Nucleic acids for inhibiting expression of gpr146 in a cell

Abstract

The present invention relates to double-stranded nucleic acid molecules that interfere with or inhibit the expression of the G protein-coupled receptor 146 (GPR146) gene. The present invention further relates to the therapeutic use of such inhibition, such as for the treatment of a GPR146-mediated disease, disorder or syndrome, such as hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis and / or atherosclerotic cardiovascular disease.

Description

Technical Field

[0001] This invention relates to double-stranded nucleic acid molecules that interfere with or inhibit the expression of the G protein-coupled receptor 146 (GPR146) gene. The invention further relates to the therapeutic use of such inhibition, such as for treating GPR146-mediated diseases, conditions, or syndromes, such as hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis, and / or atherosclerotic cardiovascular disease. Background Technology

[0002] Double-stranded RNAs (dsRNAs) capable of binding to expressed mRNAs via complementary base pairing have been shown to block gene expression through a mechanism known as RNA interference (RNAi) (Fire et al., 1998, Nature. Feb. 19, 1998; 391(6669): 806-11 and Elbashir et al., 2001, Nature. May 24, 2001; 411(6836): 494-8). Short dsRNAs guide gene-specific posttranscriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades messenger RNA that is sufficiently complementary or homologous to the silencing trigger loaded into the RISC complex. Interfering RNAs such as siRNAs, antisense RNAs, and microRNAs prevent protein formation by inhibiting gene translation through gene silencing (i.e., by degrading mRNA molecules to suppress protein translation). Gene silencers are becoming increasingly important in medical treatment applications.

[0003] According to Watts and Corey's article in the *Journal of Pathology* (2012; Vol. 226, pp. 365-379), algorithms exist for designing nucleic acid silencing triggers, but all of these algorithms have significant limitations. Multiple experimental methods may be required to identify effective siRNAs because the algorithms do not consider factors such as the tertiary structure of the target mRNA or the involvement of RNA-binding proteins. Therefore, discovering effective nucleic acid silencing triggers with minimal off-target effects is a complex process. For drug development of these high-charge molecules, the following are essential: they must be economically synthesized, distributed to target tissues, enter cells, and exert their effects within acceptable toxicity limits.

[0004] High cholesterol is a major risk factor for cardiovascular disease. For patients with atherosclerotic cardiovascular disease, lowering cholesterol can reduce cardiovascular mortality.

[0005] GPR146 is an orphan G protein-coupled receptor (GPCR) located in the plasma membrane of various tissues and organs, including the liver and adipose tissue. GPCRs typically signal via Gαs, Gαi / o, Gαq / 11, Gα12 / 13, or Gβ / γ, and regulate cAMP levels, Ca2+ mobilization, ERK / MAPK activity, or small G protein RhoA activity (Cheng et al., 2010. Curr. Chem. Genomic. Dec. 21:4:84-91). GPR146 has been shown to promote the ERK1 / 2 signaling cascade in hepatocytes, which in turn increases SREBP2 activity and VLDL secretion rate, while acting independently of LDL receptor activity (Yu et al., 2019. Cell. Vol. 179, No. 6, pp. 1276-1288). Genetic variation measurements of single nucleotide polymorphisms revealed that rs1997243 is a pathogenic variant that increases GPR146 expression, leading to elevated blood cholesterol in humans. Animal models showed that Gpr146 is primarily involved in the regulation of cholesterol levels through the liver, and that gpr146 knockout mice exhibited reduced blood cholesterol and triglyceride levels (Han et al., 2020. Cell Research. 30, 363-365).

[0006] WO2021174056A1 discloses siRNA targeting GPR146 for the treatment of GPR146-related conditions, such as hypercholesterolemia and atherosclerosis.

[0007] Preclinical mechanistic studies suggest that RNA interference-mediated off-target effects may be the driving force behind the hepatotoxicity of GalNAc-siRNA conjugates. These off-target effects can be driven by the binding of RISC-loaded siRNA to off-target transcripts via base pairing between the siRNA guide strand seed region (nucleotides 2-8) and complementary sites in the 3'-untranslated region of mRNA. This non-catalytic mechanism essentially mimics post-transcriptional silencing of endogenous miRNAs and can lead to translational repression and / or mRNA destabilization at suprapharmacological levels of RISC-loaded siRNAs, with the reduction in mRNA levels accounting for the majority (66% to >90%) of mammalian miRNA-mediated post-transcriptional repression (Schlegel et al., Nucleic Acid Research 2022; 50(12), 6656-6670).

[0008] Therefore, one object of the present invention is to provide effective and safe compounds and (pharmaceutical) compositions for treating GPR146-mediated diseases, conditions or syndromes (e.g., hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis and / or atherosclerotic cardiovascular disease). Summary of the Invention

[0009] One aspect of the present invention is a double-stranded nucleic acid for inhibiting the expression of G protein-coupled receptor 146 (GPR146), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified corresponding sequence of the first strand comprises a sequence of at least 15 nucleotides, which differs from any of the first strand sequences shown in Tables 5a, 1, 5b, 2 or 5c by no more than 3 nucleotides.

[0010] Therefore, the nucleic acid described herein is a double-stranded nucleic acid capable of inhibiting GPR146 expression (preferably inhibiting GPR146 expression in cells) and can be used as a therapeutic or diagnostic agent, for example, in relevant therapeutic or diagnostic methods.

[0011] The nucleic acid of the present invention comprises a first strand and a second strand, or is composed of a first strand and a second strand, and the first strand typically contains a sequence sufficiently complementary to GPR146 mRNA to mediate RNA interference.

[0012] One aspect relates to compositions comprising nucleic acids disclosed herein and solvents (preferably water) and / or delivery media and / or physiologically acceptable excipients and / or carriers and / or salts and / or diluents and / or buffers and / or preservatives.

[0013] One aspect relates to compositions comprising the nucleic acids and therapeutic agents disclosed herein, said therapeutic agents being selected, for example, oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides.

[0014] One aspect relates to the nucleic acids disclosed herein or compositions containing said nucleic acids, used as therapeutic or diagnostic agents, for example, in relevant methods.

[0015] One aspect relates to the nucleic acids disclosed herein or compositions containing said nucleic acids for the prevention or treatment of diseases, symptoms or syndromes.

[0016] One aspect relates to the use of the nucleic acids disclosed herein or compositions containing said nucleic acids in the prevention or treatment of diseases, symptoms or syndromes.

[0017] One aspect relates to the use of the nucleic acids disclosed herein or compositions containing said nucleic acids in the preparation of medicaments for the prevention or treatment of diseases, symptoms or syndromes.

[0018] One aspect relates to the compositions disclosed herein, used as pharmaceuticals.

[0019] One aspect relates to a method for preventing or treating a disease, condition, or syndrome, comprising administering a pharmaceutically effective dose or amount of the nucleic acid or composition disclosed herein to a subject requiring treatment. Preferably, the nucleic acid or composition is administered to the subject subcutaneously, intravenously, or orally, rectally, pulmonaryly, intramuscularly, or intraperitoneally. Detailed Implementation

[0020] This invention relates to a nucleic acid and compositions thereof, said nucleic acid being double-stranded and containing a sequence homologous to the GPR146 expression RNA transcript. These nucleic acids, their conjugates, and compositions comprising them can be used for the prevention and treatment of various diseases, conditions, and syndromes in which reduced expression of the GPR146 gene product is desired.

[0021] One aspect of the invention is a double-stranded nucleic acid for inhibiting the expression of G protein-coupled receptor 146 (GPR146), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 nucleotides, which differs from any of the first strand sequences shown in Tables 5a, 1, 5b, 2 or 5c by no more than 3 nucleotides.

[0022] The unmodified corresponding sequence of the first strand sequence may comprise a sequence of at least 15 consecutive nucleotides from any of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or Table 5c. The unmodified corresponding sequence of the first strand sequence may comprise a sequence of at least 16 consecutive nucleotides from any of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or Table 5c. The unmodified corresponding sequence of the first strand sequence may comprise a sequence of at least 17 consecutive nucleotides from any of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or Table 5c. The unmodified corresponding sequence of the first strand sequence may comprise a sequence of at least 18 consecutive nucleotides from any of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or Table 5c.

[0023] One aspect of the invention is a double-stranded nucleic acid for inhibiting GPR146 expression (preferably inhibiting GPR146 expression in cells), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified corresponding sequence of the first strand sequence comprises a sequence of at least 15 nucleotides, which differs from any of the first strand sequences shown in Table 5a by no more than 3 nucleotides. These nucleic acids have particular advantages in that they are active in a variety of species relevant to preclinical and clinical development and / or have few associated off-target effects. Having few associated off-target effects means that the nucleic acid specifically inhibits the intended target and does not significantly inhibit other genes, or only inhibits one or a few other genes at a therapeutically acceptable level.

[0024] The first chain can also be called the antisense chain. The second chain can also be called the meaningful chain.

[0025] In the context of this invention, inhibiting GPR146 expression should be understood as meaning that nucleic acids can reduce the expression level of GPR146.

[0026] Optionally, particularly if the nucleic acid or conjugated nucleic acid of the present invention is administered to a subject, the level of inhibition can be measured in different cell populations or tissues or organs or body fluids (such as blood or plasma).

[0027] For example, the unmodified corresponding sequence of the first strand sequence can be a sequence containing at least 15 nucleotides, which differs from any of the first strand sequences listed in Table 1 by no more than 3 nucleotides:

[0028] Table 1

[0029] 53 54 55 56 99 100 125 126 161 162 231 232 285 286 383 384

[0030] For example, the unmodified corresponding sequence of the first strand sequence may contain at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 nucleotide sequences that differ from any of the first strand sequences listed in Table 1 or Table 5a by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably without any nucleotide differences.

[0031] The unmodified corresponding sequence of the first strand sequence may comprise a sequence of at least 15 consecutive nucleotides from any of the first strand sequences listed in Table 1 or Table 5a. The unmodified corresponding sequence of the first strand sequence may comprise preferably at least 16 consecutive nucleotides, more preferably at least 17 consecutive nucleotides, even more preferably at least 18 consecutive nucleotides, and most preferably all 19 nucleotides, provided that there are no nucleotide differences from any of the first strand sequences listed in Table 1 or Table 5a.

[0032] Preferably, the unmodified corresponding sequence of the first strand of the nucleic acid consists of one of the first strand sequences shown in Table 1 or Table 5a. However, this sequence can be modified by many nucleic acid modifications that do not alter the identity of the nucleotide. For example, modifications to the backbone or sugar residues of the nucleic acid do not change the identity of the nucleotide because the base itself is the same as in the reference sequence. However, it should be understood that when a nucleotide is modified, it may be referred to as a "modified nucleotide".

[0033] For example, the unmodified corresponding sequence of the first strand of a nucleic acid may consist of one of the first strand sequences shown in Table 1 or Table 5a, optionally modified by one or more of the nucleic acid modifications described.

[0034] In this document, a nucleic acid comprising a sequence according to a reference sequence means that the nucleic acid comprises a sequence of consecutive nucleotides in the order defined in the reference sequence. As used herein, the term “reference sequence” will generally be understood to mean a sequence present in, for example, at least one table disclosed herein (such as Table 5a, Table 1, Table 5b, Table 2, or Table 5c).

[0035] In this document, when referring to a reference sequence that contains nucleotides, is substantially composed of nucleotides, or is composed of nucleotides, the reference is not limited to sequences having unmodified nucleotides. The same reference also covers identical nucleotide sequences in which one or more (such as two, three, four, five, six, seven, or more, including all) nucleotides are modified by modifications such as, for example, 2'-OMe, 2'-F, ligands, linkers, 3' or 5' modifications, or any other modifications. It also refers to sequences in which two or more nucleotides are interconnected by native phosphodiester bonds or any other bonds such as thiophosphate or dithiophosphate bonds.

[0036] Double-stranded nucleic acids are nucleic acids in which the first and second strands hybridize with each other at least a portion of their length, and thus are capable of forming double-stranded regions under physiological conditions (such as in PBS, at 37°C, with a concentration of 1 μM per strand). The first and second strands are preferably capable of hybridizing with each other, and thus form double-stranded regions over a region of at least 15 nucleotides, preferably 16, 17, 18, or 19 nucleotides. The first and second strands can form double-stranded regions of 17-25 nucleotides in length. For example, the first and second strands can be capable of hybridizing with each other, and thus form double-stranded regions over a region of at least 19 nucleotides. The first and second strands can form double-stranded regions of 19 nucleotides in length. The double-stranded region can consist of 17-25 consecutive nucleotide base pairs.

[0037] The double-stranded region comprises nucleotide base pairings between the two strands, preferably based on Watson-Crick base pairings and / or wobbling base pairings (such as GU base pairings). It is not necessary for all nucleotides of both strands within the double-stranded region to be base-paired with each other to form the double-stranded region. A certain number of mismatches, deletions, or insertions between the nucleotide sequences of the two strands are acceptable. Unpaired nucleotides at either end of the first or second strand, or at either end of the double-stranded nucleic acid, are also possible. The double-stranded nucleic acid is preferably a physiologically stable double-stranded nucleic acid and preferably has a melting temperature (Tm) of 45°C or higher, preferably 50°C or higher, and more preferably 55°C or higher (e.g., at a concentration of 1 μM per strand in PBS).

[0038] The double-chain region (formed between the first and second chains) can consist of one and only one double-chain region.

[0039] A physiologically stable double-stranded nucleic acid is a double-stranded nucleic acid with a Tm of 45°C or higher, preferably 50°C or higher, and more preferably 55°C or higher (e.g., at a concentration of 1 μM per strand in PBS).

[0040] The first and second strands are preferably capable of forming double-stranded regions (i.e., complementary to each other) on at least i) a portion of their length (preferably at least 15 nucleotides of their length), ii) the entire length of the first strand, iii) the entire length of the second strand, or iv) the entire length of both the first and second strands. The strands being complementary to each other at a specific length means that the strands can pair with each other at that length via Watson-Crick or swing base pairing. Each nucleotide at that length does not necessarily need to be able to pair with its corresponding base in the other strand throughout the given length, as long as a physiologically stable double-stranded nucleotide can be formed. However, in some embodiments, it is preferred that each nucleotide at that length can pair with its corresponding base in the other strand throughout the given length.

[0041] In the context of the nucleic acid according to the invention, a certain number of mismatches, deletions or insertions between the first strand and the target sequence or between the first strand and the second strand are tolerable and even have the potential to increase RNA interference (e.g., inhibition) activity in some cases.

[0042] The inhibitory activity of the nucleic acid according to the present invention depends on the formation of a double-stranded region between all or part of the first strand and a portion of the target nucleic acid. The portion of the target nucleic acid forming the double-stranded region with the first strand is the target nucleic acid sequence, or simply the target sequence. The double-stranded region is defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence (including both the first and last base pairs). The double-stranded region formed between the first and second strands need not be identical to the double-stranded region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a double-stranded structure with both the second strand and the target sequence, at least under physiological conditions.

[0043] The complementarity between the first strand and the target sequence can be complete (i.e., the first strand has 100% identity with the target sequence, with no nucleotide mismatches, insertions, or deletions).

[0044] The complementarity between the first strand and the complementary sequence of the target sequence can range from about 75% to about 100%. More specifically, the complementarity can be at least 75%, 80%, 85%, 90%, or 95%, as well as intermediate values, provided that the nucleic acid can reduce or inhibit the expression of GPR146.

[0045] Nucleic acids with less than 100% complementarity between the first strand and the target sequence can reduce GPR146 expression to the same level as nucleic acids with complete complementarity between the first strand and the target sequence. Optionally, they can reduce GPR146 expression to levels of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the reduction achieved by nucleic acids with complete complementarity.

[0046] The nucleic acid disclosed herein can be an isolated nucleic acid.

[0047] The nucleic acid disclosed herein can be a nucleic acid that, in which:

[0048] (a) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any first strand sequence in Table 5a by no more than 3 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5a by no more than 3 nucleotides.

[0049] (b) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any first strand sequence in Table 5a by no more than 2 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5a by no more than 2 nucleotides.

[0050] (c) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any first strand sequence in Table 5a by no more than 1 nucleotide, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5a by no more than 1 nucleotide.

[0051] (d) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a;

[0052] (e) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to the 2nd to 18th nucleotides from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to the 2nd to 18th nucleotides from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a;

[0053] (f) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a;

[0054] (g) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a;

[0055] (h) The unmodified corresponding sequence of the first chain sequence includes the sequence of any first chain sequence with a given SEQ ID No. shown in Table 5a, and optionally wherein the unmodified corresponding sequence of the second chain sequence includes the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a;

[0056] (i) The unmodified corresponding sequence of the first chain sequence consists substantially of any of the first chain sequences shown in Table 5a having a given SEQ ID No., and optionally wherein the unmodified corresponding sequence of the second chain sequence consists substantially of the sequence of the corresponding second chain sequence shown in Table 5a having a given SEQ ID No.;

[0057] (j) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises or is substantially composed of the sequence of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a;

[0058] (k) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the unmodified corresponding sequence of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 5a, and wherein the unmodified corresponding sequence of the first strand sequence further has one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 5a.

[0059] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or consists substantially of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a;

[0060] (l) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5a; and

[0061] The unmodified sequence corresponding to the first strand contains a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consisting of or composed of said continuous region; and

[0062] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or consists substantially of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a;

[0063] (m) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the unmodified corresponding sequence of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 5a, and wherein the unmodified corresponding sequence of the first strand sequence further has one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 5a.

[0064] The unmodified sequence corresponding to the first strand contains a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consisting of or composed of said continuous region; and

[0065] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or consists substantially of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a;

[0066] (n) The unmodified corresponding sequence of the first chain sequence consists of any first chain sequence with a given SEQ ID No. shown in Table 5a, and optionally the unmodified corresponding sequence of the second chain sequence consists of the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a;

[0067] (o) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) exist on a single strand, wherein the unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand can hybridize with each other, thereby forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; or

[0068] (p) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) are on two separate strands that can hybridize with each other and thus form a double-stranded nucleic acid with a double-stranded region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0069] In (l) or (m), the unmodified corresponding sequence of the first strand sequence may comprise, substantially consist of, or consist of a continuous region of 19-25 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861. In (l) or (m), the unmodified corresponding sequence of the first strand sequence may comprise, preferably, a continuous region of 20-24 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consist of, or consist of, the continuous region.

[0070] For example, the nucleic acid disclosed herein can be a nucleic acid in which:

[0071] (a) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any first strand sequence in Table 1 by no more than 3 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 1 by no more than 3 nucleotides.

[0072] (b) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any of the first strand sequences in Table 1 by no more than 2 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 1 by no more than 2 nucleotides.

[0073] (c) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any first strand sequence in Table 1 by no more than 1 nucleotide, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 1 by no more than 1 nucleotide.

[0074] (d) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 1;

[0075] (e) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 1;

[0076] (f) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 1;

[0077] (g) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 1;

[0078] (h) The unmodified corresponding sequence of the first chain sequence includes the sequence of any first chain sequence with a given SEQ ID No. shown in Table 1, and optionally the unmodified corresponding sequence of the second chain sequence includes the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 1;

[0079] (i) The unmodified corresponding sequence of the first chain sequence consists substantially of any of the first chain sequences shown in Table 1 having a given SEQ ID No., and optionally wherein the unmodified corresponding sequence of the second chain sequence consists substantially of the sequence of the corresponding second chain sequence shown in Table 1 having a given SEQ ID No.;

[0080] (j) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises or substantially consists of the sequence of the corresponding second strand sequence having a given SEQ ID No. shown in Table 1;

[0081] (k) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the unmodified corresponding sequence of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 1, and wherein the unmodified corresponding sequence of the first strand sequence further has one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 1.

[0082] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or is substantially composed of the corresponding second chain sequence with a given SEQ ID No. shown in Table 1;

[0083] (l) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 1, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 1; and

[0084] The unmodified sequence corresponding to the first strand contains a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consisting of or composed of said continuous region; and

[0085] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or is substantially composed of the corresponding second chain sequence shown in Table 1 having a given SEQ ID No.;

[0086] (m) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the unmodified corresponding sequence of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 1, and wherein the unmodified corresponding sequence of the first strand sequence further has one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 1.

[0087] The unmodified sequence corresponding to the first strand contains a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consisting of or composed of said continuous region; and

[0088] Optionally, the unmodified corresponding sequence of the second chain sequence comprises or is substantially composed of the corresponding second chain sequence shown in Table 1 having a given SEQ ID No.;

[0089] (n) The unmodified corresponding sequence of the first chain sequence consists of any first chain sequence with a given SEQ ID No. shown in Table 1, and optionally the unmodified corresponding sequence of the second chain sequence consists of the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 1;

[0090] (o) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) exist on a single strand, wherein the unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand can hybridize with each other, thereby forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; or

[0091] (p) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) are on two separate strands that can hybridize with each other and thus form a double-stranded nucleic acid with a double-stranded region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0092] In (l) or (m), the unmodified corresponding sequence of the first strand sequence may comprise, substantially consist of, or consist of a continuous region of 19-25 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861. In (l) or (m), the unmodified corresponding sequence of the first strand sequence may comprise, preferably, a continuous region of 20-24 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consist of, or consist of, the continuous region.

[0093] The “corresponding” second chain refers to the second chain that exists in the same doubly as the first chain given in Tables 5a, 5b, or 5c, or the corresponding second chain sequence listed in Table 1 or Table 2 (as the case may be). That is, the first chain and its corresponding second chain are designated as chain “A” and chain “B” of the doubly with a given doubly ID in Tables 5a, 5b, or 5c, or described in Tables 1 and 2 accordingly.

[0094] On one hand, if the 5' terminal nucleotide of the first chain is a nucleotide other than A or U, then that nucleotide is substituted with A or U. Preferably, if the 5' terminal nucleotide of the first chain is a nucleotide other than U, then that nucleotide is substituted with U, and more preferably with U having a 5' vinylphosphonate ester.

[0095] When the nucleic acid of the present invention does not contain the complete sequence of the reference first strand and / or second strand (e.g., as given in Tables 1, 2, 5a, 5b or 5c), or when one or two strands differ from the corresponding reference sequence by one, two or three nucleotides, the nucleic acid preferably retains at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 100% of the GPR146 inhibitory activity compared to the inhibitory activity of the corresponding nucleic acid containing the complete first and second strand reference sequences in comparable experiments.

[0096] Nucleic acids capable of hybridization under physiological conditions are those that can form base pairs (preferably Watson-Crick or swing base pairs) between at least a portion of opposing nucleotides in the strand, thereby forming at least a double-stranded region. Such double-stranded nucleic acids are preferably stable under physiological conditions (e.g., in PBS, at 37°C, 1 μM per strand), meaning that the two strands maintain mutual hybridization under these conditions. The Tm of the double-stranded nucleotides is preferably 45°C or higher, more preferably 50°C or higher, and even more preferably 55°C or higher.

[0097] One aspect of the invention relates to a nucleic acid for inhibiting GPR146 expression, wherein the nucleic acid comprises a first sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all nucleotides differing from any of the unmodified first-strand corresponding sequences in Table 5a or Table 1 by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably without any nucleotide difference, the first sequence being capable of hybridizing with a target gene transcript (such as mRNA) under physiological conditions. Preferably, the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all nucleotides differing from any of the corresponding unmodified second-strand corresponding sequences in Table 5a or Table 1 by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably without any nucleotide difference, the second sequence being capable of hybridizing with the first sequence under physiological conditions, and preferably the nucleic acid is a siRNA capable of inhibiting GPR146 expression via the RNAi pathway.

[0098] One aspect involves any double-stranded nucleic acid disclosed in Tables 1, 2, 5a, 5b, or 5c, wherein each double-stranded nucleic acid can be identified by a given double-stranded ID and is preferably used to inhibit GPR146 expression, provided that the double-stranded nucleic acid can inhibit GPR146 expression. These nucleic acids are all siRNAs. Inhibition occurs through targeted degradation of the post-transcriptional target gene mRNA transcript. The siRNA forms part of a RISC complex. The RISC complex specifically targets the target RNA through sequence complementarity of the first (antisense) strand with the target sequence.

