] Example 3. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected in 10 μL volumes according to Table 14 below, with either aCSF (artificial cerebrospinal fluid), 50 μg of the compound formulated in aCSF, or 200 μg of the compound formulated in aCSF: [Table 14-1] [Table 14-2]

[0280] Each group received the drug in four mice (n=4), with the exception of Group 1, which received it in three mice (n=3). On day 1, mice were injected intracerebroventricularly. On day 12, the mice were euthanized, and the brain and left half of the thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 15 below: [Table 15]

[0281] As shown in Table 15, most treatment groups showed a dose-dependent improvement in knockdown compared to the aCSF-treated group in both tissues. AC002089 showed the greatest knockdown in the thoracic spinal cord at both dose levels: 50 μg (approximately 35% reduction of ATXN2) and 200 μg (approximately 66% reduction of ATXN2). AC002089 also showed the greatest reduction of ATXN2 in the cerebellum at both dose levels.

[0282] Example 4. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 16 below: [Table 16]

[0283] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 17 below: [Table 17]

[0284] As shown in Table 17, all treatment groups showed improved knockdown compared to the aCSF-treated group in all tissues. With the exception of AC02091, all RNAi agents administered targeted ATXN2 at position 499. All groups showed impressive knockdown, with AC002463 demonstrating the greatest reduction in ATXN2 in the thoracic spinal cord and cerebellum.

[0285] Example 5. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 18 below: [Table 18]

[0286] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 19 below: [Table 19]

[0287] As shown in Table 19, all treatment groups showed improved knockdown in the cerebellum compared to the aCSF-treated group. Each RNAi agent administered to groups 2-14 targeted position 499 of hATXN2.

[0288] Example 6. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 20 below: [Table 20]

[0289] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 21 below: [Table 21]

[0290] As shown in Table 21, all treatment groups showed improved knockdown compared to the aCSF treatment group in at least one of the selected tissues. AD11534 (targeting position 499 of hATXN2) showed the greatest reduction in ATXN2 in the thoracic spinal cord and cortex.

[0291] Example 7. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 22 below: [Table 22]

[0292] Three mice (n=3) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 23 below: [Table 23]

[0293] As shown in Table 23, all treatment groups showed improved knockdown compared to the aCSF-treated group in both tissues. AD11650 showed the greatest reduction in ATXN2 in the thoracic spinal cord, and AD11629 showed the greatest reduction in ATXN2 in the cerebellum.

[0294] Example 8. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 24 below: [Table 24]

[0295] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 25 below: [Table 25-1] [Table 25-2]

[0296] As shown in Table 25, all treatment groups showed improved knockdown compared to the aCSF treatment group in all selected tissues, with AD11916 showing the greatest knockdown in both the cortex and cerebellum.

[0297] Example 9. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or 100 μg of a compound formulated in aCSF, in an injection volume of 10 μL, according to Table 26 below: [Table 26]

[0298] Three mice (n=3) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 27 below: [Table 27]

[0299] As shown in Table 27, all treatment groups showed improved knockdown compared to the aCSF treatment group in at least two of the selected tissues. AD12008 showed significant knockdown in the cortex.

[0300] Example 10. In vivo knockdown of ATXN2 in cynomolgus monkeys On day 1 of the study, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing 60 mg of AD13051 in aCSF, according to Table 28 below: [Table 28]

[0301] In Group 1 (control), three monkeys (n=3) were administered the drug; in Groups 2, 3, and 4 (trigger treatment), six monkeys were administered the drug; and in Group 5 (trigger treatment), five monkeys (n=5) were administered the drug. The monkeys were injected intrathecally on day 1. On day 29 of the study, animals from Groups 1 and 2 were euthanized, and brain and spinal cord tissue was collected from each animal. On day 85 of the study, animals from Group 3 were euthanized, and brain and spinal cord tissue was collected from each animal. On day 168 of the study, animals from Group 4 were euthanized, and brain and spinal cord tissue was collected from each animal. On day 253 of the study, animals from Group 5 were euthanized, and brain and spinal cord tissue was collected from each animal.

[0302] Intrathecal injection in NHP is a challenging procedure, and misadministration, resulting in improper needle placement and leakage of the test sample, is commonly observed due to limited space and reachability. To account for misadministration in protein and expression level analyses, misadministration criteria were defined, including the exclusion of animals that were improperly administered.