[0099] One aspect relates to a double-stranded nucleic acid capable of inhibiting GPR146 expression (preferably inhibiting GPR146 expression in cells) for use as a therapeutic or diagnostic agent, for example, in a related therapeutic or diagnostic method, wherein the nucleic acid preferably comprises a first strand and a second strand or is composed of a first strand and a second strand, and preferably wherein the first strand contains a sequence sufficiently complementary to GPR146 mRNA to mediate RNA interference.

[0100] The nucleic acids described herein are capable of inhibiting the expression of GPR146. Inhibition can be complete, i.e., residual expression is 0%. Inhibition of GPR146 expression can be partial, i.e., inhibition of GPR146 expression can be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or intermediate values, of the GPR146 expression level in the absence of the nucleic acids of this invention. The level of inhibition can be measured by comparing a treated sample with an untreated sample or with a control (such as, for example, siRNA that does not target GPR146). Inhibition can be measured by measuring the levels of GPR146 mRNA and / or protein, or the levels of biomarkers or indicators associated with the presence or activity of GPR146. This can be measured in vitro in cells treated with the nucleic acids described herein. Optionally or additionally, inhibition can be measured in cells (such as hepatocytes), tissues (such as liver tissue), organs (such as the liver), or body fluids (such as blood, serum, lymph, or any other body part or fluid) obtained from a subject previously treated with the nucleic acids disclosed herein. Preferably, inhibition of GPR146 expression is determined by comparing the GPR146 mRNA level measured in GPR146-expressing cells after in vitro treatment with the double-stranded RNA disclosed herein for at least 24 or 48 hours (e.g., 7 or 14 days) under ideal conditions (see appropriate concentrations and conditions in the examples) with the GPR146 mRNA level measured in control cells under the same conditions, either untreated, simulated treated, or treated with control double-stranded RNA.

[0101] One aspect of the present invention relates to a nucleic acid in which a first strand and a second strand are present on a single-stranded nucleic acid, the single-stranded nucleic acid looping around such that the first strand and the second strand can hybridize with each other, thereby forming a double-stranded nucleic acid having double-stranded regions.

[0102] Preferably, the first and second strands of the nucleic acid are separate strands. Each of the two separate strands is preferably 17-25 nucleotides in length, more preferably 18-25 nucleotides in length. The lengths of the two strands can be the same or different. The length of the first strand can be 17-25 nucleotides, preferably 18-24 nucleotides, and its length can be 18, 19, 20, 21, 22, 23, or 24 nucleotides. Most preferably, the length of the first strand is 19 nucleotides. The length of the second strand can independently be 17-25 nucleotides, preferably 18-24 nucleotides, and its length can be 18, 19, 20, 21, 22, 23, or 24 nucleotides. More preferably, the length of the second strand is 18, 19, or 20 nucleotides, and most preferably 19 nucleotides.

[0103] Preferably, the first and second strands of the nucleic acid form a double-stranded region of 17-25 nucleotides in length. More preferably, the double-stranded region is 18-24 nucleotides in length. The length of the double-stranded region can be 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In the most preferred embodiment, the double-stranded region is 18 or 19 nucleotides in length. The double-stranded region is defined herein as the region from the 5' end nucleotide of the first strand that pairs with a base of the second strand to the 3' end nucleotide of the first strand that pairs with a base of the second strand, and includes both the aforementioned 5' and 3' end nucleotides. The double-stranded region may contain nucleotides from either strand or both strands that do not pair with a nucleotide base of the other strand. It may contain one, two, three, or four such nucleotides on the first and / or second strands. However, preferably, the double-stranded region consists of 17-25 consecutive nucleotide base pairs. That is, preferably, it contains 17-25 consecutive nucleotides on both strands, all of which pair with nucleotide bases in the other strand. More preferably, the double-stranded region consists of 18 or 19 consecutive nucleotide base pairs, most preferably 18.

[0104] In each of the embodiments disclosed herein, the nucleic acid may have blunt ends at both ends; have a protruding end at one end and a blunt end at the other end; or have protruding ends at both ends.

[0105] Nucleic acids may have a protruding end at one end and a blunt end at the other. Nucleic acids may have protruding ends at both ends. Nucleic acids may have blunt ends at both ends. Nucleic acids may have blunt ends at the 5' end of the first strand and the 3' end of the second strand, or blunt ends at the 3' end of the first strand and the 5' end of the second strand.

[0106] Nucleic acids may include overhangs at either the 3' or 5' end. Nucleic acids may have a 3' overhang on the first strand. Nucleic acids may have a 3' overhang on the second strand. Nucleic acids may have a 5' overhang on the first strand. Nucleic acids may have a 5' overhang on the second strand. Nucleic acids may have overhangs at both the 5' and 3' ends of the first strand. Nucleic acids may have overhangs at both the 5' and 3' ends of the second strand. Nucleic acids may have a 5' overhang on the first strand and a 3' overhang on the second strand. Nucleic acids may have a 3' overhang on the first strand and a 5' overhang on the second strand. Nucleic acids may have a 3' overhang on the first strand and a 3' overhang on the second strand. Nucleic acids may have a 5' overhang on both the first and second strands.

[0107] The 3' or 5' overhang of the second or first strand may consist of 1, 2, 3, 4, or 5 nucleotides in length. Optionally, the overhang may consist of 1 or 2 nucleotides, and may or may not be modified.

[0108] In one embodiment, the 5' end of the first strand is a single-stranded overhang of one, two, or three nucleotides, preferably a single-stranded overhang of one nucleotide.

[0109] Nucleic acid modification

[0110] The nucleic acids discussed in this article include unmodified RNA as well as modified RNA (e.g., for improved efficacy or stability). Unmodified RNA refers to a molecule whose components (i.e., the sugar, base, and phosphate moieties) are the same or substantially the same as those found in nature (e.g., naturally occurring in the human body). As used herein, the term "modified nucleotide" refers to a nucleotide in which one or more components (i.e., the sugar, base, and phosphate moieties) differ from those found in nature. The term "modified nucleotide" may also, in some cases, refer to molecules that are not strictly nucleotides because they lack the essential components of a nucleotide (such as the sugar, base, or phosphate moieties) or substitutes for said essential components. Nucleic acids containing such modified nucleotides should still be understood as nucleic acids, even if one or more nucleotides of the nucleic acid have been replaced by modified nucleotides lacking the essential components of a nucleotide or substitutes for said essential components.

[0111] The modification of nucleic acids according to the present invention generally provides a powerful tool for overcoming potential limitations, including but not limited to the inherent in vitro and in vivo stability and bioavailability of natural RNA molecules. Nucleic acids according to the present invention can be chemically modified. Modified nucleic acids can also minimize the possibility of inducing interferon activity in the human body. Modification can further enhance the functional delivery of nucleic acids to target cells. Preferably, the modified nucleic acids of the present invention may contain one or more chemically modified ribonucleotides on either or both of the first or second strand. The ribonucleotides may contain chemical modifications of bases, sugars, or phosphate moieties. Ribonucleic acids can be modified by substitution or insertion of nucleic acid or base analogues.

[0112] Throughout this description of the invention, "identical or common modification" means that any nucleotide is modified with the same group, whether A, G, C, or U, using a group such as a methyl (2'-OMe) or fluorine (2'-F) group. For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, and 2'-F-dG are considered identical or common modifications, as are 2'-OMe-rU, 2'-OMe-rA, 2'-OMe-rC, and 2'-OMe-rG. Conversely, 2'-F modification is a different modification than 2'-OMe modification.

[0113] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, such as a 2'-F modified nucleotide.

[0114] The modified nucleotide can be a nucleotide with a glycosyl modification. The 2' hydroxyl group (OH) can be modified or replaced by a variety of different "oxygen" or "deoxy" substituents.

[0115] Examples of "oxygen" - 2' hydroxyl modifications include alkoxy or aryloxy groups (OR, such as R=H, alkyl (such as methyl), cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O). n CH2CH2OR; "locked" nucleic acid (LNA), wherein the 2' hydroxyl group is connected to the 4' carbon of the same ribose, for example, via a methylene bridge; O-AMINE (AMINE=NH2, alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy, O(CH2)nAMINE (e.g. AMINE=NH2, alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino).

[0116] "Deoxygenation" modifications include hydrogen, halogens, amino groups (e.g., NH2, alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); NH(CH2CH2NH) n CH2CH2-AMINE (AMINE = NH2, alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may optionally be substituted with, for example, amino functional groups. Other substituents in some embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluorine.

[0117] The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, the modified nucleotide may contain sugars such as arabinose.

[0118] Modified nucleotides may also include “base-free” sugars, which lack a nucleobase at the C-1' position. These base-free sugars may further contain modifications on one or more of the constituent sugar atoms.

[0119] The 2' modification can be used in combination with one or more phosphate nucleoside linkage modifications (e.g., thiophosphate or dithiophosphate).

[0120] One or more nucleotides of the nucleic acid of the present invention may be modified. The nucleic acid may contain at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in a double-stranded region. The modified nucleotide may be outside the double-stranded region, i.e., in a single-stranded region. The modified nucleotide may be on the first strand and may be outside the double-stranded region. The modified nucleotide may be on the second strand and may be outside the double-stranded region. The 3' terminal nucleotide of the first strand may be a modified nucleotide. The 3' terminal nucleotide of the second strand may be a modified nucleotide. The 5' terminal nucleotide of the first strand may be a modified nucleotide. The 5' terminal nucleotide of the second strand may be a modified nucleotide.

[0121] The nucleic acid of the present invention may have one modified nucleotide, or the nucleic acid of the present invention may have about 2-4 modified nucleotides, or the nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides, about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, about 24-26 modified nucleotides, or about 26-28 modified nucleotides. In each case, the nucleic acid containing the modified nucleotides retains at least 50% of its activity compared to the same nucleic acid without the modified nucleotides, and vice versa. Compared to the same nucleic acid without the modified nucleotide, the nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of its activity and intermediate values, or may have more than 100% of the activity of the same nucleic acid without the modified nucleotide.

[0122] The modified nucleotide can be purine or pyrimidine. At least half of the purines can be modified. At least half of the pyrimidines can be modified. All purines can be modified. All pyrimidines can be modified. The modified nucleotide can be selected from the group consisting of: 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl (2'-OMe) modified nucleotides, 2'-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-deoxy-2'-fluoro (2'-F) modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, nucleotides containing 5'-thiophosphate groups, nucleotides containing 5'-phosphate or 5'-phosphate analogs, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid bis(decylamide) groups.

[0123] Nucleic acids may include nucleotides containing modified bases, wherein the bases are selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azpyrimidine, 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutox osine), 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosyl guanosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyl adenosine, β-D-mannosyl guanosine, uridine-5-oxyacetic acid, and 2-thiocytidine.

[0124] Many modifications described herein and occurring within nucleic acids will be repeated within polynucleotide molecules, such as base modifications, phosphate moiety modifications, or modifications of non-linked O nucleotides of the phosphate moiety. In some cases, modifications will occur at all possible positions / nucleotides of the polynucleotide, but in many cases this will not be the case. Modifications may occur only at the 3' or 5' end positions, or only in terminal regions, such as at the position of the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded regions of the nucleic acids of the present invention, or only in the single-stranded regions of the nucleic acids of the present invention. Thiophosphate or dithiophosphate modifications at non-linked O positions may occur only at one or both ends, or only in terminal regions, for example, at the position of the terminal nucleotide or in the last 2, 3, 4, or 5 nucleotides of the strand, or may occur in double-stranded and / or single-stranded regions, particularly at the ends. The 5' and / or 3' ends may be phosphorylated.

[0125] The stability of the nucleic acid of this invention can be increased by including specific bases at the overhang, or by including modified nucleotides at the single-stranded overhang (e.g., 5' or 3' overhangs or both). Purine nucleotides may be included in the overhang. All or part of the bases at the 3' or 5' overhang may be modified. Modifications may include modifications at the 2'OH group of the ribose, the use of deoxyribonucleotides instead of ribonucleotides, and modifications of phosphate groups, such as thiophosphates or dithiophosphates. The overhang need not be homologous to the target sequence.

[0126] Nucleases can hydrolyze the phosphodiester bonds of nucleic acids. However, chemical modifications to nucleic acids can impart improved properties and make oligonucleotides more stable to nucleases.

[0127] As used herein, modified nucleic acids may include one or more of the following:

[0128] (i) A change, such as substitution, of one or two non-linked phosphate oxygens and / or one or more linking phosphate oxygens (also referred to as links even at the 5' and 3' ends of the nucleic acids of the present invention);

[0129] (ii) Changes in the composition of ribose (e.g., the 2' hydroxyl group on the ribose), such as substitution;

[0130] (iii) The phosphate portion is replaced by a "dephosphoric" linker;

[0131] (iv) Modification or substitution of naturally occurring bases;

[0132] (v) Substitution or modification of the ribose-phosphate backbone; and

[0133] (vi) Modification of the 3' or 5' end of the first and / or second chain, such as removal, modification or substitution of the terminal phosphate group, or concatenation of a portion (e.g., a fluorescently labeled portion) to the 3' or 5' end of one or both chains.

[0134] The terms “substitution,” “modification,” and “alteration” refer to differences from naturally occurring molecules.

[0135] The specific modifications will be discussed in more detail below.

[0136] Nucleic acids may contain one or more modified nucleotides on the second and / or first strand. Alternating nucleotides may be modified to form modified nucleotides.

[0137] As described herein, “alternation” means occurring one after another in a regular manner. In other words, alternation means occurring in a sequential, repetitive manner. For example, if one nucleotide is modified, the next consecutive nucleotide is not modified, and subsequent consecutive nucleotides are modified, and so on. One nucleotide may be modified by a first modification, the next consecutive nucleotide may be modified by a second modification, and subsequent consecutive nucleotides may be modified by a first modification, and so on, where the first and second modifications are different. Unless otherwise stated, the term “alternation” as used herein should be understood to mean complete alternation. In other words, the alternation pattern spans the entirety of the relevant strand (e.g., the first strand). For example, complete alternation may include all odd-numbered nucleotides containing the first modification and all even-numbered nucleotides not being modified. Alternatively, complete alternation may include all odd-numbered nucleotides containing the first modification and all even-numbered nucleotides containing the second modification. Complete alternation may include all even-numbered nucleotides containing the first modification and all odd-numbered nucleotides containing the second modification.

[0138] Some representative modified nucleic acid sequences of the present invention are shown in the embodiments. These embodiments are intended to be representative and not limiting.

[0139] In one aspect of the nucleic acid, at least the 2nd and 14th nucleotides of the first strand are modified, preferably by a first common modification, wherein the nucleotides are numbered sequentially starting from the 1st nucleotide at the 5' end of the first strand. The first modification is preferably 2'-F.

[0140] In one aspect, at least one, several, or preferably all even-numbered nucleotides of the first strand are modified, preferably by a first common modification, wherein the nucleotides are numbered sequentially starting from the first nucleotide at the 5' end of the first strand. The first modification is preferably 2'-F.

[0141] In one aspect, at least one, several, or preferably all odd-numbered nucleotides of the first strand are modified, with the nucleotides numbered sequentially starting from the first nucleotide at the 5' end of the first strand. Preferably, they are modified by a second modification. If the nucleic acid also contains a first modification (e.g., a first modification of the 2nd and 14th nucleotides of the first strand or of all even-numbered nucleotides), the second modification is preferably different from the first modification. The first modification is preferably any 2' ribose modification that is the same size as or smaller than the 2'-OH group, or a locked nucleic acid (LNA), or a non-locked nucleic acid (UNA), or a 2'-fluoroarabinonucleotide (FANA) modification. A 2' ribose modification that is the same size as or smaller than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halogen, or 2'-NH2. The second modification is preferably any 2' ribose modification that is larger in volume than the 2'-OH group. 2' ribose modifications with a volume greater than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid can reduce the expression of the target gene to at least the same extent as the same nucleic acid without modification under comparable conditions. The first modification is preferably 2'-F and / or the second modification is preferably 2'-OMe.

[0142] In the context of this disclosure, the size or volume of substituents (such as 2' ribose modifications) is preferably measured in van der Waals volume.

[0143] In one aspect, at least one, several, or preferably all nucleotides in the second strand corresponding to even-numbered nucleotides in the first strand are modified, preferably by a third modification. Preferably, in the same nucleic acid, the 2nd and 14th nucleotides or all even-numbered nucleotides in the first strand are modified by a first modification. Alternatively or additionally, the odd-numbered nucleotides in the first strand are modified by a second modification. Preferably, the third modification is different from the first modification and / or the third modification is the same as the second modification. The first modification is preferably any 2' ribose modification with the same or smaller volume as the 2'-OH group, or locked nucleic acid (LNA), or unlocked nucleic acid (UNA), or 2'-fluoroarabinonucleotide (FANA) modification. A 2' ribose modification with the same or smaller volume as the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halogen, or 2'-NH2. The second and / or third modification is preferably any 2' ribose modification with a volume larger than the 2'-OH group. 2' ribose modifications with a volume greater than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid can reduce the expression of the target gene to at least the same extent as the same nucleic acid without modification under comparable conditions. The first modification is preferably 2'-F and / or the second and / or third modification is preferably 2'-OMe. Nucleotides on the first strand are numbered consecutively starting from the first nucleotide at the 5' end of the first strand.

[0144] The nucleotides in the second strand that correspond to, for example, the even-numbered nucleotides in the first strand are the second-strand nucleotides that pair with the even-numbered nucleotides in the first strand.

[0145] In one aspect, at least one, several, or preferably all nucleotides in the second strand corresponding to odd-numbered nucleotides in the first strand are modified, preferably by a fourth modification. Preferably, in the same nucleic acid, the 2nd and 14th nucleotides or all even-numbered nucleotides in the first strand are modified by a first modification. Alternatively or additionally, the odd-numbered nucleotides in the first strand are modified by a second modification. Alternatively or additionally, all nucleotides in the second strand corresponding to even-numbered nucleotides in the first strand are modified by a third modification. The fourth modification is preferably different from the second modification and preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2' ribose modification with the same or smaller volume as the 2'-OH group, or locked nucleic acid (LNA), or unlocked nucleic acid (UNA), or 2'-fluoroarabinonucleotide (FANA) modification. A 2' ribose modification with the same or smaller volume as the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halogen, or 2'-NH2. The second and / or third modification is preferably any 2' ribose modification with a volume larger than the 2'-OH group. 2' ribose modifications with a volume greater than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid can reduce the expression of the target gene to at least the same extent as the same nucleic acid without modification under comparable conditions. The first and / or fourth modifications are preferably 2'-OMe modifications and / or the second and / or third modifications are preferably 2'-F modifications. Nucleotides on the first strand are numbered consecutively starting from the first nucleotide at the 5' end of the first strand.

[0146] In one aspect of the nucleic acid, the nucleotides in the second strand corresponding to the 11th or 13th nucleotide, or the 11th and 13th nucleotides, or the 11th-13th nucleotides in the first strand are modified by a fourth modification. Preferably, all nucleotides in the second strand except those corresponding to the 11th or 13th nucleotide, or the 11th and 13th nucleotides, or the 11th-13th nucleotides in the first strand are modified by a third modification. Preferably, in the same nucleic acid, the 2nd and 14th nucleotides, or all even-numbered nucleotides in the first strand, are modified by a first modification. Alternatively or additionally, the odd-numbered nucleotides in the first strand are modified by a second modification. The fourth modification is preferably different from the second modification and preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2' ribose modification that is the same size as or smaller than the 2'-OH group, or locked nucleic acid (LNA), or unlocked nucleic acid (UNA), or 2'-fluoroarabinonucleotide (FANA) modification. 2' ribose modifications of the same or smaller size as the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halogen, or 2'-NH2. Second and / or third modifications are preferably any 2' ribose modification larger than the 2'-OH group. 2' ribose modifications larger than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid can reduce the expression of the target gene to at least the same extent as the same nucleic acid without modification under comparable conditions. First and / or fourth modifications are preferably 2'-OMe modifications and / or second and / or third modifications are preferably 2'-F modifications. Nucleotides on the first strand are numbered consecutively starting from the first nucleotide at the 5' end of the first strand.

[0147] In one aspect of nucleic acids, all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides in the second strand corresponding to the even-numbered nucleotides of the first strand are modified by a third modification, and all nucleotides in the second strand corresponding to the odd-numbered nucleotides of the first strand are modified by a fourth modification, wherein the first and / or fourth modifications are 2'-F and / or the second and / or third modifications are 2'-OMe.

[0148] In one aspect of nucleic acids, all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand corresponding to positions 11-13 of the first strand are modified by a fourth modification, and all nucleotides of the second strand except those corresponding to positions 11-13 of the first strand are modified by a third modification, wherein the first and fourth modifications are 2'-F, and the second and third modifications are 2'-OMe. In one embodiment of this aspect, the 3' terminal nucleotide of the second strand is a reverse RNA nucleotide (i.e., the nucleotide is linked to the 3' end of the strand via its 3' carbon, rather than the usual 5' carbon linkage). When the 3' terminal nucleotide of the second strand is a reverse RNA nucleotide, the reverse RNA nucleotide is preferably an unmodified nucleotide, meaning it does not contain any modifications compared to its native nucleotide counterpart. Specifically, the reverse RNA nucleotide is preferably a 2'-OH nucleotide. Preferably, in this aspect, when the 3' terminal nucleotide of the second strand is a reverse RNA nucleotide, the nucleic acid is blunt-ended at least at the end containing the 5' end of the first strand.

[0149] One aspect of the present invention is a nucleic acid as disclosed herein for inhibiting GPR146 gene expression (preferably inhibiting GPR146 gene expression in cells), wherein the first strand comprises modified or unmodified nucleotides at multiple positions to facilitate RISC processing of the nucleic acid.

[0150] In one aspect, “facilitating RISC processing” means that nucleic acids can be processed by RISC, for example, any modifications present will allow nucleic acids to be processed by RISC, and preferably, will facilitate RISC processing, appropriately enabling siRNA activity to occur.

[0151] One aspect of the invention is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand starting from the 5' end are not modified by 2'-OMe modification, and the nucleotides on the second strand corresponding to positions 11 or 13, or positions 11 and 13, or positions 11, 12 and 13 of the first strand are not modified by 2'-OMe modification (in other words, they are not modified or are modified by a modification other than 2'-OMe).

[0152] In one aspect, the nucleotide on the second strand corresponding to position 13 of the first strand is a nucleotide that forms a base pair with position 13 of the first strand (starting from the 5' end).

[0153] In one aspect, the nucleotide on the second strand corresponding to position 11 of the first strand is a nucleotide that forms a base pair with position 11 of the first strand (starting from the 5' end).

[0154] In one aspect, the nucleotide on the second strand corresponding to position 12 of the first strand is a nucleotide that forms a base pair with position 12 of the first strand (starting from the 5' end).

[0155] For example, in a 19-mer double-stranded blunt-terminal nucleic acid, position 13 of the first strand (starting from the 5' end) will pair with position 7 of the second strand (starting from the 5' end). Position 11 of the first strand (starting from the 5' end) will pair with position 9 of the second strand (starting from the 5' end). This nomenclature can be applied to other positions on the second strand.

[0156] In one aspect, in the case of partially complementary first and second strands, the nucleotides on the second strand that "correspond" to the position on the first strand do not necessarily form a base pair (if the position is a mismatch), but the principle of nomenclature still applies.

[0157] One aspect is the nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 of the first strand starting from the 5' end are not modified by 2'-OMe modification, and the nucleotides on the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand are modified by 2'-F modification.

[0158] One aspect is the nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 of the first strand starting from the 5' end are modified by 2'-F modification, and the nucleotides on the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand are not modified by 2'-OMe modification.

[0159] One aspect is the nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 of the first strand starting from the 5' end are modified by 2'-F modification, and the nucleotides on the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand are modified by 2'-F modification.

[0160] One aspect is nucleic acids as disclosed herein, wherein more than 50% of the nucleotides of the first and / or second strand contain 2'-OMe modifications, such as more than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the nucleotides of the first and / or second strands containing 2'-OMe modifications, preferably measured as a percentage of the total nucleotides of both the first and second strands.

[0161] One aspect is nucleic acids as disclosed herein, in which more than 50% of the nucleotides of the first and / or second strand contain naturally occurring RNA modifications, such as where more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the nucleotides of the first and / or second strand contain such modifications, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include 2'-OMe and other 2' sugar modifications, particularly 2'-H modifications that result in DNA nucleotides.

[0162] One aspect is nucleic acids as disclosed herein, which contain no more than 20%, such as no more than 15%, such as no more than 10% of 2'-modified nucleotides having non-2'-OMe modification on the first and / or second strands, preferably calculated as a percentage of the total nucleotides of both the first and second strands.

[0163] One aspect is the nucleic acid as disclosed herein, wherein the number of nucleotides with non-2'-OMe modifications in the first and / or second strands does not exceed 7, more preferably not more than 5, and most preferably not more than 3.

[0164] One aspect is nucleic acids as disclosed herein, which contain no more than 20% (such as no more than 15% or no more than 10%) of 2'-F modifications on the first and / or second strands, preferably as a percentage of the total nucleotides of the two strands.

[0165] One aspect is the nucleic acid as disclosed herein, wherein the number of nucleotides with 2'-F modification in the first and / or second strand does not exceed 7, more preferably not more than 5, and most preferably not more than 3.

[0166] One aspect is the nucleic acid disclosed herein, in which all nucleotides except those at positions 2 and 14 from the 5' end of the first strand and those on the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand are modified by 2'-OMe modification. Preferably, the nucleotides not modified by 2'-OMe are fluorinated at the 2' position (2'-F modification).

[0167] In some embodiments, a preferred aspect is a nucleic acid as disclosed herein, wherein all nucleotides of the nucleic acid are modified at the 2' position of the sugar. Preferably, these nucleotides are modified by 2'-F modification if the modification is not 2'-OMe modification.

[0168] In one aspect, the nucleic acid is modified on the first strand by alternating 2'-OMe and 2'-F modifications, with positions 2 and 14 (starting from the 5' end) being modified by 2'-F. Preferably, alternation should be understood to mean alternation throughout the entire strand.

[0169] Preferably, the second strand is modified with 2'-F at the second-strand nucleotide corresponding to position 11, or position 13, or positions 11 and 13, or positions 11-13 of the first strand. Preferably, the second strand is modified with 2'-F at positions 11-13, starting from the first position of the complementary (double-stranded) region from the 3' end, and the remaining modifications are naturally occurring modifications, preferably 2'-OMe. At least in this case, the complementary region begins at the first position of the second strand with the corresponding nucleotide from the first strand, regardless of whether the two nucleotides can base-pair with each other.

[0170] In one aspect of nucleic acids, each nucleotide in the first and second strands is a modified nucleotide.

[0171] As described in this article, the term "odd number" refers to a number that is not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11, etc. As described in this article, the term "even number" refers to a number that is divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14, etc.

[0172] Unless otherwise expressly stated, the nucleotides of the first strand are numbered consecutively starting from the first nucleotide at the 5' end of the first strand. The nucleotides of the second strand are numbered consecutively starting from the first nucleotide at the 3' end of the second strand.

[0173] One or more nucleotides on the first and / or second strand may be modified to form modified nucleotides. One or more odd-numbered nucleotides on the first strand may be modified. One or more even-numbered nucleotides on the first strand may be modified by at least a second modification, wherein the second modification differs from the modification on one or more odd-numbered nucleotides. At least one of the modified even-numbered nucleotides may be adjacent to at least one of the modified odd-numbered nucleotides.

[0174] In the nucleic acid of the present invention, a plurality of odd-numbered nucleotides in the first chain may be modified. A plurality of even-numbered nucleotides in the first chain may be modified by a second modification. The first chain may contain adjacent nucleotides modified by a common modification. The first chain may also contain adjacent nucleotides modified by a second different modification (i.e., the first chain may contain nucleotides that are adjacent to each other and modified by a first modification, and other nucleotides that are adjacent to each other and modified by a second modification different from the first modification).

[0175] One or more odd-numbered nucleotides of the second strand (wherein the nucleotides are numbered consecutively starting from the first nucleotide at the 3' end of the second strand) may be modified by a modification different from that of odd-numbered nucleotides of the first strand (wherein the nucleotides are numbered consecutively starting from the first nucleotide at the 5' end of the first strand), and / or one or more even-numbered nucleotides of the second strand may be modified by the same modification as the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to one or more modified odd-numbered nucleotides. Multiple odd-numbered nucleotides of the second strand may be modified by common modification and / or multiple even-numbered nucleotides may be modified by the same modification present on the odd-numbered nucleotides of the first strand. Multiple odd-numbered nucleotides of the second strand may be modified by a modification different from that of the odd-numbered nucleotides of the first strand.

[0176] The second chain may contain adjacent nucleotides modified by common modification, which may be a modification different from the modification of odd-numbered nucleotides in the first chain.

[0177] In some aspects of the nucleic acids of the present invention, each odd-numbered nucleotide in the first strand and each even-numbered nucleotide in the second strand may be modified by common modification, and each even-numbered nucleotide in the first strand may be modified by different modification, and each odd-numbered nucleotide in the second strand may be modified by different modification.

[0178] The nucleic acid of the present invention may have a first chain of modified nucleotides, wherein the first chain of modified nucleotides is offset by at least one nucleotide relative to the second chain of unmodified or differently modified nucleotides.

[0179] In some respects, one or more or every odd-numbered nucleotide in the first strand may be modified, and one or more or every even-numbered nucleotide in the second strand may be modified. One or more or every alternating nucleotide in either or both strands may be modified by a second modification. One or more or every even-numbered nucleotide in the first strand may be modified, and one or more or every even-numbered nucleotide in the second strand may be modified. One or more or every alternating nucleotide in either or both strands may be modified by a second modification. One or more or every odd-numbered nucleotide in the first strand may be modified, and one or more odd-numbered nucleotides in the second strand may be modified by a common modification. One or more or every alternating nucleotide in either or both strands may be modified by a second modification. One or more or every even-numbered nucleotide in the first strand may be modified, and one or more or every odd-numbered nucleotide in the second strand may be modified by a common modification. One or more or every alternating nucleotide in either or both strands may be modified by a second modification.

[0180] The nucleic acids of the present invention may comprise single-stranded or double-stranded constructs containing at least two alternating modified regions in one or both strands. These alternating regions may contain up to about 12 nucleotides, but preferably about 3 to about 10 nucleotides. The alternating nucleotide regions may be located at the ends of one or both strands of the nucleic acid of the present invention. The nucleic acid may contain 4 to about 10 alternating nucleotides at each end (3' and 5'), and these regions may be separated by about 5 to about 12 consecutive unmodified or differently or commonly modified nucleotides.

[0181] The odd-numbered nucleotides of the first strand may be modified by a first modification, and the even-numbered nucleotides may be modified by a second modification. The second strand may contain adjacent nucleotides modified by a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. One or more nucleotides of the second strand may be modified by a second modification. One or more nucleotides with a second modification may be adjacent to each other and adjacent to a nucleotide with the same modification as the odd-numbered nucleotides of the first strand. The first strand may also contain a phosphate thioester bond between the two nucleotides at the 3' and 5' ends, or a phosphate dithioester bond between the two nucleotides at the 3' end. The second strand may contain a phosphate thioester or phosphate dithioester bond between the two nucleotides at the 5' end. The second strand may also be conjugated to a ligand at the 5' end.

[0182] The nucleic acid of the present invention may comprise a first strand containing adjacent nucleotides modified by a common modification. One or more such nucleotides may be adjacent to one or more nucleotides that may be modified by a second modification. One or more nucleotides having the second modification may be adjacent. The second strand may contain adjacent nucleotides modified by a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified by the second modification. One or more nucleotides having the second modification may be adjacent. The first strand may also contain a phosphate thioester bond between two nucleotides at the 3' and 5' ends, or a phosphate dithioester bond between two nucleotides at the 3' end. The second strand may contain a phosphate thioester or phosphate dithioester bond between two nucleotides at the 3' end. The second strand may also be conjugated to a ligand at the 5' end. Nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 on the first strand (numbered from 5' to 3' on the first strand and from 3' to 5' on the second strand) may be modified by the second modification. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the first strand can be modified by a second modification. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 on the second strand can be modified by a second modification. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the second strand can be modified by a second modification. For the purposes of the nucleic acid of this invention, the nucleotides are numbered from 5' to 3' on the first strand and from 3' to 5' on the second strand.

[0183] Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the first strand can be modified. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 on the first strand can be modified using a second modification. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 on the second strand can be modified using a second modification. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the second strand can be modified using a second modification.

[0184] Obviously, if the length of the first and / or second strand is less than 25 nucleotides (e.g., 19 nucleotides), then there are no nucleotides numbered 20, 21, 22, 23, 24, and 25 available for modification. Those skilled in the art will accordingly understand that the above description applies to shorter strands.

[0185] One or more modified nucleotides on the first strand can pair with modified nucleotides on the second strand that share the same modification. One or more modified nucleotides on the first strand can pair with modified nucleotides on the second strand that have a different modification. One or more modified nucleotides on the first strand can pair with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand can pair with unmodified nucleotides on the first strand. In other words, alternating nucleotides can be aligned on both strands, such that, for example, all modifications in alternating regions on the second strand pair with the same modifications in the first strand, or alternatively, the modification can be shifted by one nucleotide, so that a common modification in an alternating region on one strand pairs with a different modification (i.e., a second or further modification) in the other strand. Another option is to have different modifications on each strand.

[0186] Modifications on the first strand can be offset by one nucleotide relative to the modified nucleotides on the second strand, so that the co-modified nucleotides do not pair with each other.

[0187] One or more modifications may be independently selected from the group consisting of: 3'-terminal deoxythymidine, 2'-OMe, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification, morpholino modification, aminophosphate modification, 5'-thiophosphate group modification, 5'-phosphate or 5'-phosphate analog modification, and cholesterol derivative or dodecanoic acid bisdecamide group modification, and / or the modified nucleotide may be any of the following: locked nucleotide, baseless nucleotide, or nucleotide containing a non-natural base.

[0188] At least one modification may be 2'-OMe and / or at least one modification may be 2'-F. Other modifications as described herein may be present on the first and / or second strands.

[0189] The nucleic acid of the present invention may contain reverse RNA nucleotides at one or more ends of the strand. Such reverse nucleotides provide stability to the nucleic acid. Preferably, the nucleic acid contains reverse nucleotides at least at the 3' end and / or the 5' end of the first and / or second strand. More preferably, the nucleic acid contains reverse nucleotides at the 3' end of the second strand. Most preferably, the nucleic acid contains reverse RNA nucleotides at the 3' end of the second strand, and this nucleotide is preferably reverse A. A reverse nucleotide is a nucleotide that is attached to the 3' end of the nucleic acid via its 3' carbon (instead of its 5' carbon as is usually the case), or a nucleotide that is attached to the 5' end of the nucleic acid via its 5' carbon (instead of its 3' carbon as is usually the case). The reverse nucleotide is preferably present at the end of the strand, not as a protruding end, but opposite to the corresponding nucleotide in the other strand. Therefore, the nucleic acid preferably has a blunt end at the end containing the reverse RNA nucleotide. The presence of a reverse RNA nucleotide at the end of the strand preferably means that the last nucleotide at the end of the strand is a reverse RNA nucleotide. Nucleic acids having such nucleotides are stable and readily synthesized. The reverse RNA nucleotide is preferably an unmodified nucleotide, meaning that it does not contain any modifications compared to its native nucleotide counterpart. Specifically, the reverse RNA nucleotide is preferably a 2'-OH nucleotide.

[0190] The nucleic acid of the present invention may contain one or more nucleotides modified with 2'-H at the 2' position, and thus have DNA nucleotides within the nucleic acid. The nucleic acid of the present invention may contain DNA nucleotides at the 2nd and / or 14th positions of the first strand, counting from the 5' end of the first strand. The nucleic acid may contain DNA nucleotides on the second strand at positions corresponding to the 11th, 13th, 11th and 13th, or 11th-13th positions of the first strand.

[0191] In one aspect, each nucleic acid of the present invention contains no more than one DNA nucleotide.

[0192] The nucleic acid of the present invention may contain one or more LNA nucleotides. The nucleic acid of the present invention may contain an LNA nucleotide at position 2 and / or position 14 of the first strand, counting from the 5' end of the first strand. The nucleic acid may contain LNA at positions on the second strand corresponding to positions 11, 13, 11 and 13, or 11-13 of the first strand.

[0193] Some representative modified nucleic acid sequences of the present invention are shown in the embodiments. These embodiments are intended to be representative and not limiting.

[0194] In some preferred embodiments, the nucleic acid may include a first modification and a second or further modification, each independently selected from the group consisting of 2'-OMe and 2'-F modifications. The nucleic acid may include a 2'-OMe modification (which may be the first modification) and a 2'-F modification. The nucleic acid of the present invention may also include thiophosphate or dithiophosphate modifications and / or deoxygenation modifications, which may be present in or between the terminal 2 or 3 nucleotides at each or either end of each or both strands.

[0195] In one aspect of nucleic acids, at least one nucleotide of the first and / or second strand is a modified nucleotide, wherein if the first strand contains at least one modified nucleotide:

[0196] (i) at least one or both of the nucleotides at positions 2 and 14 of the first strand are modified by the first modification; and / or

[0197] (ii) At least one, several, or all even-numbered nucleotides of the first strand are modified by a first modification; and / or

[0198] (iii) At least one, several, or all odd-numbered nucleotides of the first strand are modified by a second modification; and / or

[0199] If the second chain contains at least one modified nucleotide:

[0200] (iv) At least one, several, or all nucleotides in the second strand corresponding to even-numbered nucleotides in the first strand are modified by a third modification; and / or

[0201] (v) At least one, several, or all nucleotides in the second strand corresponding to odd-numbered nucleotides in the first strand are modified by a fourth modification; and / or

[0202] (vi) At least one, several, or all of the nucleotides in the second strand corresponding to the 11th or 13th nucleotide, or the positions of the 11th and 13th nucleotides, or the positions of the 11th to 13th nucleotides in the first strand are modified by a fourth modification; and / or

[0203] (vii) At least one, several, or all nucleotides in the second strand, except those corresponding to the 11th or 13th nucleotide, or the 11th and 13th nucleotides, or the 11th-13th nucleotides in the first strand, are modified by a third modification.

[0204] The nucleotides on the first strand are numbered consecutively starting from the first nucleotide at the 5' end of the first strand.

[0205] The preferred modifications are at least one of the following:

[0206] (a) The first modification is preferably different from the second and third modifications;

[0207] (b) The first modification is preferably the same as the fourth modification;

[0208] (c) The second and third modifications are preferably the same;

[0209] (d) The first modification is preferably 2'-F modification;

[0210] (e) The second modification is preferably 2'-OMe modification;

[0211] (f) The third modification is preferably a 2'-OMe modification; and / or

[0212] (g) The fourth modification is preferably 2'-F modification; and

[0213] The nucleic acid is selectively conjugated with a ligand.

[0214] One aspect is a double-stranded nucleic acid for inhibiting GPR146 expression (preferably inhibiting GPR146 expression in cells), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified corresponding sequence of the first strand sequence comprises a sequence of at least 15 nucleotides, which differs from any of the first strand sequences shown in Table 5a or Table 1 by no more than 3 nucleotides, wherein all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides in the second strand corresponding to the even-numbered nucleotides of the first strand are modified by a third modification, and all nucleotides in the second strand corresponding to the odd-numbered nucleotides of the first strand are modified by a fourth modification, wherein the first and fourth modifications are 2'-F, and the second and third modifications are 2'-OMe.

[0215] One aspect is a double-stranded nucleic acid for inhibiting GPR146 expression (preferably inhibiting GPR146 expression in cells), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified corresponding sequence of the first strand sequence comprises a sequence of at least 15 nucleotides, which differs from any of the first strand sequences shown in Table 5a or Table 1 by no more than 3 nucleotides, wherein all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides in the second strand corresponding to positions 11-13 of the first strand are modified by a fourth modification, and all nucleotides in the second strand except those corresponding to positions 11-13 of the first strand are modified by a third modification, wherein the first and fourth modifications are 2'-F, and the second and third modifications are 2'-OMe.

[0216] The 3' and 5' ends of oligonucleotides can be modified. Such modifications can occur at the 3' or 5' end of the molecule, or at both ends. They can include modifications or substitutions of the entire terminal phosphate group or one or more atoms of the phosphate group. For example, the 3' and 5' ends of the oligonucleotide can be conjugated to other functional molecular entities, such as labeled moieties, like fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dyes) or protecting groups (e.g., sulfur-, silicon-, boron-, or ester-based protecting groups). The functional molecular entity can be linked to a sugar via a phosphate group and / or a linker. The terminal atom of the linker can be attached to or replace the linker atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Optionally, the linker can be attached to or replace the terminal atom of a nucleotide substitute (e.g., PNA). These spacers or linkers can include, for example, —(CH2). n —、—(CH2) n N—、—(CH2) n O—、—(CH2) n S—、—(CH2CH2O) n CH2CH2O— (e.g., n=3 or 6), without basic sugars, amides, carboxyl groups, amines, oxamines, oximines, thioethers, disulfides, thioureas, sulfonamides, or morpholino compounds, or biotin and fluorescein reagents. The 3' end may be an -OH group.

[0217] Other examples of end-modification include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, bile acids, adamantane acetic acid, 1-pyrene butyrate, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), and peptide conjugates (e.g., antennapedia peptides). peptide), Tat peptide), alkylating agent, phosphate group, amino group, mercapto group, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, diimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, tetraza macrocyclic Eu3+ complex).

[0218] Terminal modifications can also be used to monitor distribution, and in such cases, the added group may include a fluorophore, such as fluorescein or Alexa dye. Terminal modifications can also be used to enhance uptake; useful modifications for this purpose include cholesterol. Terminal modifications can also be used to crosslink RNA agents to another part.

[0219] Terminal modifications can be added for a variety of reasons, including regulating activity or resistance to degradation. Terminal modifications for regulating activity include modifying the 5' end with phosphate or a phosphate analog. The nucleic acids of the present invention may be 5' phosphorylated on the first or second strand or contain a phosphoryl analog at the 5' end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P—O-5'); 5'-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5'); 5'-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O) P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5'-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-monothiophosphate (thiophosphate; (HO)2(S)P—O-5'); 5'-monodithiophosphate ( Dithiophosphates; (HO)(HS)(S)P—O-5'), 5'-thiophosphates ((HO)2(O)P—S-5'); any other combination of oxygen / sulfur-substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphates ((HO)2(O)P—NH-5', (HO)(NH2)(O)P—O-5'), 5'-alkylphosphonates (alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)—O-5' (where R is alkyl), (OH)2(O)P-5'-CH2-), 5'-vinylphosphonates, 5'-alkyl ether phosphonates (alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)—O-5' (where R is alkyl ether)).

[0220] Certain portions may be attached to the 5' end of the first or second strand. These include basoribose-free moieties, basodeoxyribose-free moieties, modifications of basoribose-free and basodeoxyribose-free moieties including 2'-O alkyl modifications; reverse basoribose-free and basodeoxyribose moieties and their modifications, C6-imino-Pi; mirror nucleotides, including L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs, including 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; and 5'-amino-alkyl phosphates. Esters; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-dehydrated hexitol nucleotide; α-nucleotide; threo-pentafuranosyl nucleotide; acyclic 3',4'-open cyclic nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-reverse baseless moiety; 1,4-butanediol phosphate; 5'-amino; and bridged or unbridged methylphosphonates and 5'-mercaptoyl moieties.

[0221] In each sequence described herein, the C-terminal "–OH" portion may replace the C-terminal "–NH2" portion, and vice versa.

[0222] The present invention also provides nucleic acids according to any aspect of the invention described herein, wherein the first strand has a terminal 5' (E)-vinylphosphonate nucleotide at its 5' end. This terminal 5' (E)-vinylphosphonate nucleotide is preferably linked to a second nucleotide in the first strand via a phosphodiester bond. Preferably, the terminal 5' (E)-vinylphosphonate (“vp”) nucleotide is uridine (“vp-U”).

[0223] The first strand of a nucleic acid may contain formula (I):

[0224]

[0225] Wherein '(vp)-' is 5' (E)-vinylphosphonate, 'N' is nucleotide, 'po' is phosphodiester bond, and n is 1 to (total number of nucleotides in the first chain - 2), preferably n is 1 to (total number of nucleotides in the first chain - 3), more preferably n is 1 to (total number of nucleotides in the first chain - 4).

[0226] Preferably, the terminal 5' (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably (vp)-U.

[0227] Terminal 5' (E)-vinylphosphonate nucleotides are nucleotides in which the phosphate group at the 5' end of the ribose has been replaced by an E-vinylphosphonate group:

[0228]

[0229] Nucleotides with a phosphate at the 5' end of the ribose

[0230]

[0231] Nucleotides with a terminal 5' (E)-vinylphosphonate ester at the 5' end of the ribose

[0232] In one aspect, the first chain has a terminal 5' (E)-vinylphosphonate nucleotide at its 5' end, which is linked to a second nucleotide in the first chain via a phosphodiester bond, and the first chain contains a) more than one phosphodiester bond; b) phosphodiester bonds between at least three terminal 5' nucleotides and / or c) phosphodiester bonds between at least four terminal 5' nucleotides.

[0233] In one aspect, the first and / or second strand of the nucleic acid contains at least one phosphate thioester (ps) and / or at least one phosphate dithioester (ps2) bond located between two nucleotides.

[0234] In one aspect, the first and / or second strands of the nucleic acid contain more than one thiophosphate and / or more than one dithiophosphate bond.

[0235] In one aspect, the first and / or second strand of the nucleic acid contains a phosphate thioester or dithioester bond between two terminal 3' nucleotides, or a phosphate thioester or dithioester bond between three terminal 3' nucleotides. Preferably, the bonds between other nucleotides in the first and / or second strands are phosphodiester bonds.

[0236] In one aspect, the first and / or second strand of the nucleic acid contains a phosphate thioester bond located between two terminal 5' nucleotides or between three terminal 5' nucleotides.

[0237] In one aspect, the nucleic acid of the present invention comprises one or more phosphate-thioester or phosphate-dithioester modifications at one or more ends of the first and / or second strand. Optionally, each or any end of the first strand may comprise one, two, or three phosphate-thioester or phosphate-dithioester modified nucleotides (nucleoside bonds). Optionally, each or any end of the second strand may comprise one, two, or three phosphate-thioester or phosphate-dithioester modified nucleotides (nucleoside bonds).

[0238] In one aspect, the nucleic acid contains phosphate thioester bonds between the terminal two or three 3' and / or 5' nucleotides of the first and / or second strands. Preferably, the nucleic acid contains phosphate thioester bonds between each of the terminal three 3' and terminal three 5' nucleotides of the first and second strands. Preferably, all remaining bonds between the nucleotides of the first and / or second strands are phosphodiester bonds.

[0239] In one aspect, the nucleic acid comprises dithiophosphate bonds between each of two, three, or four terminal nucleotides at the 3' end of the first strand, and / or dithiophosphate bonds between each of two, three, or four terminal nucleotides at the 3' end of the second strand, and / or dithiophosphate bonds between each of two, three, or four terminal nucleotides at the 5' end of the second strand, and bonds other than dithiophosphate bonds between the two, three, or four terminal nucleotides at the 5' end of the first strand.

[0240] In one aspect, the nucleic acid contains phosphate thioester bonds between the terminal three 3' nucleotides of the first and second strands and between the terminal three 5' nucleotides. Preferably, all remaining bonds between the nucleotides of the first and / or second strands are phosphodiester bonds.

[0241] In one aspect, nucleic acids:

[0242] (i) There are phosphate thioester bonds between the three 3' nucleotides at the end of the first strand and between the three 5' nucleotides at the end;

[0243] (ii) Conjugate a trianthal ligand to the 3' or 5' nucleotide of the second strand;

[0244] (iii) A phosphate thioester bond is present between the three terminal nucleotides of the second strand opposite the end of the conjugate to the trianthal ligand; and

[0245] (iv) Optionally, all remaining bonds between nucleotides of the first and / or second strand are phosphodiester bonds.