[0303] Mis-dosing criteria were based solely on the tissue distribution of the siRNA compound. Cynomolgus monkeys were determined to have received a mis-dosing if approximately 50% or more of the analyzed brain tissue areas had compound concentrations lower than 25% of the group average, and were excluded from the analysis. Of the 26(26) NHPs that received the test sample, 10(10) animals were identified as mis-dosed, and their protein expression level data were excluded from the analysis. Due to mis-dosing, 3 animals were excluded from Group 2, 2 animals from Group 3, 3 animals from Group 4, and 2 animals from Group 5.

[0304] Tissues were lysed in RIPA buffer (Thermo Fisher, #89901) and protease / phosphatase inhibitors were added (Halt, #87786). The total protein concentration for each sample was calculated using BCA (Pierce Protein Assay Kit #23225), and all samples were diluted to 5 mg / ml or an appropriate concentration using RIPA lysis buffer. 3 μL of each diluted tissue sample was combined with 1.5 μL of 5× Sample Mix (from JESS® EZ Standard Pack 3, #PS-ST03EZ-8) and 3 μL of 0.1× Sample Buffer (final sample concentration 2 mg / mL). The samples were then spun down, boiled at 70°C for 10 minutes, spun down again, and cooled on ice. The samples were gently mixed by pipetting up and down before loading into JESS plates (JESS 66-440 kDa isolation module, #SM-W008). The JESS plates were loaded according to the Simple Western instructions. Briefly, 3 μL of sample (2 mg / mL) was loaded per well of the plate. The ATXN2 primary antibody (BD Biosciences, #611378) was diluted 1:400 and α-actinin (CellSignaling, #6487) was diluted 1:100 and loaded into the plates. Secondary antibodies, including anti-mouse (for ATXN2, from the anti-mouse detection module from JESS, #DM-002) and anti-rabbit (for α-actinin, from the anti-rabbit detection module from JESS, #DM-001), were also loaded. After adding all reagents according to the Simple Western instructions, the plates were loaded into the instrument and executed using the JESS default RePlex protocol (onboard instrument protocol, JESS Replex module, RP-001).

[0305] The average results for each group relative to Group 1 are shown in Table 29 below: [Table 29-1] [Table 29-2]

[0306] The decline in ATXN2 protein in CNS tissue samples peaked on day 29 of the study, with an average decline of approximately 80–90% across all major brain regions evaluated. This overall decline of 80–90% observed on day 29 began to recover over time, reaching approximately 50–70% on day 85, approximately 50% on day 169, and less than 50% on day 253, although the exact values ​​varied across specific tissues. Focusing on the cerebellum, the primary brain region affected in SCA2 patients, ATXN2 protein declined by approximately 83% on day 29, 50% on day 85, 64% on day 169, and approximately 8% on day 253. The lumbar spinal cord, closest to the injection site, showed the strongest and most consistent decline in ATXN2 protein over time, with declines of approximately 95% on day 29, 88% on day 85, 81% on day 169, and 75% on day 253.

[0307] Example 11. In vivo knockdown of ATXN2 in cynomolgus monkeys On day 1 of the study, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing 5 mg, 15 mg, or 30 mg of AD13051 in aCSF, according to Table 28 below: [Table 30]

[0308] Four monkeys (n=4) were administered the treatment in Group 1 (control), and six monkeys each were administered the treatment in Groups 2, 3, and 4 (trigger treatment). The treatment was administered intrathecally to the monkeys on day 1. On day 29 of the study, the animals were euthanized, and brain and spinal cord tissue was collected from each animal.

[0309] Intrathecal injection in NHP is a challenging procedure, and misadministration, resulting in improper needle placement and leakage of the test sample, is commonly observed due to limited space and reachability. To account for misadministration in protein and expression level analyses, misadministration criteria were defined, including the exclusion of animals that were improperly administered.

[0310] The mis-dosing criterion was based solely on the tissue distribution of the siRNA compound. Cynomolgus monkeys were determined to have received a mis-dosing if approximately 50% or more of the analyzed brain tissue areas had compound concentrations lower than 25% of the group mean, and were excluded from the analysis. Of the 26(26) NHPs that received the test sample, 10(10) animals were identified as mis-dosed, and their protein expression level data were excluded from the analysis. Due to mis-dosing, 3 animals were excluded from Group 2, 1 animal from Group 3, and 3 animals from Group 4.