[0246] In one aspect, nucleic acids:

[0247] (i) The first strand has a terminal 5' (E)-vinylphosphonate nucleotide at its 5' end;

[0248] (ii) It has a phosphate thioester bond between the three terminal 3' nucleotides of the first and second chains and between the three terminal 5' nucleotides of the second chain, or it has a phosphate dithioester bond between the two terminal 3' nucleotides of the first and second chains and between the two terminal 5' nucleotides of the second chain; and

[0249] (iii) Optionally, all remaining bonds between nucleotides of the first and / or second strand are phosphodiester bonds.

[0250] Compared to molecules containing thiophosphates at the same position, the use of dithiophosphate bonds in the nucleic acids of this invention reduces stereochemical variations within the nucleic acid molecular population. Thiophosphate bonds introduce chiral centers and make it difficult to control which non-linked oxygen is replaced by sulfur. The use of dithiophosphates ensures the absence of chiral centers in this bond, thereby reducing or eliminating any variations within the nucleic acid molecular population, depending on the number of dithiophosphates and thiophosphate bonds used in the nucleic acid molecule.

[0251] In one aspect, the nucleic acid comprises a dithiophosphate bond between two terminal nucleotides at the 3' end of the first strand, a dithiophosphate bond between two terminal nucleotides at the 3' end of the second strand, and a dithiophosphate bond between two terminal nucleotides at the 5' end of the second strand, and includes bonds other than dithiophosphate bonds between two, three, or four terminal nucleotides at the 5' end of the first strand. Preferably, the first strand has a terminal 5' (E)-vinylphosphonate nucleotide at its 5' end. This terminal 5' (E)-vinylphosphonate nucleotide is preferably linked to a second nucleotide in the first strand via a phosphodiester bond. Preferably, all bonds between nucleotides of the two strands are phosphodiester bonds, except for the bonds between the two terminal nucleotides at the 3' end of the first strand and the bonds between the two terminal nucleotides at the 3' and 5' ends of the second strand.

[0252] In one aspect, the nucleic acid contains phosphate-thioester bonds between the three terminal 3' nucleotides on the first strand and / or between the three terminal 5' nucleotides, and / or between the three terminal 3' nucleotides on the second strand and / or between the three terminal 5' nucleotides, provided that no dithioester bonds are present at the terminal. The absence of dithioester bonds at the terminal means that the bonds between the two terminal nucleotides, or preferably between the three terminal nucleotides, of the nucleic acid in question are bonds other than dithioester bonds.

[0253] In one aspect, all nucleic acid bonds between nucleotides of the two strands are phosphodiester bonds, except for the bonds between the two terminal nucleotides at the 3' end of the first strand and the bonds between the two terminal nucleotides at the 3' and 5' ends of the second strand.

[0254] Other phosphate bond modifications are also possible.

[0255] Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), or carbon (bridged methylene phosphonate). Substitution can occur at the terminal oxygen. Replacing non-linked oxygen with nitrogen is also possible.

[0256] The phosphate group can also be replaced by a phosphorus-free connector.

[0257] Examples of alternatives to the phosphate group include siloxanes, carbonates, carboxymethyl groups, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thioformacetals, methylacetals, oximes, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino. In some embodiments, substitutions may include methylene carbonylamino and methylene methylimino groups.

[0258] The phosphate linker and ribose can be replaced with nuclease-resistant nucleotides. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside substitutes. In some embodiments, PNA substitutes may be used.

[0259] In one aspect, the nucleic acid (preferably a siRNA that inhibits GPR146 expression, said inhibition preferably by RNAi, and preferably in cells) comprises one or more or all of the following:

[0260] (i) Modified nucleotides;

[0261] (ii) Modified nucleotides, other than 2'-OMe modified nucleotides, preferably 2'-F modified nucleotides, at positions 2 and 14 from the 5' end of the first strand having the given SEQ ID No.;

[0262] (iii) The first strand having a given SEQ ID No. is a nucleotide modified with 2'-OMe, with each odd-numbered nucleotide starting from 1 at the 5' end;

[0263] (iv) The first strand having a given SEQ ID No. is a nucleotide modified with 2'-F for each even-numbered nucleotide starting from 1 at the 5' end;

[0264] (v) The nucleotides in the second chain corresponding to the 11th and / or 13th or 11-13th positions of the first chain having a given SEQ ID No. are modified by a modification other than 2'-OMe modification, preferably wherein one or both or all of these positions contain 2'-F modification;

[0265] (vi) Reverse nucleotide, preferably a 3'-3' bond at the 3' end of the second strand having the given SEQ ID No.;

[0266] (vii) One or more thiophosphate bonds;

[0267] (viii) One or more dithiophosphate bonds; and / or

[0268] (ix) The first chain having a given SEQ ID No. has a terminal 5' (E)-vinylphosphonate nucleotide at its 5' end, wherein the terminal 5' (E)-vinylphosphonate nucleotide is preferably uridine and is preferably linked to a second nucleotide in the first chain via a phosphodiester bond.

[0269] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 5b by no more than 3 nucleotides.

[0270] The nucleic acid disclosed herein can be a nucleic acid that, in which:

[0271] (a) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than 3 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than 3 nucleotides;

[0272] (b) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than 2 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than 2 nucleotides;

[0273] (c) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than one nucleotide, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than one nucleotide;

[0274] (d) The first strand sequence comprises the sequence of nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b;

[0275] (e) The first strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b;

[0276] (f) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b;

[0277] (g) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b;

[0278] (h) The first chain sequence comprises any of the first chain sequences shown in Table 5b having a given SEQ ID No., and optionally wherein the second chain sequence comprises the corresponding second chain sequence shown in Table 5b having a given SEQ ID No.; or

[0279] (i) The first chain sequence consists substantially of any of the first chain sequences shown in Table 5b having a given SEQ ID No., and optionally the second chain sequence consists substantially of the sequences of the corresponding second chain sequences shown in Table 5b having a given SEQ ID No.; or

[0280] (j) The first strand sequence comprises a sequence corresponding to nucleotides 1 through 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b; and optionally wherein the second strand sequence comprises or substantially consists of the sequence of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b;

[0281] (k) The first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 5b.

[0282] Optionally, the second chain sequence comprises or consists substantially of the corresponding second chain sequence having a given SEQ ID No. shown in Table 5b;

[0283] (l) The first strand sequence comprises nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b; and wherein the first strand sequence comprises nucleotides corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 5b having the corresponding second strand sequence with a given SEQ ID No.

[0284] (m) The first strand sequence comprises a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence comprises nucleotides corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 5b having the corresponding second strand sequence with a given SEQ ID No.

[0285] (n) The first chain sequence consists of any first chain sequence shown in Table 5b having a given SEQ ID No., and optionally the second chain sequence consists of the sequence of the corresponding second chain sequence shown in Table 5b having a given SEQ ID No.;

[0286] (o) In any of the above segments (a) to (n), the first and second strands of a nucleic acid molecule exist on a single strand, wherein the first and second strands are capable of hybridization and thus form a double-stranded nucleic acid with a duplex region length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or

[0287] (p) The first and second strands of any of the above segments (a) to (n) are on two separate strands that can hybridize with each other and thus form a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0288] In (l) or (m), the first strand sequence may comprise, consist substantially of, or consist of a continuous region of 19-25 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861. In (l) or (m), the first strand sequence may comprise, preferably of 20-24 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861, or consist substantially of, the continuous region.

[0289] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 3 nucleotides.

[0290] Table 2

[0291] 805 806 807 808 813 814 815 816 819 820 829 830 833 834 853 854

[0292] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 3 nucleotides.

[0293] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 2 nucleotides.

[0294] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 1 nucleotide.

[0295] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 3 nucleotides.

[0296] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 2 nucleotides.

[0297] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 1 nucleotide.

[0298] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 3 nucleotides.

[0299] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 2 nucleotides.

[0300] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 nucleotides, and the sequence differs from any first strand sequence having a given SEQ ID No. shown in Table 2 by no more than 1 nucleotide.

[0301] The nucleic acid disclosed herein may comprise a first strand and a second strand, wherein the first strand sequence is substantially composed of or consists of either the sequence of any first strand sequence having a given SEQ ID No. shown in Table 2.

[0302] For example, the nucleic acid disclosed herein can be a nucleic acid in which:

[0303] (a) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than 3 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than 3 nucleotides;

[0304] (b) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than 2 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than 2 nucleotides;

[0305] (c) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than one nucleotide, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than one nucleotide;

[0306] (d) The first strand sequence comprises the sequence of nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2;

[0307] (e) The first strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2;

[0308] (f) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2;

[0309] (g) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2;

[0310] (h) The first chain sequence comprises a sequence of any first chain sequence in Table 2, and optionally the second chain sequence comprises a sequence of the corresponding second chain sequence in Table 2; or

[0311] (i) The first chain sequence is substantially composed of any of the first chain sequences in Table 2, and optionally the second chain sequence is substantially composed of the corresponding second chain sequence in Table 2.

[0312] (j) The first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. in Table 2; and optionally wherein the second strand sequence comprises or is substantially composed of the sequence corresponding to the second strand sequence having a given SEQ ID No. in Table 2;

[0313] (k) The first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 2, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 2.

[0314] Optionally, the second chain sequence comprises or consists substantially of the corresponding second chain sequence shown in Table 2 having a given SEQ ID No.;

[0315] (l) The first strand sequence comprises nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and wherein the first strand sequence comprises a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID No. 861, substantially consisting of said continuous region or consisting of said continuous region; and optionally wherein the second strand sequence comprises nucleotides having a given SEQ ID No. shown in Table 2. The sequence corresponding to the second chain sequence of No. is composed of or substantially composed of the sequence described above;

[0316] (m) The first strand sequence comprises a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No., and wherein the first strand sequence comprises nucleotides corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 2 having the corresponding second strand sequence with a given SEQ ID No.

[0317] (n) The first chain sequence consists of any first chain sequence in Table 2, and optionally the second chain sequence consists of the sequence corresponding to the second chain sequence in Table 2.

[0318] (o) In any of the above segments (a) to (n), the first and second strands of a nucleic acid molecule exist on a single strand, wherein the first and second strands are capable of hybridization and thus form a double-stranded nucleic acid with a duplex region length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or

[0319] (p) The first and second strands of any of the above segments (a) to (n) are on two separate strands that can hybridize with each other and thus form a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0320] In (l) or (m), the first strand sequence may comprise, consist substantially of, or consist of a continuous region of 19-25 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861. In (l) or (m), the first strand sequence may comprise, preferably of 20-24 nucleotides in length complementary to the GPR146 transcript of SEQ ID NO. 861, or consist substantially of, the continuous region.

[0321] All characteristics of nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0322] heterogeneous part

[0323] The nucleic acids of this invention can be conjugated to heterologous moieties. Such nucleic acids may also be referred to as conjugated compounds or conjugated nucleic acids. A heterologous moiety is a portion of any non-nucleic acid molecule capable of inhibiting GPR146 expression. A heterologous moiety can be or may contain peptides (or polypeptides), sugars (or polysaccharides), lipids, different nucleic acids, or any other suitable molecule. Any given nucleic acid can be conjugated to multiple heterologous moieties, which may be the same or different.

[0324] A single heterologous part may contain one or more functional parts (such as the target described in more detail below), each part optionally covalently bound to a nucleic acid via a linker.

[0325] The heterologous moiety or its functional components can, for example, be used to modulate bioavailability or pharmacokinetics. For instance, it can increase the in vivo half-life. Optionally, the heterologous moiety (or its functional components) may contain a targeting agent. Efficient delivery of oligonucleotides (particularly the double-stranded nucleic acids of the present invention) to cells in vivo is important and requires specific targeting and adequate protection from the extracellular environment (particularly serum proteins). One approach to achieving specific targeting is to conjugate a targeting agent to a nucleic acid, wherein the targeting agent facilitates the targeting of the nucleic acid to target cells having cell surface receptors that bind to the targeting agent.

[0326] In this context, the term "receptor" is used to include any molecule on the surface of a target cell capable of binding a target agent, and should not be construed as implying any particular function of a cell surface receptor. A target agent can be considered a "ligand" of a cell surface receptor. The terms "target agent" and "ligand" are used interchangeably. Again, the term should not be construed as implying any particular function of a target agent or a cell surface receptor, or any particular relationship between the two molecules other than the ability of one to bind to the other.

[0327] Therefore, the target can be any part that has an affinity for the selected receptor. For example, it can be an affinity protein (such as an antibody or fragment thereof that has an affinity for the selected receptor), an aptamer, or any other suitable part. In some embodiments, the target can be a physiological ligand of the receptor.

[0328] Binding between a target agent and a receptor can facilitate the uptake of conjugated nucleic acids by target cells (e.g., via receptor internalization or any other suitable mechanism). Therefore, suitable ligands for desired receptor molecules can be used as target agents so that the conjugated nucleic acids are taken up by target cells via mechanisms such as different receptor-mediated endocytosis pathways or functionally similar processes. In other embodiments, ligands capable of mediating nucleic acid internalization into target cells via mechanisms other than receptor-mediated endocytosis can also optionally be conjugated to the nucleic acids of the present invention for cell- or tissue-specific targeting.

[0329] An example of a ligand that mediates receptor-mediated endocytosis is the GalNAc moiety described herein, which exhibits a high affinity for the desialylate glycoprotein receptor complex (ASGP-R). The ASGP-R complex comprises polymers of varying proportions of the membrane-bound ASGR1 and ASGR2 receptors, which are highly abundant on hepatocytes. One of the earliest disclosures of the use of trianthophorne as a conjugated ligand is in U.S. Patent No. 5,885,968. Conjugates having three GalNAc ligands and containing phosphate groups are known and described in Dubber et al. (Bioconjug. Chem. Jan–Feb 2003; 14(1): 239–46.). The ASGP-R complex shows an affinity for N-acetyl-D-galactosamine (GalNAc) that is 50 times greater than that for D-galactose.

[0330] The ASGP-R complex specifically recognizes the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides (Weigel, PH et al., Biochim. Biophys. Acta. 2002 Sep 19; 1572(2-3):341-63), and can be used to deliver drugs to hepatocytes expressing the receptor complex by covalently coupling galactose or galactosamine to the drug substance (Ishibashi, S.; et al., J Biol. Chem. 1994 Nov 11; 269(45):27803-6). Furthermore, binding affinity can be significantly enhanced by a multivalent effect, which is achieved through repetition of the targeting moiety (Biessen EA, et al., J Med Chem. 1995 Apr 28; 38(9):1538-46).

[0331] The ASGP-R complex is a mediator of the active uptake of a glycoprotein containing a terminal β-galactosyl group into the endosome. Therefore, ASGPR is well-suited for the targeted delivery of drug candidates (e.g., nucleic acids) conjugated to such ligands into cells expressing the receptor (Akinc et al., Mol Ther. 2010 July; 18(7):1357-64).

[0332] More typically, the ligand may contain a sugar selectively chosen to have affinity for at least one type of receptor on the target cell. In particular, the receptor is on the surface of mammalian hepatocytes, for example, the previously described hepatic desialylate glycoprotein receptor complex (ASGP-R).

[0333] The sugar can be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine, and fucose. The sugar can be N-acetylgalactosamine (GalNAc). The heteromeric moiety can contain multiple such sugars, for example, two or especially three such sugars, such as three GalNAc groups.

[0334] Therefore, the heterologous portion may comprise (i) one or more functional components, and (ii) a linker, wherein the linker conjugates the functional components to a nucleic acid as defined in either of the foregoing aspects. The linker may be a monovalent, divalent, trivalent, or tetravalent branched structure. Nucleotides may be modified as defined herein.

[0335] Therefore, the functional component can be a ligand (or a target agent). When multiple functional components are present, they can be the same or different. When the functional component is a ligand, it can be a sugar, and therefore can be (or contain) GalNAc.

[0336] In one aspect, nucleic acids are conjugated to the heterologous portion of a compound comprising formula (II):

[0337]

[0338] in:

[0339] S represents a functional component, such as a ligand, like a sugar, preferably N-acetylgalactosamine;

[0340] X 1 Represents C3-C6 alkylene groups or (-CH2-CH2-O). m (-CH2)2-, where m is 1, 2 or 3;

[0341] P is a phosphate ester or a modified phosphate ester, preferably a thiophosphate ester;

[0342] X 2 It is an alkylene group or of the formula (-CH2). n-O-CH2- alkylene ethers, where n=1-6;

[0343] A is a branch unit;

[0344] X 3 Represents a bridge-connecting unit;

[0345] The nucleic acid according to the invention is conjugated to X via a phosphate ester or a modified phosphate ester (preferably a thiophosphate ester). 3 .

[0346] In formula (II), the branching unit "A" is preferably branched into three branches to accommodate three sugar ligands. The branching unit is preferably covalently linked to the remaining tethered portion of the ligand and the nucleic acid. The branching unit may contain branched aliphatic groups, including groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxyamino groups. The branching unit may contain groups selected from alkyl and ether groups.

[0347] Branch unit A may have a structure selected from the following:

[0348] and

[0349] Each A1 independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0350] Branch units may have a structure selected from the following:

[0351] ; and

[0352] Each A1 independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0353] Branch units may have a structure selected from the following:

[0354] and

[0355] Where A1 is O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0356] Branch units can have the following structures:

[0357] .

[0358] Branch units can have the following structures:

[0359] .

[0360] Branch units can have the following structures:

[0361] .

[0362] Alternatively, branch unit A may have a structure selected from the following:

[0363] ,

[0364] in:

[0365] R 1 It is hydrogen or C1-C10 alkylene;

[0366] And R 2 It is a C1-C10 alkylene group.

[0367] Optionally, the branching unit consists of only carbon atoms.

[0368] “X 3 "The part is a bridging unit. The bridging unit is linear and covalently bound to the branching unit and nucleic acid."

[0369] X 3 Optional from -C1-C 20 Alkylene -, -C2-C 20 alkenyl-, formula-(C1-C 20 alkylene)-O-(C1-C 20 alkylene ethers of alkylene group (-alkylene), -C(O)-C1-C 20 Alkylene-, -C0-C4 alkylene(Cy)C0-C4 alkylene-, where Cy represents a substituted or unsubstituted 5- or 6-membered cycloalkylene, arylene, heterocyclic or heteroaryl ring, -C1-C4 alkylene-NHC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)NH-C1-C4 alkylene-, -C1-C4 alkylene-SC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)S-C1-C4 alkylene-, -C1-C4 alkylene-OC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)O-C1-C4 alkylene- and -C1-C6 alkylene-SS-C1-C6 alkylene-.

[0370] X 3 It can be expressed as -(C1-C 20 alkylene)-O-(C1-C 20 Alkylene ethers (alkylene). X 3 It can be expressed as -(C1-C 20 alkylene)-O-(C4-C 20 alkylene ethers of (C4-C)-, wherein... 20Alkylene is attached to Z. X 3 The following groups can be freely selected: -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C6H 12 -and-CH2-O-C8H 16 -, especially -CH2-O-C4H8-, -CH2-O-C6H 12 -and-CH2-O-C8H 16 - where in each case the -CH2- group is attached to A.

[0371] In one aspect, nucleic acids are conjugated to the heterologous portion of formula (III):

[0372]

[0373] in:

[0374] S represents a functional component, such as a ligand, like a sugar, preferably GalNAc;

[0375] X 1 Represents C3-C6 alkylene groups or (-CH2-CH2-O). m (-CH2)2-, where m is 1, 2 or 3;

[0376] P is a phosphate ester or a modified phosphate ester, preferably a thiophosphate ester;

[0377] X 2 It is a C1-C8 alkylene group;

[0378] A is selected from the following branch units:

[0379]

[0380] X 3 For bridging units;

[0381] The nucleic acid according to the invention is conjugated to X via a phosphate ester or a modified phosphate ester (preferably a thiophosphate ester). 3 .

[0382] Branch unit A can have the following structure:

[0383] .

[0384] Branch unit A can have the following structure:

[0385] , where X 3 Connected to a nitrogen atom.

[0386] X 3 It can be C1-C 20Alkylene. Preferably, X 3 Choose from the following groups: -C3H6-, -C4H8-, -C6H 12 -and-C8H 16 -, especially -C4H8-, -C6H 12 -and-C8H 16 -

[0387] In one aspect, nucleic acids are conjugated to ligands of compounds containing formula (IV):

[0388]

[0389] in:

[0390] S represents a functional component, such as a ligand, like a sugar, preferably GalNAc;

[0391] X 1 Represents C3-C6 alkylene groups or (-CH2-CH2-O). m (-CH2)2-, where m is 1, 2 or 3;

[0392] P is a phosphate ester or a modified phosphate ester, preferably a thiophosphate ester;

[0393] X 2 It is an alkylene ether of the formula -C3H6-O-CH2-;

[0394] A is a branch unit;

[0395] X 3 Alkylene ethers selected from the group consisting of: -CH2-O-CH2-, -CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H 10 -,-CH2-O-C6H 12 -、-CH2-O-C7H 14 -and-CH2-O-C8H 16 - where in each case the -CH2- group is attached to A,

[0396] And X 3 The nucleic acid according to the invention is conjugated to a phosphate ester or a modified phosphate ester (preferably a thiophosphate ester).

[0397] The branching unit may contain carbon. Preferably, the branching unit is carbon.

[0398] X 3 The following groups can be freely selected: -CH2-O-C4H8-, -CH2-O-C5H 10 -,-CH2-O-C6H12 -、-CH2-O-C7H 14 -and-CH2-O-C8H 16 - Preferably, X 3 Choose freely -CH2-O-C4H8-, -CH2-O-C6H 12 -and-CH2-O-C8H 16 - A group that consists of.

[0399] X 1 It can be (-CH2-CH2-O)(-CH2)2-. X 1 It can be (-CH2-CH2-O)2(-CH2)2-. X 1 It can be (-CH2-CH2-O)3(-CH2)2-. Preferably, X 1 It is (-CH2-CH2-O)2(-CH2)2-. Optionally, X 1 Represents C3-C6 alkylene groups. X 1 It can be propylidene. X 1 It can be butylated. X 1 It can be pentylene. X 1 It can be hexanediol. Preferably, the alkyl group is a linear alkylene group. In particular, X 1 It can be butylene.

[0400] X 2 Alkylene ethers with the formula -C3H6-O-CH2-, i.e., C3 alkoxymethylene, or –CH2CH2CH2OCH2-.

[0401] For any of the above aspects, when P represents a modified phosphate ester group, P can be expressed as:

[0402]

[0403] Where Y 1 and Y 2 Each independently represents =O, =S, and -O. - , -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2R x -OCH2C(S)OR x and –OR x , where R x Represents C1-C6 alkyl groups, and wherein This indicates that it is connected to the rest of the compound.

[0404] A modified phosphate ester is a phosphate ester group in which one or more non-linked oxygen atoms are replaced. Examples of modified phosphate ester groups include thiophosphates, dithiophosphates, selenophosphates, boron phosphates / esters, boron phosphates, hydrogen phosphonates, aminophosphates, alkyl or aryl phosphonates, and triphosphates. In dithiophosphates, both non-linked oxygen atoms are replaced by sulfur. One, each, or both non-linked oxygen atoms in the phosphate ester group may independently be any of S, Se, B, C, H, N, or OR (R being alkyl or aryl).

[0405] Phosphate esters can also be modified by replacing the linking oxygen with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene phosphonate). Substitution can occur at the terminal oxygen. Replacing non-linked oxygen with nitrogen is possible.

[0406] For example, Y 1 It can represent -OH and Y 2 It can represent =O or =S; or

[0407] Y 1 Can represent -O - And Y 2 It can represent =O or =S;

[0408] Y 1 It can represent =O and Y 2 It can represent –CH3, -SH, -OR x Or –BH3;

[0409] Y 1 Can represent =S and Y 2 Can represent –CH3, OR x Or –SH.

[0410] Those skilled in the art will understand that, in certain circumstances, Y 1 and Y 2 There will be a delocalization effect between them.

[0411] Preferably, the modified phosphate group is a thiophosphate group. The thiophosphate group includes dithiophosphate (i.e., where Y...). 1 Representative = S and Y 2 Representative – S - ) and monothiophosphate (i.e., where Y 1 Representative -O - And Y 2 Representative = S, or where Y 1 Representative = O and Y 2 Representative – S - Preferably, P is a monothiophosphate. The inventors have discovered that conjugates having a thiophosphate group replacing a phosphate group have improved potency and duration of action in vivo.

[0412] P can also be an ethyl phosphate (i.e., where Y is an ethyl phosphate ester). 1 Representative = O and Y 2 (Represents OCH2CH3).

[0413] Ligands (e.g., sugars) can be selected to have affinity for at least one type of receptor on target cells. In particular, receptors are located on the surface of mammalian hepatocytes, such as the hepatic desialylate glycoprotein receptor complex (ASGP-R).

[0414] For any of the above or below aspects, the sugar may be selected from N-acetyl groups having one or more of the following: galactosamine, mannose, galactose, glucose, glucosamine, and fructose. Typically, the ligands used in this invention may include N-acetylgalactosamine (GalNAc). Preferably, the compounds of this invention may have three ligands, each ligand preferably comprising N-acetylgalactosamine.

[0415] "GalNAc" refers to 2-(acetamido)-2-deoxy-D-galactopyranose, often referred to as N-acetylgalactosamine in the literature. When "GalNAc" or "N-acetylgalactosamine" is mentioned, it includes both β-type: 2-(acetamido)-2-deoxy-β-D-galactopyranose and α-type: 2-(acetamido)-2-deoxy-α-D-galactopyranose. In some embodiments, β-type: 2-(acetamido)-2-deoxy-β-D-galactopyranose and α-type: 2-(acetamido)-2-deoxy-α-D-galactopyranose are used interchangeably. Preferably, the compounds of the present invention comprise β-type, 2-(acetamido)-2-deoxy-β-D-galactopyranose.

[0416]

[0417] 2-(acetamido)-2-deoxy-D-galactopyranose

[0418]

[0419] 2-(acetamido)-2-deoxy-β-D-galactopyranose

[0420]

[0421] 2-(acetamido)-2-deoxy-α-D-galactopyranose

[0422] In one aspect, nucleic acids are conjugated nucleic acids, wherein the nucleic acid is conjugated to a heterologous portion having one of the following structures, which may be referred to as a "tri-antennae ligand" for ease of reference:

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432] Where Z is any nucleic acid as defined in this paper.

[0433] In some embodiments, nucleic acid Z is conjugated to a trianthal ligand via a phosphate or thiophosphate group that links the trianthal ligand to the 3' or 5' position of the sugar of the terminal nucleotide of said nucleic acid Z, particularly the 3' or 5' position of the ribose.

[0434] In some implementations, the heterologous portion (“triantennae”) is conjugated to the 3’ position of the terminal nucleotide ribose of the second (sense) strand of Z (also referred to as strand “B” in Tables 5a, 5b, 5c).

[0435] In other embodiments, the heterologous portion (“triantennae”) is conjugated to the 5’ position of the terminal nucleotide ribose of the second (sense) strand of Z (also referred to as strand “B” in Tables 5a, 5b, 5c).

[0436] In other embodiments, the heterologous portion (“triantennae”) is conjugated to the 3’ position of the terminal nucleotide ribose of the first (antisense) strand of Z (which is also referred to as strand “A” in Tables 5a, 5b, 5c).

[0437] Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a trianticular ligand having one of the following structures:

[0438]

[0439] ,

[0440] Where Z is any nucleic acid as defined in this paper.

[0441] In a preferred embodiment, nucleic acid Z is conjugated to a trianthal ligand via a phosphate ester or thiophosphate group, which connects the trianthal ligand to the 3' or 5' position of the terminal nucleotide ribose of nucleic acid Z.

[0442] Preferably, the trianthal ligand is conjugated to the 5' position of the terminal nucleotide ribose of the second (sense) chain of Z (also referred to as chain "B" in Tables 5a, 5b, 5c).

[0443] The heteromorphic portion of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein, may be attached to any position at the 3' end of the first (antisense) strand and / or the 3' and / or 5' end of the second (sense) strand. The nucleic acid may contain more than one heteromorphic portion of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein. However, a single heteromorphic portion of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein, is preferred because a single such portion is sufficient to effectively target the nucleic acid to the target cell. Preferably, in this case, at least the last two, preferably at least the last three, and more preferably at least the last four nucleotides of the nucleic acid terminal connecting the ligand are linked by a phosphodiester bond.

[0444] Preferably, the 5' end of the first (antisense) strand is not connected to the heterologous portion, because connection at this position could potentially interfere with the biological activity of the nucleic acid.

[0445] Nucleic acid with a single heterologous moiety (e.g., a heterologous moiety of formula (II), (III), or (IV) or any of the tritenoid ligands disclosed herein) at the 5' end of the strand is easier and therefore cheaper to synthesize than the same nucleic acid with the same group at the 3' end. Therefore, preferably, a single heterologous moiety (e.g., a heterologous moiety of formula (II), (III), or (IV) or any of the tritenoid ligands disclosed herein) is covalently attached to (conjugated to) the 5' end of the second strand of the nucleic acid.

[0446] In one aspect, the first chain of a nucleic acid is a compound of formula (V):

[0447]

[0448] Where b is preferably 0 or 1; and

[0449] Compounds with the second chain of formula (VI):

[0450]

[0451] in:

[0452] c and d are independently preferred to be 0 or 1;

[0453] Z1 and Z2 are the first and second strands of the nucleic acid, respectively;

[0454] Y can be either O or S independently;

[0455] n is independently 0, 1, 2, or 3; and

[0456] L1 is a linker for connecting ligands, wherein L1 is the same or different in formulas (V) and (VI), and is the same or different in formulas (V) and (VI) when L1 appears more than once in the same formula, wherein L1 is preferably formula (VII).

[0457] Furthermore, b+c+d is preferably 2 or 3.

[0458] Preferably, L1 in equations (V) and (VI) is equation (VII):

[0459]

[0460] in:

[0461] L is selected from the group consisting of the following or preferably the group consisting of the following:

[0462] -(CH2) r -C(O)-, where r=2-12;

[0463] -(CH2-CH2-O) s -CH2-C(O)-, where s=1-5;

[0464] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, where t is independently 1-5;

[0465] -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1-5; and

[0466] -(CH2) v -NH-C(O)-, where v=2-12; and

[0467] The end C(O) (if present) is connected to X in equation (VII), or to W1 in equation (VII) if X does not exist, or to V in equation (VII) if W1 does not exist.

[0468] W1, W3, and W5 are either absent or selected from the group consisting of, or preferably from, the following:

[0469] -(CH2) r -, where r = 1-7;

[0470] -(CH2) s -O-(CH2) s -, where s independently ranges from 0 to 5;

[0471] -(CH2)t -S-(CH2) t -, where t is independently 0-5;

[0472] X is absent or selected from the group consisting of or preferably the group consisting of: NH, NCH3 or NC2H5;

[0473] V is selected from the group consisting of the following or preferably the group consisting of the following:

[0474] CH, N, , , , or ;

[0475] If B is present, it is either a modified or natural nucleobase.

[0476] In one aspect, the first chain is a compound of formula (VIII):

[0477]

[0478] Where b is preferably 0 or 1; and

[0479] The second chain is a compound of formula (IX):

[0480]

[0481] Where c and d are independently preferably 0 or 1;

[0482] in:

[0483] Z1 and Z2 are the first and second strands of the nucleic acid, respectively;

[0484] Y can be either O or S independently;

[0485] R1 is H or methyl;

[0486] n is independently preferably 0, 1, 2, or 3; and

[0487] L is the same or different in equations (VIII) and (IX), and is the same or different in equations (VIII) and (IX) when L appears more than once in the same equation, and is selected from the group consisting of or preferably the group consisting of:

[0488] -(CH2) r -C(O)-, where r=2-12;

[0489] -(CH2-CH2-O) s -CH2-C(O)-, where s=1-5;

[0490] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, where t is independently 1-5;

[0491] -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1-5; and

[0492] -(CH2) v -NH-C(O)-, where v=2-12; and

[0493] The terminal C(O) (if present) is attached to an NH group (the NH group of the linker, not the NH group of the target ligand);

[0494] Furthermore, b+c+d is preferably 2 or 3.

[0495] In one aspect, the first chain of a nucleic acid is a compound of formula (X):

[0496]

[0497] Where b is preferably 0 or 1; and

[0498] The second chain is a compound of formula (XI):

[0499]

[0500] in:

[0501] c and d are independently preferred to be 0 or 1;

[0502] Z1 and Z2 are the first and second RNA strands of the nucleic acid, respectively;

[0503] Y can be either O or S independently;

[0504] n is independently preferably 0, 1, 2, or 3; and

[0505] L2 is the same or different in equations (X) and (XI), and is the same or different in the parts enclosed in b, c, and d, and is selected from the group consisting of or preferably the group consisting of:

[0506] ; ;and ;or

[0507] n is 0 and L2 is:

[0508]

[0509] Furthermore, the terminal OH group is absent, thus forming the following part:

[0510] ;

[0511] in:

[0512] F represents saturated branched or unbranched chains (such as unbranched chains) C 1-8 Alkyl (e.g., C10) 1-6 An alkyl chain wherein one of the carbon atoms is optionally replaced by an oxygen atom, provided that the oxygen atom is separated from another heteroatom (e.g., an O or N atom) by at least two carbon atoms;

[0513] L is the same or different in formulas (X) and (XI), and is selected from the group consisting of or preferably the group consisting of:

[0514] -(CH2) r -C(O)-, where r=2-12;

[0515] -(CH2-CH2-O) s -CH2-C(O)-, where s=1-5;

[0516] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, where t is independently 1-5;

[0517] -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1-5; and

[0518] -(CH2) v -NH-C(O)-, where v=2-12; and

[0519] The terminal C(O) (if present) is attached to an NH group (the NH group of the linker, not the NH group of the target ligand);

[0520] Furthermore, b+c+d is preferably 2 or 3.

[0521] In one aspect, in any nucleic acid of formulas (V) and (VI), (VIII) and (IX), or (X) and (XI), b is 0, c is 1, and d is 1; b is 1, c is 0, and d is 1; b is 1, c is 1, and d is 0; or b is 1, c is 1, and d is 1. Preferably, b is 0, c is 1, and d is 1; b is 1, c is 0, and d is 1; or b is 1, c is 1, and d is 1. Most preferably, b is 0, c is 1, and d is 1.

[0522] In one aspect, Y is O in any nucleic acid of formulas (V) and (VI), (VIII) and (IX), or (X) and (XI). In another aspect, Y is S. In a preferred aspect, Y is independently selected from O or S at different positions in the formula.

[0523] In one aspect, in any nucleic acid of formulas (VIII) and (IX), R1 is H or methyl. In another aspect, R1 is H. In the third aspect, R1 is methyl.

[0524] In one aspect, in any nucleic acid of formulas (V) and (VI), (VIII) and (IX), or (X) and (XI), n is 0, 1, 2, or 3. Preferably, n is 0.

[0525] In any nucleic acid of formulas (X) and (XI), examples of the F portion include (CH2). 1-6 For example, (CH2) 1-4 For example, CH2, (CH2)4, (CH2)5, or (CH2)6, or CH2O(CH2). 2-3 For example, CH2O(CH2)CH3.

[0526] In one aspect, L2 in equations (X) and (XI) is:

[0527] .

[0528] In one aspect, L2 is:

[0529] .

[0530] In one aspect, L2 is:

[0531] .

[0532] In one aspect, L2 is:

[0533] .

[0534] In one aspect, n is 0 and L2 is:

[0535]

[0536] Furthermore, the terminal OH group is absent, resulting in the following part:

[0537] ;

[0538] Where Y is O or S.

[0539] In one aspect, the L in the nucleic acids of formulas (V) and (VI) or (VIII) and (IX) or (X) and (XI) is selected from the group consisting of, or preferably from, the group consisting of:

[0540] -(CH2) r -C(O)-, where r=2-12;

[0541] -(CH2-CH2-O) s -CH2-C(O)-, where s=1-5;

[0542] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, where t is independently 1-5;

[0543] -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1-5; and

[0544] -(CH2) v -NH-C(O)-, where v=2-12;

[0545] The terminal C(O) is attached to an NH group.

[0546] Preferably, L is -(CH2). r -C(O)-, where r=2-12, more preferably r=2-6, even more preferably r=4 or 6, for example 4.

[0547] Preferably, L is:

[0548] .

[0549] Within the portions enclosed by b, c, and d, the L2 values ​​in the nucleic acids of formulas (X) and (XI) are generally the same. Between the portions enclosed by b, c, and d, the L2 values ​​may be the same or different. In one embodiment, the L2 values ​​in the portion enclosed by c are the same as those in the portion enclosed by d. In one embodiment, the L2 values ​​in the portion enclosed by c are different from those in the portion enclosed by d. In one embodiment, the L2 values ​​in the portions enclosed by b, c, and d are the same, for example, when the linker portion is a serine-derived linker portion.

[0550] The serine-derived linker moiety can be based on any stereochemical serine, i.e., a combination derived from L-serine isomers, D-serine isomers, racemic serine, or other isomers. In a preferred aspect of the invention, the serine-GalNAc moiety (SerGN) has the following stereochemistry:

[0551]

[0552] That is, building blocks based on solid-phase supported (S)-serine alcohol-phosphoramide or (S)-serine alcohol succinate derived from L-serine isomers.

[0553] In a preferred embodiment, the first chain of the nucleic acid is a compound of formula (VIII) and the second chain of the nucleic acid is a compound of formula (IX), wherein:

[0554] b is 0;

[0555] c and d are both 1.

[0556] When n is 0,

[0557] Z1 and Z2 are the first and second strands of the nucleic acid, respectively.

[0558] Y is S,

[0559] R1 is H, and

[0560] L is -(CH2)4-C(O)-, where the terminal C(O) of L is connected to the N atom of the linker (i.e., a possible N atom that is not the target ligand).

[0561] In another preferred aspect, the first chain of the nucleic acid is a compound of formula (V) and the second chain of the nucleic acid is a compound of formula (VI), wherein:

[0562] b is 0,

[0563] c and d are both 1.

[0564] When n is 0,

[0565] Z1 and Z2 are the first and second strands of the nucleic acid, respectively.

[0566] Y is S,

[0567] L1 is equation (VII), where:

[0568] W1 is -CH2-O-(CH2)3-.

[0569] W3 is -CH2-.

[0570] W5 does not exist.

[0571] V is CH.

[0572] X is NH, and

[0573] L is -(CH2)4-C(O)-, where the terminal C(O) of L is attached to the N atom of X in formula (VII).

[0574] In another preferred aspect, the first chain of the nucleic acid is a compound of formula (V) and the second chain of the nucleic acid is a compound of formula (VI), wherein:

[0575] b is 0,

[0576] c and d are both 1.

[0577] When n is 0,

[0578] Z1 and Z2 are the first and second strands of the nucleic acid, respectively.

[0579] Y is S,

[0580] L1 is equation (VII), where:

[0581] W1, W3, and W5 do not exist.

[0582] V is ,

[0583] X does not exist, and

[0584] L is -(CH2)4-C(O)-NH-(CH2)5-C(O)-, where the terminal C(O) of L is attached to the N atom of V in formula (VII).

[0585] In one aspect, nucleic acids are conjugated to trianthal ligands having the following structure:

[0586]

[0587] The nucleic acid is conjugated to the trianthal ligand via the phosphate ester group of the ligand.

[0588] a) The 3' position of the terminal nucleotide ribose of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, and 5c), or

[0589] b) The 5' position of the terminal nucleotide ribose of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, and 5c), or

[0590] c) The 3' position of the terminal nucleotide ribose of the first (antisense) strand of Z (also referred to as strand "A" in Tables 5a and 5b).

[0591] In one aspect of nucleic acids, hepatocytes are cells targeted by nucleic acids with ligands.

[0592] Of any of the ligands containing GalNAc mentioned above, GalNAc can be replaced by any other targeting ligand, such as those mentioned herein, particularly mannose, galactose, glucose, glucosamine, and fucose.

[0593] In one aspect, nucleic acids are conjugated to a heterologous portion, said heterologous portion comprising lipids, and more preferably cholesterol.

[0594] In one aspect, the double-stranded nucleic acid used to suppress GPR146 expression is one of the double strands shown in Table 5c, which can be mentioned by its double strand ID number.

[0595] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3172, EM3175, EM3098, EM3090, EM3031, EM3203, EM3204, EM3207, EM3208, EM3210, EM3215, EM3217, EM3227, EM3056, EM3751, EM3177, EM3187, EM3049, EM3096, EM3097, EM3164, EM3172, and EM3175.

[0596] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3203, EM3204, EM3207, EM3208, EM3210, EM3215, EM3217 and EM3227.

[0597] In a preferred aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is a double-stranded nucleic acid having a double-stranded ID number EM3203, EM3204, EM3207, EM3208, EM3210, EM3215, EM3217 or EM3227.

[0598] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3172, EM3175, EM3098, EM3090, or EM3031.

[0599] In one aspect, the double-stranded nucleic acid used to suppress GPR146 expression is selected from double-stranded ID numbers EM3056, EM3751, EM3177 and EM3187.

[0600] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3031, EM3049, EM3096, EM3097, EM3098, EM3164, EM3172 and EM3175.

[0601] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3203, EM3208, EM3207 and EM3215.

[0602] In one aspect, the double-stranded nucleic acid used to suppress GPR146 expression is selected from double-stranded ID numbers EM3203, EM3207 and EM3210.

[0603] In one aspect, the double-stranded nucleic acid used to suppress GPR146 expression is selected from double-stranded ID numbers EM3223, EM3210 and EM3207.

[0604] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3207, EM3210, EM3204, EM3208, EM3210, EM3203 and EM3217.

[0605] In one aspect, the double-stranded nucleic acid used to inhibit GPR146 expression is selected from double-stranded ID numbers EM3203, EM3204, EM3207, EM3208, EM3210, EM3215, EM3217 and EM3227.

[0606] Compositions, uses and methods

[0607] This invention also provides compositions comprising the nucleic acids of this invention. The nucleic acids and compositions can be used alone or in combination with other substances as therapeutic or diagnostic agents. For example, one or more nucleic acids of this invention can be combined with a delivery medium (such as liposomes) and / or excipients (such as carriers, diluents). Other substances such as preservatives and stabilizers may also be added. Pharmaceutically acceptable salts or solvates of any nucleic acid of this invention are also within the scope of this invention. Methods for delivering nucleic acids are known in the art and are within the knowledge of those skilled in the art.

[0608] The compositions disclosed herein are, in particular, pharmaceutical compositions. Such compositions are suitable for administration to a subject.

[0609] In one aspect, the composition comprises a nucleic acid disclosed herein or a pharmaceutically acceptable salt or solvate thereof, and a solvent (preferably water) and / or a delivery medium and / or a physiologically acceptable excipient and / or a carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative.

[0610] Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, or parenteral administration (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal). The formulation may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical industry. Subcutaneous or transdermal administration may be particularly suitable for the compounds described herein.

[0611] The effective dose of the nucleic acid of this invention for prevention or treatment will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining the dose are well known to those skilled in the medical field. The dose and method of administration can be adjusted to achieve optimal efficacy and may depend on factors such as weight, diet, concomitant medications, and others well known to those skilled in the medical field. The most suitable dosage and dosing regimen for human use can be guided by the results obtained from this invention and can be confirmed in appropriately designed clinical trials.

[0612] Effective dosages and treatment regimens can be determined using routine methods, starting with low doses in laboratory animals and then increasing the dose while monitoring the effects, and also systematically modifying the dosing regimen. Clinicians may consider many factors when determining the optimal dose for a given subject. These considerations are known to those skilled in the art.

[0613] The nucleic acids or their salts of the present invention can be formulated into pharmaceutical compositions prepared for storage or administration, the pharmaceutical compositions typically containing a preventive or therapeutically effective amount of the nucleic acids or their salts of the present invention in a pharmaceutically acceptable carrier.

[0614] The nucleic acids or conjugated nucleic acids of the present invention can also be administered in combination with other therapeutic compounds (alone or simultaneously, e.g., as a combined unit dose). The present invention also includes compositions comprising one or more nucleic acids according to the present invention, wherein the nucleic acids are in physiologically / pharmaceuticalally acceptable excipients (such as stabilizers, preservatives, diluents, buffers, etc.).

[0615] In one aspect, the composition comprises a nucleic acid disclosed herein and a therapeutic agent selected from the group consisting of oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides. Preferably, the therapeutic agent is an agent that targets GPR146, preferably inhibiting its expression or activity. Preferably, the therapeutic agent is one of: a) a peptide that inhibits the expression or activity of GPR146, or b) an antibody that specifically binds to GPR146 or one of its subunits or a proteolytic cleavage product under physiological conditions.

[0616] In some embodiments, the therapeutic agent is selected from the group consisting of: 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors / statins—simvastatin, atorvastatin, rosuvastatin, pravastatin, pitavastatin, fluvastatin, lovastatin; 2-azacyclobutanone—ezetimibe; bile acid sequestrants (cholestyramine, colestipol, colesvelam); proprotein convertase subtilisin / Kexin type 9 (PCSK9) inhibitors (alirocumab, evolocumab, and inclisiran); fibrates (fenofibrate, clofibrate, and gemfibrozil); omega-3 fatty acids (ethyl eicosapentaenoate, ethyl omega-3-acid, omega-3-carboxylic acid, ethyl omega-3-acid) and niacin / nicotinic acid. In some embodiments, two or more nucleic acids of the present invention with different sequences may be applied simultaneously or sequentially.

[0617] In another aspect, the present invention provides compositions (e.g., pharmaceutical compositions) comprising one or a combination of different nucleic acids of the present invention and at least one pharmaceutically acceptable carrier.

[0618] The dosage levels of the therapeutic agents and compositions of the present invention can be determined experimentally by those skilled in the art. In one aspect, a unit dose may comprise about 0.01 mg / kg to about 100 mg / kg body weight of nucleic acid or conjugated nucleic acid. Optionally, the dose may be 10 mg / kg to 25 mg / kg body weight, or 1 mg / kg to 10 mg / kg body weight, or 0.05 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to 1 mg / kg body weight, or 0.1 mg / kg to 0.5 mg / kg body weight, or 0.5 mg / kg to 1 mg / kg body weight. Optionally, the dosage may be from about 0.5 mg / kg to about 10 mg / kg body weight, or from about 0.6 mg / kg to about 8 mg / kg body weight, or from about 0.7 mg / kg to about 7 mg / kg body weight, or from about 0.8 mg / kg to about 6 mg / kg body weight, or from about 0.9 mg / kg to about 5.5 mg / kg body weight, or from about 1 mg / kg to about 5 mg / kg body weight, or from about 1 mg / kg body weight, or from about 3 mg / kg body weight, or from about 5 mg / kg body weight, wherein “about” means a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, even more preferably up to 5%, and most preferably 0%, relative to the value shown. The dosage level may also be calculated using other parameters, such as, for example, body surface area.

[0619] The dosage units of these nucleic acids preferably comprise about 1 mg / kg to about 5 mg / kg body weight, or about 1 mg / kg to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 5 mg / kg body weight. Compared with untreated subjects or subjects treated with control nucleic acids under comparable conditions, the levels of GPR146 mRNA in the liver and / or the levels of GPR146 protein in plasma or blood of subjects treated with the dosage units of nucleic acids preferably decreased by at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% at the maximal effect time point.

[0620] Dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative) and may be adjusted during treatment. In some prophylactic applications, relatively low doses are administered at relatively infrequent intervals over relatively long periods. Some subjects may continue treatment throughout their lives. In some therapeutic applications, relatively high doses may be administered at relatively short intervals until disease progression slows or until the patient shows partial or complete remission of disease symptoms. Afterward, the patient may switch to an appropriate prophylactic dosing regimen.

[0621] The actual dose level of the nucleic acid (alone or in combination with one or more other active ingredients) in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and route of administration without causing harmful side effects to the subject or patient. The selected dose level will depend on a variety of factors, such as pharmacokinetic factors, including the activity of the specific nucleic acid or composition used, the route of administration, the time of administration, the excretion rate of the specific nucleic acid used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health, and medical history of the subject or patient being treated, and similar factors well known in the medical field.

[0622] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in lyophilized form.

[0623] The pharmaceutical composition may be a unit dosage form. In such a form, the composition is dispensed into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be a packaged article containing discrete amounts of the article, such as packaged tablets, capsules, and powder in vials or ampoules. The unit dosage form may also be the capsule, sac, or tablet itself, or may be appropriate quantities of any of these packaged forms. It may be provided in a single-dose injectable form, such as in a pen syringe. The composition may be formulated for any suitable route and manner of administration.