[0311] Tissues were lysed in RIPA buffer (Thermo Fisher, #89901) and protease / phosphatase inhibitors were added (Halt, #87786). The total protein concentration for each sample was calculated using BCA (Pierce Protein Assay Kit #23225), and all samples were diluted to 5 mg / ml or an appropriate concentration using RIPA lysis buffer. 3 μL of each diluted tissue sample was combined with 1.5 μL of 5× Sample Mix (from JESS® EZ Standard Pack 3, #PS-ST03EZ-8) and 3 μL of 0.1× Sample Buffer (final sample concentration 2 mg / mL). The samples were then spun down, boiled at 70°C for 10 minutes, spun down again, and cooled on ice. The samples were gently mixed by pipetting up and down before loading into JESS plates (JESS 66-440 kDa isolation module, #SM-W008). The JESS plates were loaded according to the Simple Western instructions. Briefly, 3 μL of sample (2 mg / mL) was loaded per well of the plate. The ATXN2 primary antibody (BD Biosciences, #611378) was diluted 1:400 and α-actinin (CellSignaling, #6487) was diluted 1:100 and loaded into the plates. Secondary antibodies, including anti-mouse (for ATXN2, from the anti-mouse detection module from JESS, #DM-002) and anti-rabbit (for α-actinin, from the anti-rabbit detection module from JESS, #DM-001), were also loaded. After adding all reagents according to the Simple Western instructions, the plates were loaded into the instrument and executed using the JESS default Replex protocol (onboard instrument protocol, JESS Replex module, RP-001).

[0312] The average results for each group relative to Group 1 are shown in Table 31 below: [Table 31-1] [Table 31-2]

[0313] The greatest decrease in ATXN2 protein was observed in the spinal cord closest to the intrathecal injection site, with a 75% decrease at 5 mg in the lumbar region and approximately 90% decreases at 15 mg and 30 mg doses, respectively. In the cervical and thoracic regions, a 35–40% decrease at 5 mg and 60–70% decreases at 15 mg and 30 mg doses were observed. In the cerebellar and major cortical regions analyzed in this study, the greatest decrease in ATXN2 protein was already achieved at the lowest dose of 5 mg, with approximately a 58% decrease in the cerebellum and approximately 67%, 75%, and 54% decreases in the frontal, temporal, and motor cortices, respectively.

[0314] Example 12. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or one of the compounds formulated in aCSF in doses of 18.75 μg, 37.5 μg, 75 μg, 150 μg, or 300 μg in 10 μL of an injection volume, according to Table 26 below: [Table 32-1]

[0315] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, cerebellum, and brainstem. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 33 below: [Table 33-1]

[0316] As shown in Table 33, AD11916 and AD13051, respectively, showed a dose-dependent increase in knockdown in all assayed tissues, with particularly high knockdown in the thoracic spinal cord. AD13051 ligated to the LP-293 PK / PD modulator generally showed increased ATXN2 knockdown compared to AD11916 ligated to LP-183.

[0317] Example 13. In vivo knockdown of ATXN2 in mice On day 1 of the study, C57bl / 6 mice were intraventrally injected with either aCSF (artificial cerebrospinal fluid) or one of the compounds formulated in aCSF in doses of 18.75 μg, 37.5 μg, 75 μg, 150 μg, or 300 μg in 10 μL of an injection volume, according to Table 26 below: [Table 32-2]

[0318] Four mice (n=4) were administered the drug to each group. On day 1, the mice were injected intracerebroventricularly. On day 8, the mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, cerebellum, and brainstem. The samples were analyzed for ATXN2 knockdown by qPCR. The mean results for each group are shown in Table 33 below: [Table 33-2-1] [Table 33-2-2]

[0319] As shown in Table 33, AD12008 and AD13052, respectively, showed a dose-dependent increase in knockdown in all assayed tissues, with particularly high knockdown in the thoracic spinal cord. AD13052, ligated to the LP-293 PK / PD modulator, generally showed increased ATXN2 knockdown compared to AD12008 ligated to LP-183.

[0320] Example 13. In vivo knockdown of ATXN2 in mice A transgenic mouse model, BAC-Q22, expressing the hATXN2 gene with a normal number of 22 CAG repeats, was used in this experiment. On day 1 of the study, mice were administered ICV injections of aCSF or AD13051 at different concentrations (Table 34). Clinical observations were performed at 3 and 24 hours post-administration, and daily health checks were conducted throughout the study. On day 15 of the study, the animals' body weight was measured, and they were then humanely euthanized by administration of isoflurane followed by transcardiac perfusion. The thoracic spinal cord and cerebellum were collected for downstream analysis. [Table 34]

[0321] RNA was extracted from frozen tissue and purified using the Qiagen RNeasy Mini Kit (catalog number 74106) according to the standard protocol. Reverse transcription was performed using the ThermoFisher High-Capacity cDNA Reverse Transcription Kit (catalog number 4368814) according to the manufacturer's instructions. qPCR was performed using the Quantstudio 12K Flex Real-Time PCR System (Applied Biosystems) with human ATXN2 Taqman Assay ID Hs00268077_m1 and mouse β-actin (mACTB) Taqman Assay ID Mm02619580_g1 (ThermoFisher).