[0624] The therapeutic agents and pharmaceutical compositions of the present invention can be administered to mammalian subjects in pharmaceutically effective doses. Mammals may be selected from humans, non-human primates, apes or prosimians, dogs, cats, horses, cattle, pigs, goats, sheep, mice, rats, hamsters, hedgehogs, and guinea pigs, or other relevant species. Accordingly, “GPR146” as used herein refers to a nucleic acid or protein (if naturally or artificially expressed therein) in any of the above-mentioned species, but preferably this terminology refers to a human nucleic acid or protein.

[0625] The pharmaceutical compositions of the present invention can be administered alone or in combination with one or more other therapeutic or diagnostic agents. Combination therapies may include the nucleic acids of the present invention in combination with at least one other therapeutic agent selected based on the specific patient, disease, or condition being treated. Examples of other such agents include, in particular, therapeutically active small molecules or peptides, single-chain antibodies, classical antibodies or fragments thereof, or nucleic acid molecules that regulate the expression of one or more additional genes, as well as similar modulating therapeutic agents that may complement or otherwise benefit therapeutic or preventative treatment regimens.

[0626] Pharmaceutical compositions are generally sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, alcohols such as ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixture. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants according to formulation chemistry well known in the art. In some embodiments, isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride may be desired in the composition. The absorption of injectable compositions can be prolonged by including agents that delay absorption (e.g., monostearate and gelatin) in the composition.

[0627] One aspect of the invention is the nucleic acid or composition disclosed herein, used as a therapeutic agent. The nucleic acid or composition is preferably used for the prevention or treatment of diseases, symptoms, or syndromes.

[0628] The present invention provides nucleic acids for use alone or in combination with one or more additional therapeutic agents in pharmaceutical compositions for the treatment or prevention of conditions, diseases and symptoms that respond to inhibition of GPR146 expression.

[0629] One aspect of the present invention is the use of the nucleic acids or compositions disclosed herein in the prevention or treatment of diseases, symptoms or syndromes.

[0630] The nucleic acid and pharmaceutical compositions of the present invention can be used to treat a variety of conditions, symptoms, or diseases. Treatment with the nucleic acid of the present invention preferably results in the depletion of GPR146 in the body, preferably in the liver and / or blood. Therefore, the nucleic acid of the present invention and compositions comprising the nucleic acid of the present invention can be used in methods for treating a variety of pathological conditions, wherein inhibition of GPR146 expression can be beneficial. The present invention provides a method for treating a disease, symptom, or syndrome, comprising the step of administering a preventive or therapeutically effective amount of the nucleic acid of the present invention to a subject in need.

[0631] Therefore, the present invention provides a method for preventing or treating a disease, symptom, or syndrome, the method comprising the step of administering a therapeutically effective amount of the nucleic acid of the present invention or a pharmaceutical composition comprising the nucleic acid of the present invention to a subject in need (e.g., a patient).

[0632] The most desirable therapeutically effective dose is a dose that will produce the desired efficacy for a specific treatment chosen by a person skilled in the art for a given subject in need. This dose will vary depending on a variety of factors understood by a skilled practitioner, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general condition, responsiveness to a given dose, and type of medication), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. A person skilled in the clinical and pharmacological fields will be able to determine the therapeutically effective dose experimentally, i.e., by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. See, for example, Remington: The Science and Practice of Pharmacy 21st Ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0633] In some embodiments, the nucleic acid and pharmaceutical compositions of the present invention can be used to treat or prevent diseases, symptoms, or syndromes.

[0634] In some embodiments, the present invention provides a method for preventing or treating a disease, symptom, or syndrome in a mammalian subject (such as a human), the method comprising the step of administering a preventive or therapeutically effective amount of the nucleic acid disclosed herein to the subject in need.

[0635] Applying a "therapeutic dose" of the nucleic acid of this invention can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease.

[0636] The nucleic acids of this invention can be beneficial for the treatment or diagnosis of diseases, conditions, or syndromes that can be diagnosed or treated using the methods described herein. The treatment and diagnosis of other diseases, conditions, or syndromes are also considered to fall within the scope of this invention.

[0637] One aspect of the invention is a method for preventing or treating a disease, symptom, or syndrome, comprising administering to an individual requiring treatment a pharmaceutically effective dose or amount of the nucleic acid or composition disclosed herein, preferably administered to the subject subcutaneously, intravenously, or orally, rectally, pulmonaryly, intramuscularly, or intraperitoneally. Preferably, it is administered subcutaneously.

[0638] Diseases, symptoms, or syndromes are typically GPR146-mediated and are associated with abnormal and / or excessive activation (hyperactivation) of GPR146 and / or overexpression, ectopic expression, localization, or accumulation of GPR146.

[0639] GPR146-mediated diseases, conditions, or syndromes may include hyperlipidemia, hypercholesterolemia, atherosclerosis and / or atherosclerotic cardiovascular disease, aortic stenosis, ischemic stroke, ischemic heart disease / coronary artery disease, peripheral artery disease, pulmonary hypertension, diabetes, diabetic nephropathy, diabetic neuropathy, and / or diabetic retinopathy.

[0640] Hypercholesterolemia can include acquired hypercholesterolemia and / or familial hypercholesterolemia (FH).

[0641] GPR146-mediated diseases, conditions, or syndromes may be selected from the group comprising, and preferably, the following: hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis and / or atherosclerotic cardiovascular disease, aortic stenosis, ischemic stroke, ischemic heart disease / coronary artery disease, peripheral artery disease, pulmonary hypertension, diabetes, diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy.

[0642] GPR146-mediated diseases, conditions, or syndromes may be selected from the group comprising, and preferably, the following: hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis, and / or atherosclerotic cardiovascular disease.

[0643] Preferably, the GPR146-mediated disease, condition, or syndrome includes hypercholesterolemia. More preferably, the GPR146-mediated disease, condition, or syndrome includes familial hypercholesterolemia (FH). Familial hypercholesterolemia may be selected from (i) heterozygous FH, (ii) homozygous FH, and (iii) autosomal recessive hypercholesterolemia.

[0644] The nucleic acids or compositions disclosed herein may be used in treatment regimens comprising: once or twice a week, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every three months, every four months, every five months, every six months, or in regimens with different dosing frequencies (such as combinations of the above intervals). The nucleic acids or compositions may be used subcutaneously, intravenously, or via any other route of application such as oral, rectal, pulmonary, or intraperitoneal. Preferably, they are used subcutaneously.

[0645] Inhibition levels are preferably measured under selected conditions, as these conditions demonstrate the maximum effect of the nucleic acid or conjugate nucleic acid of the present invention on the target mRNA (here: GPR146 mRNA) levels in cells treated in vitro or in vivo. Inhibition levels can be measured, for example, after treatment with the nucleic acid or conjugate nucleic acid of the present invention at concentrations of 0.03 nM to 10 µM (preferably 0.1 nM, 0.5 nM, 1 nM, 10 nM, 100 nM, or 1000 nM) for in vitro testing, or at amounts of 1 nmol to 100 µmol for in vivo testing of mouse or non-human primate samples, or at amounts of 0.5 µmol to 100 µmol, preferably 8 µmol to 80 µmol, for in vivo testing of human samples, for 24 hours, 48 ​​hours, 1 week, 2 weeks, 4 weeks, 8 weeks, or 12 weeks. These conditions may vary for different nucleic acids or conjugate nucleic acids of the present invention. Examples of suitable conditions for determining inhibition levels are described in the Examples section below.

[0646] Compared to untreated cells and / or subjects, GPR146 expression can be suppressed by 15% to 100% in cells and / or subjects treated with or receiving the nucleic acids or compositions disclosed herein, but at least by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or an intermediate value. The level of suppression may allow for the treatment of diseases associated with GPR146 expression or overexpression, or may be used for further research into the function and physiological role of the GPR146 gene product. The level of suppression is preferably measured in the liver or blood or kidney of subjects treated with the nucleic acids or compositions, preferably in liver samples (which may also be referred to as liver biopsies).

[0647] The level of inhibition of GPR146 expression can be measured by RT-qPCR. For example, the level of inhibition of GPR146 expression can be measured in liver biopsy samples, such as by RT-qPCR, in liver biopsies of subjects treated with nucleic acids or compositions. Cholesterol and / or triglyceride levels can also be measured.

[0648] One aspect is the use of the nucleic acids or compositions disclosed herein in the preparation of medicaments for treating diseases, conditions, or syndromes (such as those listed above or other pathological conditions associated with elevated GPR146 levels (preferably elevated GPR146 levels in the blood, liver, or kidneys), or other therapeutic methods that aim to inhibit GPR146 expression). A medicament is a pharmaceutical composition.

[0649] Each nucleic acid of the present invention, and its pharmaceutically acceptable salts and solvates, constitute a separate embodiment of the present invention.

[0650] The present invention also includes methods for preventing or treating diseases, symptoms, or syndromes (such as those listed above), comprising administering to an individual in need a composition containing the nucleic acids or compositions described herein. The nucleic acids or compositions may be administered, for example, in treatment regimens comprising: twice weekly, once weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve weeks or longer, or in regimens with different dosing frequencies (such as combinations of the above intervals). Nucleic acids or conjugated nucleic acids may be used subcutaneously or intravenously or via other routes of administration such as oral, rectal, or intraperitoneal.

[0651] The nucleic acids of the present invention can be administered via any suitable route of administration known in the art, including but not limited to aerosol, enteric, nasal, ocular, oral, parenteral, rectal, vaginal, or percutaneous (e.g., topical application of creams, gels, or ointments, or via percutaneous patches). “Parenteral administration” is generally associated with injection at or in connection with the intended site of action, including infraorbital, infusion, intra-arterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or intratracheal administration.

[0652] Using chemical modification patterns of nucleic acids to confer stability to nucleases in serum and make, for example, subcutaneous application feasible.

[0653] Solutions or suspensions intended for intradermal or subcutaneous application typically include one or more of the following: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and / or tonic modifiers such as, for example, sodium chloride or dextrose. pH may be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide) or with buffers such as citrates, phosphates, or acetates. Such formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0654] Sterile injectable solutions can be prepared by incorporating the desired amount of nucleic acid with one or a combination of the components described above (as needed) into a suitable solvent, followed by sterile microfiltration. Dispersions can be prepared by incorporating the active compound into a sterile medium containing a dispersion medium and optionally other components (such as those described above). In the case of sterile powders used to prepare sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying (lyophilization), which yields powders of the active ingredient and any other desired components from a sterile filtered solution.

[0655] When a preventive or therapeutically effective amount of the nucleic acid of the present invention is administered, for example, via intravenous, skin, or subcutaneous injection, the nucleic acid will be in the form of a pyrogen-free, parenteral-acceptable aqueous solution. Methods for preparing parenteral-acceptable solutions, taking into account appropriate pH, isotonicity, stability, etc., are well known to those skilled in the art. Preferred pharmaceutical compositions for intravenous, skin, or subcutaneous injection will, in addition to the nucleic acid, contain an isotonic medium, such as sodium chloride injection, Ringer's solution, dextran injection, dextran and sodium chloride injection, lactated Ringer's solution, or other mediators known in the art. The pharmaceutical compositions of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those skilled in the art.

[0656] The amount of nucleic acid that can be combined with a carrier material to produce a single dosage form will vary depending on a number of factors, including the subjects being treated and the specific method of administration. Typically, it will be the amount of the composition that produces an appropriate therapeutic effect in a given situation. Typically, in percentage terms, this amount ranges from about 0.01% to about 99% nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% nucleic acid combined with a pharmaceutically acceptable carrier.

[0657] Nucleic acids can be prepared using carriers that protect the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or commonly known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0658] Dosing regimens can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered, multiple fractionated doses can be administered over time, or the dose can be proportionally reduced or increased according to the specific circumstances of each case and the particular condition of the treatment. When administered to subjects or patients, it is particularly advantageous to formulate the parenteral composition in unit dosage form for ease of administration and dose uniformity. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dose to a subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect. The specifications of the unit dosage form of the present invention depend on the specific characteristics of the active compound and the specific therapeutic or preventive effect to be achieved, as well as the treatment and sensitivity of any individual patient.

[0659] The nucleic acids or compositions of the present invention can be produced using conventional methods in the art, including chemical synthesis, such as solid-phase chemical synthesis.

[0660] The nucleic acids or compositions of the present invention can be administered using one or more of a variety of medical devices known in the art. For example, in one embodiment, the nucleic acids of the present invention can be administered using a needle-free subcutaneous injection device. Examples of implants and modules well known in the art that can be used with the present invention include, for example, implantable microinfusion pumps for controlled-rate delivery; devices for transdermal administration; infusion pumps for delivery at precise infusion rates; variable-flow implantable infusion devices for continuous drug delivery; and permeation drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.

[0661] In some embodiments, the nucleic acids or compositions of the present invention may be formulated to ensure desired distribution in vivo. To target specific in vivo sites, the therapeutic compounds or compositions of the present invention may be formulated, for example, in liposomes, which may contain one or more portions selectively transported to specific cells or organs, thereby enhancing targeted drug delivery.

[0662] The invention is characterized by high specificity at both the molecular and tissue-targeted delivery levels. The sequences of the nucleic acids of the invention are highly specific to their targets, meaning they do not inhibit the expression of genes they are not designed to target, or only minimally inhibit the expression of genes they are not designed to target, and / or only inhibit the expression of a small number of genes they are not designed to target. Further levels of specificity are achieved when the nucleic acids are linked to ligands that are specifically recognized and internalized by a particular cell type. This is, for example, when the nucleic acids are linked to ligands containing a GalNAc moiety, which is specifically recognized and internalized by hepatocytes. This results in the nucleic acids inhibiting the expression of their targets only in cells targeted by the ligand to which they are linked. These two levels of specificity potentially confer a better safety profile than existing available treatments. In some embodiments, the invention therefore provides additional specificity for target gene knockdown via RNA interference by linking to nucleic acids of the invention containing one or more GalNAc moieties or containing one or more other moieties that confer cell type or tissue-specific internalization.

[0663] The nucleic acids described herein can be formulated into liposomes in combination with lipids. Such formulations are described in the art as lipid complexes. Lipid / liposome compositions can be used to aid in the delivery of the nucleic acids of the present invention to target cells. The lipid delivery systems described herein can be used as alternatives to conjugated ligands. When the nucleic acids of the present invention are used via lipid delivery systems or ligand conjugated delivery systems, the modifications described herein may be present.

[0664] Such lipid complexes may comprise lipid compositions comprising:

[0665] i) Cationic lipids or their pharmaceutically acceptable salts;

[0666] ii) Steroids;

[0667] iii) Phosphatidylethanolamine phospholipids; and / or

[0668] iv) PEGylated lipids.

[0669] Cationic lipids can be amino cationic lipids.

[0670] Cationic lipids may have formula (XII):

[0671]

[0672] Or its pharmaceutically acceptable salt, wherein:

[0673] X represents O, S, or NH;

[0674] R 1 and R 2 Each independently represents C4-C22 Straight-chain or branched alkyl chains or C4-C chains with one or more double bonds 22 Straight-chain or branched alkenyl chains, wherein the alkyl or alkenyl chains optionally contain intercalated ester, amide or disulfide bonds;

[0675] When X represents S or NH, R 3 and R 4 Each independently represents a hydrogen, methyl, ethyl, monoamine, or polyamine moiety, or R 3 and R 4 Together they form a heterocyclic base ring;

[0676] When X represents O, R 3 and R 4 Each independently represents a hydrogen, methyl, ethyl, monoamine, or polyamine moiety, or R 3 and R 4 Together they form a heterocyclic base ring, or R 3 Represents hydrogen and R 4 It represents C(NH)(NH2).

[0677] Cationic lipids may have formula (XIII):

[0678]

[0679] Or its pharmaceutically acceptable salt.

[0680] Cationic lipids can have formula (XIV):

[0681]

[0682] Or its pharmaceutically acceptable salt.

[0683] The content of the cationic lipid component can be from about 55 mol% to about 65 mol% of the total lipid content of the composition. In particular, the cationic lipid component is about 59 mol% of the total lipid content of the composition.

[0684] The composition may further comprise steroids. The steroids may be cholesterol. The steroid content may be from about 26 mol% to about 35 mol% of the total lipid content of the lipid composition. More specifically, the steroid content may be from about 30 mol% of the total lipid content of the lipid composition.

[0685] Phosphatidylethanolamine phospholipids can be selected from the group consisting of: 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1,2-dipalmitoyl The phospholipids are: 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate ethanolamine (POPE), 1,2-disqualoyl-sn-glycero-3-phosphate ethanolamine (DEPE), 1,2-disqualyl-sn-glycero-3-phosphate ethanolamine (DSQPE), and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate ethanolamine (SLPE). The phospholipid content can be approximately 10 mol% of the total lipid content of the composition.

[0686] The PEGylated lipids can be selected from the group consisting of 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol (DMG-PEG), and C16-ceramide-PEG. The content of the PEGylated lipids can be about 1 to 5 mol% of the total lipid content of the composition.

[0687] The content of the cationic lipid component in the composition can be from about 55 mol% to about 65 mol% of the total lipid content of the lipid composition, preferably about 59 mol% of the total lipid content of the lipid composition.

[0688] The composition may have component molar ratios selected from the following i):ii):iii):iv):55:34:10:1; 56:33:10:1; 57:32:10:1; 58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1; 64:25:10:1 and 65:24:10:1.

[0689] The composition may contain cationic lipids having the following structures:

[0690]

[0691] Steroids with the following structures:

[0692]

[0693] Phosphatidylethanolamine phospholipids have the following structure:

[0694]

[0695] And PEGylated lipids with the following structures:

[0696]

[0697] Neutral liposome compositions may be formed from, for example, myristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions may be formed from myristoyl phosphatidylglycerol, while anionic fused liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition may be formed from phosphatidylcholine (PC) such as, for example, soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0698] The positively charged synthetic cationic lipid N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that spontaneously interact with nucleic acids, forming lipid-nucleic acid complexes capable of fusing with negatively charged lipids to the cell membranes of tissue culture cells. DOTMA analogues can also be used to form liposomes.

[0699] The lipid derivatives and analogs described in this article can also be used to form liposomes.

[0700] Nucleic acid-containing liposomes can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent, thereby forming micelles with the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The nucleic acid product is then added to the micelles containing the lipid component. The cationic groups on the lipids interact with the nucleic acid and condense around the nucleic acid to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the liposome product of the nucleic acid.

[0701] If necessary, a carrier compound to facilitate condensation can be added during the condensation reaction (e.g., through controlled addition). For example, the carrier compound can be a polymer other than nucleic acids (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0702] The nucleic acid formulations of the present invention may contain surfactants. In one embodiment, the nucleic acid is formulated as an emulsion containing a surfactant.

[0703] Nonionic surfactants are surfactants that do not ionize. Examples include nonionic esters, such as ethylene glycol esters, propylene glycol esters, and glycerol esters; nonionic alkanolamides; and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers.

[0704] Surfactants that carry a negative charge when dissolved or dispersed in water are anionic surfactants. Examples include carboxylates such as soaps, acyl lactates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl hydroxyethanesulfonates, acyl taurate and sulfosuccinates, and phosphates.

[0705] Surfactants that carry a positive charge when dissolved or dispersed in water are cationic surfactants. Examples include quaternary ammonium salts and ethoxylated amines.

[0706] Surfactants capable of carrying either a positive or negative charge are amphoteric surfactants. Examples include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0707] "Micelles" are defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules face inwards, while the hydrophilic portions are in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists. Micelles can be formed by mixing an aqueous solution of nucleic acids, an alkali metal alkyl sulfate, and at least one micelle-forming compound.

[0708] Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, glyceryl monooleate, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanoylglycine and its pharmaceutically acceptable salts, glycerol, polyglycerol, lysine, polylysine, trioleic acid, polyoxyethylene ethers and their analogs, polydocalyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof.

[0709] Phenol and / or m-cresol can be added to the mixed micelle composition as stabilizers and preservatives. Isotonic agents such as glycerin can also be added.

[0710] Nucleic acid products can be incorporated into particles such as microparticles. Microparticles can be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these methods.

[0711] definition

[0712] As used herein, the terms “inhibition,” “downregulation,” or “reduction” in relation to gene expression refer to a reduction in the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule (e.g., mRNA) encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, to levels observed in the absence of the nucleic acid of the present invention or its conjugated nucleic acid, or to levels obtained with siRNA molecules that have no known homology to human transcripts (referred to herein as non-silencing controls). Such controls can be conjugated and modified in a manner similar to that of the molecules of the present invention and delivered to target cells via the same pathway. Expression after treatment with the nucleic acid of the present invention can be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 0% or an intermediate value, or to levels observed in the absence of the nucleic acid or its conjugated nucleic acid. Expression can be measured in the cells to which the nucleic acid is applied. Optionally, particularly when the nucleic acid is administered to a subject, levels can be measured in different cell populations or tissues or organs or body fluids such as blood or plasma. Inhibition levels are preferably measured under selected conditions, as these conditions demonstrate the maximum effect of nucleic acids on the levels of target mRNA in cells treated with nucleic acids in vitro. Inhibition levels can be measured, for example, after treatment with nucleic acids at concentrations of 0.038 nM to 10 µM (preferably 0.5 nM, 1 nM, 10 nM, or 100 nM) for 24 or 48 hours. These conditions may vary for different nucleic acid sequences or different types of nucleic acids (such as unmodified or modified, or conjugated to or unconjugated to ligands). Examples of suitable conditions for determining inhibition levels are described in the embodiments.

[0713] Nucleic acids are nucleotides containing two chains of nucleotides that can interfere with gene expression. Repression can be complete or partial and can lead to downregulation of gene expression in a targeted manner. Nucleic acids consist of two separate polynucleotide chains: a first chain (also called the guide chain) and a second chain (also called the follower chain). The first and second chains can be parts of the same polynucleotide molecule, which is self-complementary and folds back to form a double-stranded molecule. Nucleic acids can be siRNA molecules.

[0714] Nucleic acids may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogs that mimic nucleotides to “pair” with corresponding bases on a target sequence or complementary strand. Nucleic acids may further comprise a double-stranded portion or duplex region formed by all or part of a first strand (also referred to in the art as the guide strand) and all or part of a second strand (also referred to in the art as the lagging strand). A duplex region is defined as beginning with the first base pair formed between the first and second strands and ending with the last base pair formed between the first and second strands (including both the first and last base pairs).

[0715] A double-stranded region is a region in which two complementary or substantially complementary oligonucleotides form base pairs with each other through Watson-Crick base pairing or any other means that allows for the formation of a double strand between complementary or substantially complementary oligonucleotide chains. For example, an oligonucleotide chain with 21 nucleotide units can pair with another oligonucleotide chain with 21 nucleotide units, but only 19 nucleotides on each chain are complementary or substantially complementary, making the "double-stranded region" consist of 19 base pairs. The remaining base pairs can exist as 5' and 3' overhangs or as a single-stranded region. Furthermore, 100% complementarity is not required within a double-stranded region; substantially complementary complementarity is permitted.

[0716] Basic complementarity refers to the complementarity between strands that allows them to anneal under biological conditions. Techniques for empirically determining whether two strands can anneal under biological conditions are well known in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine whether they anneal to each other. The portions of the first and second strands forming at least one double-stranded region can be completely complementary and at least partially complementary to each other. Depending on the length of the nucleic acid, a perfect match in base complementarity between the first and second strands is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions.

[0717] As used herein, the term "unpaired nucleotide analog" refers to a nucleotide analog containing a non-base-paired portion, including but not limited to: 6-deaminoadenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribose U, N3-Me ribose T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC. In some embodiments, the unpaired nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

[0718] As used herein, the term "terminal functional group" includes, but is not limited to, halogen, alcohol, amine, carboxylic acid, ester, amide, aldehyde, ketone and ether groups.

[0719] As used herein, "protruding end" has its normal and conventional meaning in the art, referring to the single-stranded portion of a double-stranded nucleic acid that extends beyond the terminal nucleotide of the complementary strand. The term "blunt end" includes double-stranded nucleic acids where both strands terminate at the same position, regardless of whether the terminal nucleotides are base-paired. The terminal nucleotides of the first and second strands at the blunt end may be base-paired. The terminal nucleotides of the first and second strands at the blunt end may not be base-paired. The two terminal nucleotides of the first and second strands at the blunt end may be base-paired. The two terminal nucleotides of the first and second strands at the blunt end may not be base-paired.

[0720] The term "serine alcohol-derived linker moiety" refers to a linker moiety that contains the following structure:

[0721]

[0722] The O atoms in this structure are typically linked to the RNA chain, and the N atoms are typically linked to the target ligand.

[0723] The terms “patient,” “subject,” and “individual” are used interchangeably and refer to human or non-human animals. These terms include mammals such as humans, primates, livestock (e.g., cattle, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0724] As used herein, “treating” or “treatment” and its grammatical variations refer to a method for achieving a beneficial or desired clinical outcome. The term may refer to slowing the onset or progression of a condition, symptom, or disease; reducing or alleviating associated symptoms; producing complete or partial remission of the condition; or any combination of the foregoing. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, reduction or alleviation of symptoms, reduction in disease severity, stabilization of the disease state (i.e., non-deterioration), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also refer to an extension of survival compared to expected survival without treatment. Therefore, a subject requiring treatment (e.g., a person) may be a subject already suffering from the disease or symptom discussed. The term “treatment” includes suppressing or reducing the increase in the severity of a pathological state or symptoms relative to the absence of treatment and does not necessarily imply a complete cessation of the associated disease, symptom, or condition.

[0725] As used herein, the term “prevention” and its grammatical variations refer to methods for suppressing or preventing the development, progression, or onset time or rate of a condition, disease, or symptom, and may involve pathology and / or symptoms. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, prevention, suppression, or mitigation of symptoms, progression, or development of a disease, whether detectable or undetectable. Therefore, a subject requiring prevention (e.g., a person) may be a subject who does not yet have the disease or symptom discussed. The term “prevention” includes mitigating the onset of a disease relative to the absence of treatment and does not necessarily imply permanent prevention of the relevant disease, symptom, or condition. Therefore, in certain contexts, “prevention” of a condition may refer to reducing the risk of developing the condition, or preventing, suppressing, or delaying the development of symptoms associated with the condition. It should be understood that prevention can be considered a treatment or therapy.

[0726] As used herein, “effective amount,” “preventive effective amount,” “therapeutic effective amount,” or “effective dose” is the amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as prevention or treatment of a target condition or beneficial relief of symptoms associated with that condition.

[0727] As used herein, the term "pharmaceutically acceptable salt" means a salt that is harmless to a patient or subject administering the salt in question. It can be selected from, for example, acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrates, and acetates. Examples of basic salts include those in which the cation is selected from alkali metal cations (such as sodium or potassium ions), alkaline earth metal cations (such as calcium or magnesium ions), and substituted ammonium ions (such as N(R) ions). 1 (R) 2 (R) 3 (R) 4 )+ type ions, where R 1 R 2 R 3 and R 4A pharmaceutically acceptable salt is a salt that independently represents hydrogen, an optionally substituted C1-6 alkyl group, or an optionally substituted C2-6 alkenyl group. Examples of relevant C1-6 alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl. Examples of possibly relevant C2-6 alkenyl groups include vinyl, 1-propenyl, and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in Remington's Pharmaceutical Sciences, 17th edition, edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, PA, USA, 1985 (and later editions), Encyclopaedia of Pharmaceutical Technology, 3rd edition, edited by James Swarbrick, Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm.Sci. 66: 2 (1977). A pharmaceutically acceptable salt qualitatively preserves the desired biological activity of the parent compound without producing any undesirable effects associated with the compound. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids (such as hydrochloric acid, nitric acid, phosphorous acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, etc.) or non-toxic organic acids (such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include salts derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.) and non-toxic organic amines (such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0728] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical industry and described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by AR Gennaro, 1985). For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH can be used. Exemplary pH buffers include phosphates, citrates, acetates, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine (which is a preferred buffer), arginine, lysine, or acetates, or mixtures thereof. The term further covers any substance listed in the United States Pharmacopeia for use in animals (including humans). "Pharmaceutical acceptable carrier" includes any and all physiologically acceptable (i.e., compatible) solvents, dispersion media, coatings, antimicrobial agents, isotonic agents, and absorption delay agents, etc. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the chosen route of administration, the nucleic acid may be encapsulated in one or more materials designed to protect the compound from acid and other naturally occurring inactivating conditions that the nucleic acid may be exposed to when administered to a subject via a particular route of administration.

[0729] In the context of this invention, the term "solvent" refers to a complex with a defined stoichiometry formed between a solute (in this case, a nucleic acid compound or a pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in question can be, for example, water or other pharmaceutically acceptable, typically small-molecule organic substances (such as, but not limited to, acetic acid or lactic acid). When the solvent in question is water, such solvates are generally referred to as hydrates.

[0730] The invention will now be described with reference to the following non-limiting drawings and embodiments. Attached Figure Description

[0731] Figure 1 GPR146 mRNA expression in primary cynomolgus monkey hepatocytes was normalized relative to PPIB mRNA after treatment with five different concentrations of GalNAc-siRNA targeting GPR146 at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The expression is shown as a mean relative to untreated (ut). Cells treated with GPR146 nonspecific siRNA served as a control (Ctr).

[0732] Figure 2GPR146 mRNA expression in primary cynomolgus monkey hepatocytes was normalized relative to PPIB mRNA after treatment with five different concentrations of GalNAc-siRNA targeting GPR146 at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The expression is shown as a mean relative to untreated (ut). Cells treated with GPR146 nonspecific siRNA served as a control (Ctr).

[0733] Figure 3 GPR146 mRNA expression in primary human hepatocytes normalized relative to PPIB mRNA after treatment with five different concentrations of GalNAc-siRNA targeting GPR146 at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The expression is shown as the mean relative to untreated (ut). Cells treated with GPR146 nonspecific siRNA served as a control (Ctr).

[0734] Figure 4 GPR146 mRNA expression normalized relative to PPIB mRNA in human liver spheroids at 7 or 14 days after treatment with three different concentrations of GalNAc-siRNA targeting GPR146 at 100 nM, 10 nM, and 1 nM. Mean value relative to untreated (ut). Cells treated with GPR146 nonspecific siRNA served as controls (Ctr).

[0735] Figure 5 GPR146 mRNA expression normalized to ApoB mRNA in liver samples isolated on day 29 after injection of an AAV vector carrying the human GPR146 gene and on day 15 after subcutaneous injection of a GPR146-targeting GalNAc-siRNA conjugate at concentrations of 1 or 5 mg / kg. Mean values ​​are shown relative to PBS-treated controls. Data from both studies are presented.

[0736] Figure 6 GPR146 mRNA expression normalized relative to ApoB mRNA in liver samples isolated at weeks 4 and 8 following subcutaneous injection of a GalNAc-siRNA conjugate targeting GPR146 at a dose level of 3 mg / kg. Mean and standard deviation are shown.

[0737] Figure 7GPR146 mRNA expression normalized relative to PPIB mRNA was measured in liver samples isolated at weeks 4, 8, and 12 following single and multiple subcutaneous injections of GalNAc-conjugated siRNA EM3204 or multiple injections of the mediator. A single dose of 1, 3, or 9 mg / kg was administered on day 1, while three doses of 3 mg / kg were administered on days 1, 8, and 15. Means and standard deviations are shown.

[0738] Example

[0739] Example 1

[0740] In vitro PC3 cell studies demonstrating the GPR146 knockdown efficacy of siRNA tested after transfection with 0.5 nM or 10 nM siRNA.

[0741] The GPR146 knockdown efficacy of siRNAs EM3001-EM3200 (Table 5b) was determined after transfecting PC3 cells with 0.5 or 10 nM siRNA. The results are shown in Table 3 below. At 10 nM, the residual GPR146 level reached a minimum of 42% after knockdown, and at 0.5 nM, the residual GPR146 level reached a minimum of 50%. At 10 nM, the most effective siRNAs were EM3172, EM3175, EM3098, EM3090, and EM3031.

[0742] For PC3 cells transfected with siRNA, cells were seeded at a density of 30,000 cells / well in 96-well tissue culture plates (TPP, catalog number 92096, Switzerland). siRNA transfection was performed directly before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, catalog number 13778-500, Germany) according to the manufacturer's instructions. Dual-dose selection was performed in triplicate with 10 nM and 0.5 nM GPR146-siRNA, with disordered siRNA and luciferase-targeting siRNA serving as non-specific controls. Twenty-four hours after treatment, cells were lysed and RNA was isolated using the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, catalog number 61012, USA). RT-qPCR was performed using GPR146 and PPIB specific primer and probe sets and Takyon. TMOne-Step Low Rox Probe 5X MasterMix dTTP was performed on an Applied Biosystems QuantStudio6 device in single-well 384-well format. Expression differentials were calculated using the ΔΔCt (delta-delta Ct) method, and relative GPR146 expression was determined relative to the housekeeping gene PPIB. Results are presented in Table 3 as the percentage of remaining GPR146 mRNA after siRNA transfection.

[0743] Table 3: Results of dual-dose screening (10 nM and 0.5 nM) of siRNA targeting GPR146.

[0744] The identity, sequence, and modifications of the single strands forming each siRNA double helix can be seen in Tables 5a and 5b.

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751] Example 2

[0752] This study demonstrates the GPR146 knockdown efficacy of siRNA tested after transfection with 20, 4, 0.8, 0.16, 0.032, or 0.006 nM siRNA in in vitro PC3 cells.

[0753] The GPR146 knockdown efficacy of selected siRNAs (Tables 4 and 5b) was determined after transfection of PC3 cells with 20, 4, 0.8, 0.16, 0.032, or 0.006 nM siRNAs. The results are shown in Table 4. At 20 nM, the residual GPR146 level after knockdown reached a minimum of 36%, and at 4 nM, it reached a minimum of 45%. At 20 nM, the most effective siRNAs were EM3086, EM3175, EM3177, and EM3187.

[0754] For PC3 cells transfected with siRNA, cells were seeded at a density of 30,000 cells / well in 96-well tissue culture plates (TPP, catalog number 92096, Switzerland). siRNA transfection was performed directly before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, catalog number 13778-500, Germany) according to the manufacturer's instructions. Dosage-response screening was performed in triplicate with GPR146 siRNA at doses of 20, 4, 0.8, 0.16, 0.032, or 0.006 nM, with disordered siRNA and luciferase-targeting siRNA serving as non-specific controls. Twenty-four hours post-treatment, cells were lysed and RNA was isolated using the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, catalog number 61012, USA). RT-qPCR was performed using GPR146 and PPIB specific primer and probe sets and Takyon. TM One-Step Low Rox Probe5X MasterMix dTTP was performed in a singlet 384-well format on an Applied Biosystems QuantStudio6 device. Expression differentials were calculated using the ΔΔCt method, and relative GPR146 expression was determined relative to the housekeeping gene PPIB. Results are presented in Table 4 as the percentage of remaining GPR146 mRNA after siRNA transfection.

[0755] Table 4: Results of dose-response screening of siRNAs targeting GPR146 (20, 4, 0.8, 0.16, 0.032 or 0.006 nM).

[0756] The identity, sequence, and modifications of the single strands forming each siRNA double helix can be seen in Tables 5a and 5b.

[0757]

[0758]

[0759]

[0760]

[0761]

[0762] Example 3

[0763] In vitro primary cynomolgus hepatocyte study demonstrating the GPR146 knockdown potency of the tested GalNAc-siRNA conjugate.

[0764] GPR146 mRNA expression was assessed after incubation with the GalNAc-siRNA conjugate (further described in Table 5c) at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The mRNA level of the housekeeping gene PPIB served as a control.

[0765] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting GPR146 in primary cynomolgus hepatocytes, 45,000 cells per well (supplier: Life Technologies, USA) were added to siRNA in plating medium (Life Technologies, USA, catalogue A1217601, CM3000, CM4000, USA) to a final concentration of 100 nM to 0.16 nM in collagen-coated 96-well plates (Life Technologies, catalogue A1217601, CM3000, CM4000, USA). Twenty-four hours after treatment, cells were lysed and RNA was isolated using the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, catalogue 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes targeting GPR146 and PPIB. Expression differentials were calculated using the ΔΔCt method, and relative GPR146 expression normalized relative to the housekeeping gene PPIB was determined. The results are expressed as the ratio of GPR146 to PPIB mRNA relative to untreated levels and are visible in [the table / table]. Figure 1 .

[0766] At 100 nM, the residual GPR146 level after knockdown ranged from 38% to 95%. The strongest knockdown was observed in EM3203, EM3208, EM3207 and EM3215 at 100 nM, with residual GPR146 levels of 38%, 46%, 48% and 48%, respectively.

[0767] The results are presented in Table 8 as the percentage of GPR146 mRNA remaining after siRNA transfection.

[0768] Table 8: Results of dose-response screening (100, 20, 4, 0.8 or 0.16 nM) of siRNA targeting GPR146 in primary cynomolgus monkey hepatocytes.

[0769]

[0770]

[0771]

[0772]

[0773]

[0774] Example 4

[0775] In vitro primary cynomolgus hepatocyte study demonstrating the GPR146 knockdown potency of the tested GalNAc-siRNA conjugate.

[0776] GPR146 mRNA expression was assessed after incubation with the GalNAc-siRNA conjugate (further described in Table 5c) at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The mRNA level of the housekeeping gene PPIB served as a control.

[0777] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting GPR146 in primary cynomolgus hepatocytes, 50,000 cells per well (supplier: Primacyt, Germany) were added to siRNA in plating medium (Life Technologies, catalogue A1217601, CM3000, CM4000, USA) in collagen-coated 96-well plates (Life Technologies, catalogue A1217601, CM3000, CM4000, USA) to a final concentration ranging from 100 nM to 0.01 nM. Twenty-four hours after treatment, cells were lysed and RNA was isolated using the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, catalogue 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes targeting GPR146 and PPIB. Expression differentials were calculated using the ΔΔCt method, and relative GPR146 expression normalized relative to the housekeeping gene PPIB was determined. The results are expressed as a ratio of GPR146 to PPIB mRNA relative to untreated levels and are visible in Figure 2 .

[0778] Dose-dependent knockdown of GPR146 mRNA was observed in all GalNAc conjugates tested. At 100 nM, the residual GPR146 levels after knockdown ranged from 40% to 98%. The strongest knockdown was observed in EM3203, EM3207, and EM3210 at 100 nM, with residual GPR146 levels of 40%, 44%, and 44%, respectively.

[0779] The results are expressed in Table 9 as the percentage of GPR146 mRNA remaining after siRNA transfection.

[0780] Table 9: Results of dose-response screening (100, 20, 4, 0.8 or 0.16 nM) of siRNA targeting GPR146 in primary cynomolgus monkey hepatocytes.

[0781]

[0782]

[0783] Example 5

[0784] In vitro primary human hepatocyte studies demonstrating the GPR146 knockdown potency of the tested GalNAc-siRNA conjugates.

[0785] GPR146 mRNA expression was assessed after incubation with the GalNAc-siRNA conjugate (further described in Table 5c) at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. The mRNA level of the housekeeping gene PPIB served as a control.

[0786] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting GPR146 in primary human hepatocytes, 35,000 cells per well (supplier: Life Technologies, USA) were added to siRNA in plating medium (Life Technologies, USA, catalogue A1217601, CM3000, CM4000, USA) in collagen-coated 96-well plates (Life Technologies, catalogue A1217601, CM3000, CM4000, USA) to a final concentration of 100 nM to 0.16 nM. Twenty-four hours after treatment, cells were lysed and RNA was isolated using the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, catalogue 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes targeting GPR146 and PPIB. Expression differentials were calculated using the ΔΔCt method, and relative GPR146 expression normalized relative to the housekeeping gene PPIB was determined. The results are expressed as a ratio of GPR146 to PPIB mRNA relative to untreated levels and are visible in Figure 3 .

[0787] Dose-dependent knockdown of GPR146 mRNA was observed in all GalNAc conjugates tested. At 100 nM, the residual GPR146 levels after knockdown ranged from 44% to 90%. The strongest knockdown was observed in EM3223, EM3210, and EM3207 at 100 nM, with residual GPR146 levels of 44%, 46%, and 47%, respectively.

[0788] The results are presented in Table 10 as the percentage of GPR146 mRNA remaining after siRNA transfection.

[0789] Table 10: Results of dose-response screening (100, 20, 4, 0.8 or 0.16 nM) of siRNA targeting GPR146 in primary human hepatocytes.

[0790]

[0791]

[0792]

[0793]

[0794] Example 6

[0795] In vitro human liver spheroids study demonstrating the GPR146 knockdown potency of the tested GalNAc-siRNA conjugates.

[0796] GPR146 mRNA expression was assessed after incubation with GalNAc-siRNA conjugates EM3202, EM3203, EM3204, EM3207, EM3208, EM3209, EM3210, EM3215, EM3217, or EM3227 (further described in Table 5c) at 100 nM, 10 nM, and 1 nM. The mRNA level of the housekeeping gene PPIB served as a control.

[0797] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting GPR146 in hepatocytes, primary human hepatocytes were cultured in 3D structures. 250 cells per microcavity were seeded into Elplasia plates (Corning, catalog 4442, USA) to form spheroids and cultured in FCS-containing medium before treatment. On day 7, GalNAc-conjugated siRNA targeting GPR146 was added and covered with Matrigel (0.99 mg / ml, Corning, catalog 354234, USA). The medium was changed to FCS-free medium every 2–3 days. Cells were lysed at days 7 and 14 post-treatment, and RNA was isolated using the Dynabeads mRNA DIRECT kit (ThermoFisher Scientific, catalog 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes targeting GPR146 and PPIB. Expression differentials were calculated using the ΔΔCt method, and relative GPR146 expression normalized relative to the housekeeping gene PPIB was determined. Results are expressed as a ratio of GPR146 to PPIB mRNA relative to untreated levels and are visible in Figure 4 .

[0798] All GalNAc conjugates tested exhibited dose-dependent knockdown of GPR146 mRNA. At 100 nM, the residual GPR146 levels after knockdown ranged from 21% to 73% at day 7 post-treatment and from 20% to 60% at day 14 post-treatment. At day 7 post-treatment, the strongest knockdown was observed at 100 nM in EM3207, EM3210, EM3204, and EM3208, with residual GPR146 levels of 21%, 29%, 33%, and 33%, respectively. At day 14 post-treatment, the strongest knockdown was observed at 100 nM in EM3207, EM3210, EM3203, and EM3217, with residual GPR146 levels of 20%, 26%, 28%, and 30%, respectively.

[0799] The results are presented in Table 10 as the percentage of GPR146 mRNA remaining after siRNA transfection.

[0800] Table 10: Results of dose-response screening (100, 10 or 1 nM) of siRNA targeting GPR146 in human hepatocytes at 7 or 14 days after treatment.

[0801]

[0802] Example 7

[0803] This study demonstrates in vivo knockdown of human GPR146 mRNA in mouse liver tissue following AAV-mediated expression and a single subcutaneous administration of 1 or 5 mg / kg GalNAc conjugated siRNA.

[0804] Recombinant AAV particles were purchased from VectorBuilder (Chicago, USA). In short, the sequence encoding human GPR146 (including the adjacent UTR region, NM_001303473.2) was cloned downstream of the apoE / hAAT promoter in the pAAV[Exp] vector. Serotype 8 AAV was packaged in HEK293T cells and purified by PEG precipitation and CsCl gradient ultracentrifugation.

[0805] Animal experiments were conducted at Experimental Pharmacology & Oncology Berlin-Buch GmbH (Berlin, Germany) in accordance with the German Animal Protection Ethics Guidelines, July 2013 edition. AAV was diluted in phosphate-buffered saline (PBS) to 4E11 GC / mL and then administered intravenously (iv) to 8–10 week old male C57BL / 6 mice. All animals were administered at a dose of 2E12 GC / kg. Stable expression of human GPR146 was observed two weeks post-injection, as determined in previous experiments.

[0806] Two weeks after AAV administration, mice were treated with GalNAc-siRNA conjugates EM3203, EM3204, EM3207, EM3208, EM3210, EM3215, EM3217, or EM3227 (further described in Table 5c). Mice were randomly assigned to 5 groups and received a single subcutaneous dose of 1 or 5 mg / kg of siRNA dissolved in PBS, or PBS alone as a control. Viability, body weight, and behavior of mice were monitored during the study period, and no pathological findings were observed.

[0807] The study was terminated 2 weeks after siRNA administration. All animals were euthanized, and liver samples were flash-frozen and stored at -80°C until further analysis. Briefly, total RNA was isolated using the InviTrap Spin Tissue RNA Mini Kit (InvitekMolecular, catalog number 1062100300, Germany) according to the manufacturer's recommendations. To assess the integrity of the isolated RNA, automated electrophoresis was performed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). 100 ng of total RNA per reaction was used for RT-qPCR, using amplicon groups specific to human GPR146 and mouse ApoB (as a housekeeping gene). Expression differences were calculated using the ∆∆Ct method, and the relative expression of hGPR146 relative to ApoB, normalized relative to the PBS group, was used for comparisons between different siRNAs. Data from both studies are presented in […]. Figure 5 At 5 mg / kg, the residual hGPR146 mRNA level after knockdown ranged from 1% to 2%.

[0808] Example 8

[0809] The synthesis of (vp)-mU-phos was performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. The synthesis of phosphorusamide derivatives of ST41 (ST41-phos) and ST23 (ST23-phos) was performed as described in WO2017 / 174657. The synthesis of thiophosphosamides was performed as described in Caruthers, J. Org. Chem. 1996, 61, 4272-4281.

[0810] Example 9

[0811] Example compounds were synthesized according to the methods described below and methods known to those skilled in the art. Assembly of the oligonucleotide chains and linker building blocks was performed using a phosphoramidite method via solid-phase synthesis.

[0812] Downstream pyrolysis, deprotection, and purification were performed according to standard procedures well known in the art.

[0813] Oligonucleotide synthesis was performed on an AKTA oligopilot 10 using a CPG solid support loaded with commercially available 2'O-methyl RNA and 2'fluoro-2' deoxy RNA bases and phosphoramide (all standard protections, ChemGenes, LinkTech).

[0814] Auxiliary reagents were purchased from EMP Biotech and Biosolve. Synthesis was performed using 0.1 M phosphorous acid solution in anhydrous acetonitrile (<20 ppm H₂O), with benzylthiotetrazole (BTT) used as the activator (0.3 M in acetonitrile). Coupling time was 10 min. When introducing dithiophosphate bonds (PS₂) using thiophosphorous acid, repeated coupling-washing cycles exceeding 60 min were performed. Cap / OX / Cap or Cap / Thio / Cap cycles were applied (Cap: Ac₂O / NMI / Lutidine / acetonitrile, oxidant: 0.05 M I₂ in pyridine / H₂O). Thiophosphates and dithiophosphates were introduced using 50 mM EDITH (Link Technologies) in acetonitrile, a commercially available thiolation reagent. DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. After the programmed synthesis cycle, washing with diethylamine (DEA) was performed. All oligonucleotides were synthesized in DMT-off mode.

[0815] The tri-antennae GalNAc cluster (ST23 / ST41) was introduced via successive coupling of a branched trebler phosphoridamide derivative (C4XLT-phos) and a subsequent GalNAc phosphoridamide (ST23-phos). The (vp)-mU moiety was linked by the use of (vp)-mU-phos in the final synthetic cycle. (vp)-mU-phos does not provide hydroxyl groups suitable for further synthetic extension and therefore lacks a DMT group. Thus, coupling with (vp)-mU-phos resulted in the termination of the synthesis.

[0816] To remove the methyl ester masking the vinylphosphonate, CPG carrying the fully assembled oligonucleotides was dried under reduced pressure and transferred to a 20 mL PP syringe reactor equipped with a disc filter (Carl Roth GmbH) for solid-phase peptide synthesis. The CPG was then contacted at room temperature with 250 µL of TMSBr and 177 µL of pyridine in CH2Cl2 (0.5 mL / µmol of oligonucleotide bound to the solid support), and the reactor was sealed with a Luer cap. The reaction vessel was gently agitated for 2 × 15 minutes, excess reagents were discarded, and the residual CPG was washed twice with 10 mL of acetonitrile. Further downstream processing was no different from that of any other example compound.