[0322] For RNA from spinal cord tissue, qPCR was performed in four-strand units using a multiplexed hATXN2 and mACTB Taqman assay. Quantification of each transcript was determined using a standard curve. After normalizing hATXN2 to mACTB in each reaction, the average of the four-strand wells was determined for each RNA sample.

[0323] RNA from cerebellar tissue was subjected to hATXN2 and mACTB reactions separately for each animal sample, using 4-replica and 3-replica methods, respectively. Using the delta-Ct method, the Ct repeats were averaged for each sample, and then the hATXN2 expression level, normalized to mACTB, was determined.

[0324] Protein extracts were prepared by homogenizing mouse cerebellum in extraction buffer (25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl2, 0.5 M urea and protease inhibitor; Sigma; catalog no. P-8340), followed by centrifugation at 16,100 × g at 4°C for 20 minutes. Only the supernatant was used for Western blotting to determine steady-state protein levels. Protein extracts were separated by SDS-PAGE and transferred to Hybond P membranes (Amersham Bioscience Inc., USA). After blocking with 5% skim milk in 0.1% Tween® 20 / PBS, the membranes were incubated with primary antibodies in 0.1% Tween® 20 / PBS with 5% skim milk at room temperature for 2 hours or overnight at 4°C. After washing with 0.1% Tween® 20 / PBS, the membrane was incubated at room temperature for 2 hours with the corresponding secondary antibody conjugated with HRP in 5% skim milk in 0.1% Tween® 20 / PBS, and then washed again. The signal was detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore Inc., USA; catalog number WBKLSO100) according to the manufacturer's protocol. Protein intensity was determined using the ImageJ software analysis system, and the protein was quantified as a ratio to β-actin. ATXN2 monoclonal antibody (BD Biosciences Inc., catalog number 611378) was used at a ratio of 1:4000. The secondary antibody was goat anti-mouse IgG-HRP (1:5000) (Sigma Inc., catalog number A2304). ACTB monoclonal antibody was HRP conjugated and used at a ratio of 1:10,000 (Sigma-Aldrich, catalog number A3854).

[0325] Tables 35 and 36 show the decrease in hATNX2 mRNA and protein, respectively. [Table 35] [Table 36]

[0326] AD13051 generally shows a dose-dependent response in mice expressing hATXN2. AD13051 demonstrated deep ATXN2 protein knockdown at a low dose of 30 μg.

[0327] Other Embodiments While the present invention has been described in detail, it should be understood that the foregoing description is illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An RNAi agent for inhibiting the expression of the Ataxin-2 (ATXN2) gene, An antisense strand containing at least 17 consecutive nucleotides that differ by only 0 or 1 nucleotide from any one of the sequences provided in Table 2 or Table 3, An RNAi agent comprising a sense strand having a nucleotide sequence at least partially complementary to the antisense strand.

2. The RNAi agent according to claim 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence provided in Table 2 or Table 3.

3. The RNAi agent according to claim 1 or 2, wherein the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs by 0 or 1 nucleotide from any one of the sequences provided in Table 2 or Table 4, and the sense strand has a region of at least 85% complementarity with respect to the antisense strand across the 17 consecutive nucleotides.

4. The RNAi agent according to any one of claims 1 to 3, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or comprises a modified nucleoside bond.

5. The RNAi agent according to any one of claims 1 to 4, wherein all or substantially all of the nucleotides are modified nucleotides.

6. The RNAi agent according to any one of claims 4 to 5, wherein the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methylnucleotide, inverted 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinylphosphonate-containing nucleotide, cyclopropylphosphonate-containing nucleotide, and 3'-O-methylnucleotide.

7. The RNAi agent according to claim 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

8. The RNAi agent according to any one of claims 1 to 7, wherein the antisense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 3.

9. The RNAi agent according to any one of claims 1 to 8, wherein the sense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 4.

10. The RNAi agent according to claim 1, wherein the antisense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 3, and the sense strand comprises one nucleotide sequence from any of the modified sequences provided in Table 4.

11. The RNAi agent according to any one of claims 1 to 10, wherein the sense strand is 18 to 30 nucleotides long and the antisense strand is 18 to 30 nucleotides long.

12. The RNAi agent according to claim 11, wherein the sense strand and the antisense strand are each 18 to 27 nucleotides long.

13. The RNAi agent according to claim 12, wherein the sense strand and the antisense strand are each 18 to 24 nucleotides long.

14. The RNAi agent according to claim 13, wherein the sense strand and the antisense strand are each 21 nucleotides long.

15. The RNAi agent according to claim 14, wherein the RNAi agent has two blunt ends.

16. The RNAi agent according to any one of claims 1 to 15, wherein the sense strand comprises one or two terminal caps.

17. The RNAi agent according to any one of claims 1 to 16, wherein the sense strand comprises one or two inverse debase residues.

18. The RNAi agent according to claim 1, wherein the RNAi agent comprises a sense strand and an antisense strand that form a double helix having one of the double helix structures shown in Table 7, Table 8, Table 9A, or Table 10.

19. The RNAi agent according to claim 18, wherein all or substantially all of the nucleotides are modified nucleotides.

20. The RNAi agent according to claim 19, wherein all or substantially all of the nucleotides are modified nucleotides.

21. The RNAi agent according to any one of claims 19 to 20, wherein the sense strand further comprises an inverted debase residue at the 3' terminal end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

22. The RNAi agent according to any one of claims 1 to 21, wherein the RNAi agent is linked to a lipid portion.

23. The aforementioned lipid portion is as follows: 【Chemistry 20】 Selected from the group consisting of, in the formula, 【Chemistry 21】 The RNAi agent according to claim 22, wherein is a linking point with the RNAi agent.

24. The RNAi agent according to claim 22 or claim 23, wherein the lipid portion is conjugated to the sense strand.

25. The RNAi agent according to claim 24, wherein the lipid portion is conjugated to the 5' terminal end of the sense strand.

26. A composition comprising an RNAi agent according to any one of claims 1 to 25, wherein the composition further comprises a pharmaceutically acceptable additive.

27. The composition according to claim 26, further comprising a second RNAi agent capable of inhibiting the expression of the Ataxin-2 gene.

28. The composition according to any one of claims 26 to 27, further comprising one or more additional therapeutic agents.

29. The composition according to any one of claims 26 to 28, wherein the RNAi agent is a sodium salt.

30. The composition according to any one of claims 26 to 29, wherein the pharmaceutically acceptable additive is water for injection.

31. The composition according to any one of claims 26 to 29, wherein the pharmaceutically acceptable additive is buffered saline.

32. A method for inhibiting the expression of the ATXN2 gene in cells, comprising introducing an effective amount of an RNAi agent according to any one of claims 1 to 25 or a composition according to any one of claims 26 to 31 into the cells.

33. The method according to claim 32, wherein the aforementioned cells are located within the target.

34. The method according to claim 33, wherein the subject is a human subject.

35. The method according to any one of claims 32 to 34, wherein, after administration of the RNAi agent, the expression of the Ataxin-2 (ATXN2) gene is inhibited by at least about 30%.

36. A method for treating one or more symptoms or diseases associated with enhancement or elevation of membrane ATXN2 activity levels, comprising administering a therapeutically effective amount of the composition according to any one of claims 26 to 31 to a human subject in need thereof.

37. The method according to claim 36, wherein the disease is a neurodegenerative disease.

38. The method according to claim 37, wherein the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2).

39. The method according to claim 37, wherein the disease is ALS.

40. The method according to any one of claims 32 to 39, wherein the RNAi agent is administered in a dose of approximately 0.01 mg per kg of body weight of the subject to approximately 5.0 mg per kg.

41. The method according to any one of claims 32 to 40, wherein the RNAi agent is administered in a dose of approximately 0.03 mg per kg of body weight of the subject to approximately 2.0 mg per kg.

42. The method according to any one of claims 32 to 41, wherein the RNAi agent is administered in a dose of 2 or more.

43. Use of an RNAi agent according to any one of claims 1 to 25 for the treatment of a disease, disorder or condition that is at least partially mediated by mutant ATXN2 activity and / or ATXN2 gene expression.

44. Use of the composition according to any one of claims 26 to 31 for the treatment of a disease, disorder or condition that is at least partially mediated by Ataxin-2 (ATXN2) activity and / or Ataxin-2 (ATXN2) gene expression.

45. Use of the composition according to any one of claims 26 to 31 for the manufacture of a pharmaceutical product for the treatment of a disease, disorder or condition that is at least partially mediated by Ataxin-2 (ATXN2) gene expression.

46. The use according to any one of claims 43 to 45, wherein the disease is a neurodegenerative disease.

47. A method for producing an RNAi agent according to any one of claims 1 to 25, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

48. The method according to claim 47, wherein the sense chain includes a lipid portion.

49. The method according to claim 48, comprising conjugating the lipid portion to the sense chain.

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