[0817] Single-stranded oligonucleotides were cleaved from CPG by treatment with 40% aqueous methylamine (20 mM DTT if dithiophosphate bonds are present) at room temperature for 90 min. The resulting crude oligonucleotides were purified by ion-exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Fractions containing the product were combined, desalted on a size exclusion column (Zetadex, EMP Biotech), and lyophilized until further use.

[0818] All final single-chain products were analyzed by AEX-HPLC to verify their purity. The identity of each single-chain product was confirmed by LC-MS analysis.

[0819] Example 10

[0820] Each single strand was dissolved in H₂O at a concentration of 60 OD / ml. The solutions of both individual oligonucleotides were added together to the reaction vessel. Titration was performed for easy reaction monitoring. The first strand was added in 25% excess relative to the second strand, as determined by UV absorption at 260 nm. The reaction mixture was heated to 80 °C and held for 5 minutes, then slowly cooled to room temperature. Double-strand formation was monitored by ion-pairing reversed-phase HPLC. The required amount of the second strand was calculated based on the UV area of ​​the remaining single strand and added to the reaction mixture. The reaction was reheated to 80 °C and slowly cooled to room temperature. This process was repeated until less than 10% of the remaining single strand was detected.

[0821] In vivo studies showing GPR146 mRNA knockdown in non-human primates (NHP)

[0822] Example 11

[0823] The aim of this experiment was to determine the knockdown efficacy of GalNAc-siRNA conjugates targeting GPR146 on liver mRNA in non-human primates (NHP).

[0824] Specially bred cynomolgus macaques (8-19 years old, non-first-time obese males) were assigned to different treatment groups (n=4 per group). On day 1, each group was treated with a single subcutaneous injection of 3 mg GalNAc siRNA / kg body weight, while control animals received the medium subcutaneously in 0.9% saline. Liver biopsies were collected at weeks -7, 4, and 8, flash-frozen, and stored at -80°C until further analysis. Briefly, total RNA was isolated using the MagMAX mirVana Total RNA Isolation Kit (ThermoFisher, catalog number A27828) according to the manufacturer's recommendations. To assess the integrity of the isolated RNA, automated electrophoresis was performed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). 100 ng of total RNA per reaction was used for RT-qPCR, using amplicons specific to GPR146 and mouse ApoB (as a housekeeping gene). Expression differentials were calculated using the ∆∆Ct method, and relative GPR146 expression normalized to the housekeeping gene ApoB was determined. Results are expressed as the percentage of remaining GPR146 mRNA after siRNA treatment, normalized to pre-drug levels for each individual.

[0825] Following single subcutaneous administration of GalNAc-conjugated siRNAs EM3204, EM3208, EM3210, and EM3227 at a dose level of 3 mg / kg, the results showed a decrease in GPR146 mRNA over 8 weeks. Figure 6 As shown in the figure, EM3204 treatment resulted in a mean reduction of 74% in GPR146 mRNA levels in cynomolgus monkey livers at week 4 and a mean reduction of 68% at week 8. EM3208 treatment resulted in a mean reduction of 50% in GPR146 mRNA levels at week 4 and a mean reduction of 37% at week 8. EM3210 treatment resulted in a mean reduction of 35% in GPR146 mRNA levels in cynomolgus monkey livers at week 4 and a mean reduction of 24% at week 8. A single dose of EM3227 resulted in a mean reduction of 44% in GPR146 mRNA levels at week 4 and a mean reduction of 17% at week 8.

[0826] Example 12

[0827] The aim of this study was to determine the dose-dependent knockdown efficacy of GalNAc-siRNA conjugates targeting GPR146 in non-human primates (NHPs) for liver mRNA.

[0828] Specially bred cynomolgus macaques (13-20 years old, non-first-time obese males) were assigned to different treatment groups (n=4 per group). On day 1, animals were treated with a single subcutaneous injection of 1, 3, or 9 mg GalNAc siRNA / kg body weight, or three doses of 3 mg / kg on days 1, 8, and 15, while control animals received the medium, 0.9% saline, subcutaneously on days 1, 8, and 15. Liver biopsies were collected on days -7, 4, 8, and 12, flash-frozen, and stored at -80°C until further analysis. Briefly, total RNA was isolated using the MagMAX mirVana Total RNA Isolation Kit (ThermoFisher, catalog number A27828) according to the manufacturer's recommendations. To assess the integrity of the isolated RNA, automated electrophoresis was performed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). 100 ng of total RNA per reaction was used for RT-qPCR, using amplicons specific to GPR146 and mouse PPIB (as a housekeeping gene). Expression differentials were calculated using the ∆∆Ct method, and relative GPR146 expression normalized relative to the housekeeping gene PPIB was determined. Results are expressed as the percentage of remaining GPR146 mRNA after siRNA treatment, normalized to pre-drug values ​​for each individual.

[0829] The results showed a decrease in GPR146 mRNA within 12 weeks after administration of a subcutaneous dose of GalNAc-conjugated siRNA EM3204. Figure 7 As shown in the figure, in monkeys treated with a single dose of 1 mg / kg, GPR146 mRNA levels decreased by an average of 43% after week 4, 29% after week 8, and 26% after week 12. A single dose of 3 mg / kg reduced GPR146 mRNA levels by 68% after week 4, 49% after week 8, and 47% after week 12. A single dose of 9 mg / kg reduced GPR146 mRNA levels by 80% after week 4, 74% after week 8, and 63% after week 12. Three doses of EM3204 administered at a dose level of 3 mg / kg on days 1, 8, and 15 reduced GPR146 mRNA levels by 84% at week 4, 74% at week 8, and 70% at week 12.

[0830] Summary of Abbreviations – Table 4

[0831]

[0832]

[0833]

[0834] The abbreviations listed in the above abbreviation table may be used in this article. This list of abbreviations is not exhaustive, and other abbreviations and their meanings can be found throughout this article.

[0835] Summary sequence list

[0836] Table 5a – Unmodified bistrands

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847] The bistrands listed in Table 5b have the various modifications shown, and the explanations of the abbreviations used are given in Table 4. Where appropriate, the sequences of the corresponding unmodified strands in Table 5a are also indicated.

[0848] Table 5b – Modified bichains

[0849]

[0850]

[0851]

[0852]

[0853]

[0854]

[0855]

[0856]

[0857]

[0858]

[0859]

[0860]

[0861]

[0862]

[0863]

[0864]

[0865] The conjoined bichains listed in Table 5c have the various modifications shown, and the explanations of the abbreviations used are given in Table 4. Where appropriate, the sequences of the corresponding unmodified chains in Table 5a are also indicated.

[0866] Table 5c – Modified GalNAc conjoined bichains

[0867]

[0868]

[0869]

[0870]

[0871] SEQ ID No. 861

[0872] > NM_001303473.2 Homo sapiens G protein-coupled receptor 146 (GPR146), transcript variant 3, mRNA

[0873]

Claims

1. A double-stranded nucleic acid for inhibiting GPR146 expression, wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified corresponding sequence of the first strand sequence comprises a sequence of at least 15 nucleotides, said sequence differing from any of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2 or Table 5c by no more than 3 nucleotides.

2. The nucleic acid according to claim 1, wherein the first strand and the second strand are separate strands, each having a length of 18-25 nucleotides.

3. The nucleic acid according to any one of the preceding claims, wherein the first strand and the second strand form a double-stranded region of 17-25 nucleotides in length.

4. The nucleic acid according to any one of the preceding claims, wherein the first strand and the second strand form a double-stranded region of 19 nucleotides in length.

5. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid: a) Both ends are flat. b) The first chain has a protruding end at the 5' end and a flat end at the 3' end; c) The first chain has a protruding end at the 3' end and a flat end at the 5' end; d) The second chain has a protruding end at the 5' end and a flat end at the 3' end; e) The second chain has a protruding end at the 3' end and a flat end at the 5' end; f) Having protruding ends at the 5' end and the 3' end of the first chain; or g) There are protruding ends at the 5' end of the second chain and the 3' end of the second chain.

6. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is siRNA.

7. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid mediates RNA interference.

8. The nucleic acid according to any one of the preceding claims, wherein: (a) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any of the first strand sequences shown in Table 5a or Table 1 by no more than 3 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence shown in Table 5a or Table 1 by no more than 3 nucleotides. (b) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any of the first strand sequences shown in Table 5a or Table 1 by no more than 2 nucleotides, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence shown in Table 5a or Table 1 by no more than 2 nucleotides. (c) The unmodified corresponding sequence of the first strand sequence comprises a sequence that differs from any of the first strand sequences shown in Table 5a or Table 1 by no more than 1 nucleotide, and optionally the unmodified corresponding sequence of the second strand sequence comprises a sequence that differs from the corresponding second strand sequence shown in Table 5a or Table 1 by no more than 1 nucleotide. (d) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a or Table 1; (e) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a or Table 1; (f) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a or Table 1; (g) The unmodified corresponding sequence of the first strand sequence comprises the sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises the sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a or Table 1; (h) The unmodified corresponding sequence of the first chain sequence comprises a sequence of any first chain sequence having a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second chain sequence comprises a sequence of the corresponding second chain sequence having a given SEQ ID No. shown in Table 5a or Table 1; or (i) The unmodified corresponding sequence of the first chain sequence consists substantially of any of the first chain sequences with a given SEQ ID No. shown in Table 5a or Table 1, and optionally wherein the unmodified corresponding sequence of the second chain sequence consists substantially of the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a or Table 1. (j) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises or is substantially composed of the sequence of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5a or Table 1; (k) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein the first to 19 nucleotides of the unmodified corresponding sequence of the first strand sequence from the 5' end correspond to the first to 19 nucleotides from the 5' end of any first strand sequence with a given SEQ ID No. shown in Table 5a or Table 1, and wherein the unmodified corresponding sequence of the first strand sequence further has one (the 20th nucleotide from the 5' end), two (the 20th and 21st nucleotides), three (the 20th, 21st, and 22nd nucleotides), four (the 20th, 21st, 22nd, and 23rd nucleotides), five (the 20th, 21st, 22nd, 23rd, and 24th nucleotides), or six (the 20th, 21st, 22nd, 23rd, 24th, and 25th nucleotides) additional nucleotides at the 3' end of any first strand sequence with a given SEQ ID No. shown in Table 5a or Table 1. Optionally, the unmodified corresponding sequence of the second chain sequence comprises or is substantially composed of the corresponding second chain sequence having a given SEQ ID No. shown in Table 5a or Table 1; (l) The unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5a or Table 1, wherein the unmodified corresponding sequence of the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and wherein the unmodified corresponding sequence of the first strand sequence comprises a sequence corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally, the unmodified corresponding sequence of the second strand sequence comprises or consists substantially of the corresponding second strand sequence with a given SEQ ID No. shown in Table 5a or Table 1; (m) The unmodified corresponding sequence of the first strand sequence consists of a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein the first to 19 nucleotides of the unmodified corresponding sequence of the first strand sequence from the 5' end correspond to the first to 19 nucleotides of any first strand sequence with a given SEQ ID No. shown in Table 5a or Table 1, and wherein the unmodified corresponding sequence of the first strand sequence further comprises a sequence with a given SEQ ID No. shown in Table 5a or Table 1. The 3' end of any first strand sequence of SEQ ID NO. contains one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides, wherein the unmodified corresponding sequence of the first strand sequence comprises a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID NO. 861, substantially consisting of or consisting of said continuous region; and optionally wherein the unmodified corresponding sequence of the second strand sequence comprises or substantially consists of the sequence of the corresponding second strand sequence of a given SEQ ID NO. shown in Table 5a or Table 1; (n) The unmodified corresponding sequence of the first chain sequence consists of any first chain sequence with a given SEQ ID No. shown in Table 5a or Table 1, and optionally the unmodified corresponding sequence of the second chain sequence consists of the sequence of the corresponding second chain sequence with a given SEQ ID No. shown in Table 5a or Table 1; (o) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) exist on a single strand, wherein the unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand are capable of hybridization and thereby forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; or (p) The unmodified corresponding sequence of the first strand and the unmodified corresponding sequence of the second strand of any of the above segments (a) to (n) are on two separate strands that are capable of hybridization and thus form a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

9. The nucleic acid according to any one of the preceding claims, wherein at least one nucleotide of the first strand and / or the second strand is a modified nucleotide, preferably wherein the modified nucleotide is a non-naturally occurring nucleotide, such as a 2'-F modified nucleotide.

10. The nucleic acid according to any one of the preceding claims, wherein at least the 2nd and 14th nucleotides of the first strand are modified by a first modification, and the nucleotides are numbered consecutively starting from the 1st nucleotide at the 5' end of the first strand sequence.

11. The nucleic acid according to any one of the preceding claims, wherein each even-numbered nucleotide of the first strand is modified by a first modification, the nucleotides being numbered consecutively starting from the first nucleotide at the 5' end of the first strand sequence.

12. The nucleic acid according to any one of the preceding claims, wherein the odd-numbered nucleotides of the first strand are modified by a second modification, wherein the second modification is different from the first modification.

13. The nucleic acid according to any one of the preceding claims, wherein the nucleotides in the second strand corresponding to the even-numbered nucleotides of the first strand are modified by a third modification, wherein the third modification is different from the first modification.

14. The nucleic acid according to any one of the preceding claims, wherein the nucleotides in the second strand corresponding to the odd-numbered nucleotides in the first strand are modified by a fourth modification, wherein, when a second modification and / or a third modification are present, the fourth modification is different from the second modification and different from the third modification.

15. The nucleic acid according to any one of claims 1 to 12, wherein the nucleotide in the second chain corresponding to the 11th or 13th nucleotide or the 11th and 13th nucleotides or the 11th-13th nucleotides of the first chain is modified by a fourth modification, and preferably wherein the nucleotide in the second chain not modified by the fourth modification is modified by a third modification.

16. The nucleic acid according to any one of the preceding claims, wherein if the first modification and the fourth modification are present in the nucleic acid, the first modification and the fourth modification are the same, and preferably wherein if the second modification and the third modification are present in the nucleic acid, the second modification and the third modification are the same.

17. The nucleic acid according to any one of the preceding claims, wherein the first modification is a 2'-F modification; the second modification, if present in the nucleic acid, is preferably a 2'-OMe modification; the third modification, if present in the nucleic acid, is preferably a 2'-OMe modification; and the fourth modification, if present in the nucleic acid, is preferably a 2'-F modification.

18. The nucleic acid according to any one of the preceding claims, wherein each nucleotide of the first strand and the second strand is a modified nucleotide.

19. The nucleic acid according to any one of the preceding claims, wherein the first chain has a terminal 5'-(E)-vinylphosphonate nucleotide at its 5' end, and optionally wherein the terminal 5'-(E)-vinylphosphonate nucleotide is preferably linked to a second nucleotide in the first chain via a phosphodiester bond.

20. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises a phosphate thioester bond between two or three 3' nucleotides and / or 5' nucleotides at the end of the first and / or second strand, and optionally wherein the bond between the remaining nucleotides is a phosphodiester bond.

21. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises a phosphate thioester bond between each of the three 3' nucleotides at the end of the first strand.

22. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises a phosphate thioester bond between each of the three 3' nucleotides at the end of the second strand.

23. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises dithiophosphate bonds between each of the two, three, or four terminal nucleotides at the 3' end of the first strand, and / or dithiophosphate bonds between each of the two, three, or four terminal nucleotides at the 3' end of the second strand, and / or dithiophosphate bonds between each of the two, three, or four terminal nucleotides at the 5' end of the second strand, and comprises bonds other than dithiophosphate bonds between the two, three, or four terminal nucleotides at the 5' end of the first strand.

24. The nucleic acid according to any one of claims 1-20, wherein the nucleic acid comprises phosphate thioester bonds between each of the two, three, or four terminal nucleotides at the 3' end of the first strand and / or between each of the two, three, or four terminal nucleotides at the 5' end of the first strand, and / or between each of the two, three, or four terminal nucleotides at the 3' end of the second strand, and / or between each of the two, three, or four terminal nucleotides at the 5' end of the second strand.

25. The nucleic acid according to claim 20 or claim 24, wherein all bonds between the nucleotides of the two chains are phosphodiester bonds, except for the bond between the two terminal nucleotides at the 3' end of the first chain and the bond between the two terminal nucleotides at the 3' and 5' ends of the second chain.

26. The nucleic acid according to any one of the preceding claims, wherein: (a) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than 3 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than 3 nucleotides; (b) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than 2 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than 2 nucleotides; (c) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 5b by no more than one nucleotide, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 5b by no more than one nucleotide; (d) The first strand sequence comprises the sequence of nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b; (e) The first strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b; (f) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b; (g) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and optionally the second strand sequence comprises the sequence of nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b; (h) The first chain sequence comprises any of the first chain sequences shown in Table 5b having a given SEQ ID No., and optionally the second chain sequence comprises the corresponding second chain sequence shown in Table 5b having a given SEQ ID No.; (i) The first chain sequence consists essentially of any of the first chain sequences shown in Table 5b having a given SEQ ID No., and optionally the second chain sequence consists essentially of the sequence of the corresponding second chain sequence shown in Table 5b having a given SEQ ID No.; (j) The first strand sequence comprises a sequence corresponding to nucleotides 1 through 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b; and optionally wherein the second strand sequence comprises or is substantially composed of the sequence of the corresponding second strand sequence having a given SEQ ID No. shown in Table 5b; (k) The first strand sequence comprises a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b. Optionally, the second chain sequence comprises or consists substantially of the corresponding second chain sequence having a given SEQ ID No. shown in Table 5b; (l) The first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and wherein the first strand sequence comprises a sequence corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 5b having the corresponding second strand sequence with a given SEQ ID No. (m) The first strand sequence comprises a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 5b, and wherein the first strand sequence contains nucleotides corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 5b having the corresponding second strand sequence with a given SEQ ID No. (n) The first chain sequence consists of any first chain sequence shown in Table 5b having a given SEQ ID No., and optionally the second chain sequence consists of the sequence of the corresponding second chain sequence shown in Table 5b having a given SEQ ID No.; (o) The first and second strands of any of the above segments (a) to (n) exist on a single strand, wherein the first and second strands are capable of hybridization and thereby forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or (p) The first and second strands of any of the above segments (a) to (n) are on two separate strands that are capable of hybridization and thus forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

27. The nucleic acid according to any one of the preceding claims, wherein: (a) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than 3 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than 3 nucleotides; (b) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than 2 nucleotides, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than 2 nucleotides; (c) The first strand sequence comprises a sequence that differs from any first strand sequence in Table 2 by no more than one nucleotide, and optionally the second strand sequence comprises a sequence that differs from the corresponding second strand sequence in Table 2 by no more than one nucleotide; (d) The first strand sequence comprises the sequence of nucleotides 2 to 17 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 3 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2; (e) The first strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2; (f) The first strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally the second strand sequence comprises the sequence of nucleotides 2 to 19 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2; (g) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID No. shown in Table 2; (h) The first chain sequence comprises any of the first chain sequences shown in Table 2 having a given SEQ ID No., and optionally the second chain sequence comprises the corresponding second chain sequence shown in Table 2 having a given SEQ ID No.; (i) The first chain sequence consists substantially of any of the first chain sequences shown in Table 2 having a given SEQ ID No., and optionally the second chain sequence consists substantially of the sequence of the corresponding second chain sequence shown in Table 2 having a given SEQ ID No.; (j) The first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. in Table 2; and optionally wherein the second strand sequence comprises or is substantially composed of the sequence corresponding to the second strand sequence having a given SEQ ID No. in Table 2; (k) The first strand sequence comprises a sequence of 25, 24, 23, 22, 21, or 20 nucleotides in length, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence shown in Table 2 with a given SEQ ID No., and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence shown in Table 2 with a given SEQ ID No., and optionally wherein the second strand sequence comprises nucleotides shown in Table 2 with a given SEQ ID No. The sequence corresponding to the second chain sequence of No. is composed of or substantially composed of the sequence described above; (l) The first strand sequence comprises nucleotides 1 to 19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No.; and wherein the first strand sequence comprises a continuous region of 17-25 nucleotides, preferably 18-24 nucleotides, complementary to the GPR146 transcript of SEQ ID No. 861, substantially consisting of said continuous region or consisting of said continuous region; and optionally wherein the second strand sequence comprises a sequence having a given SEQ ID No. shown in Table 2. The sequence of the second chain sequence corresponding to ID No. or substantially composed of the sequence; (m) The first strand sequence comprises a sequence of length 25, 24, 23, 22, 21, or 20 nucleotides, wherein nucleotides 1-19 from the 5' end of the first strand sequence correspond to nucleotides 1-19 from the 5' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and wherein the first strand sequence further comprises one (nucleotide 20 from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any first strand sequence having a given SEQ ID No. shown in Table 2, and wherein the first strand sequence contains nucleotides corresponding to SEQ ID No. The GPR146 transcript of 861 is complementary to a continuous region of 17-25 nucleotides in length, preferably 18-24 nucleotides in length, substantially consisting of or consisting of said continuous region; and optionally wherein the second strand sequence comprises or consists substantially of the sequence shown in Table 2 having the corresponding second strand sequence with a given SEQ ID No. (n) The first chain sequence consists of any first chain sequence shown in Table 2 having a given SEQ ID No., and optionally the second chain sequence consists of the sequence of the corresponding second chain sequence shown in Table 2 having a given SEQ ID No.; (o) The first and second strands of any of the above segments (a) to (n) exist on a single strand, wherein the first and second strands are capable of hybridization and thereby forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; or (p) The first and second strands of any of the above segments (a) to (n) are on two separate strands that are capable of hybridization and thus forming a double-stranded nucleic acid with a double-stranded region length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

28. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is conjugated to a heterologous portion.

29. The nucleic acid of claim 28, wherein the heterologous portion comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker wherein the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid.

30. The nucleic acid according to claim 28 or claim 29, wherein the nucleic acid is conjugated to a heterologous portion of a compound comprising formula (II): in: S represents a functional component, such as a ligand, like a sugar, preferably said sugar being N-acetylgalactosamine; X 1 Represents C3-C6 alkylene groups or (-CH2-CH2-O). m (-CH2)2-, where m is 1, 2 or 3; P is a phosphate ester or a modified phosphate ester, preferably a thiophosphate ester; X 2 It is an alkylene group or of the formula (-CH2). n -O-CH2- alkylene ethers, where n=1-6; A is a branch unit; X 3 Represents a bridge-connecting unit; The nucleic acid defined in any one of claims 1 to 27 is conjugated to X by a phosphate ester or a modified phosphate ester, preferably a thiophosphate ester. 3 .

31. The nucleic acid according to any one of claims 28 to 30, wherein the first chain of the nucleic acid is a compound of formula (V): Where b is 0 or 1; and The second chain is a compound of formula (VI): in: c and d are independently 0 or 1; Z1 and Z2 are the first and second strands of the nucleic acid, respectively. Y can be either O or S independently; n is independently 0, 1, 2, or 3; and L1 is the linker that connects the ligand, where L1 is the same or different in equations (V) and (VI), and is the same or different in equations (V) and (VI) when L1 appears more than once in the same equation. And b+c+d is 2 or 3.

32. The nucleic acid according to any one of claims 28 to 31, wherein the nucleic acid is one of the duplexes shown in Table 5c having a given duplex ID No.

33. A composition comprising the nucleic acid of any one of the preceding claims, and a solvent and / or delivery medium and / or a physiologically acceptable excipient and / or carrier and / or salt and / or diluent and / or buffer and / or preservative.

34. The composition of claim 33, wherein the composition comprises the nucleic acid and the therapeutic agent as described in any of the preceding claims, the therapeutic agent being selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

35. The nucleic acid of any one of the preceding claims or the composition of claim 33 or 34, used as a therapeutic agent.

36. The nucleic acid of any one of claims 1 to 32 or the composition of claim 33 or 34, for use in the prevention or treatment of a disease, symptom or syndrome.

37. The nucleic acid or composition for use according to claim 36, wherein the disease, condition or syndrome is a GPR146-mediated disease, condition or syndrome.

38. The nucleic acid or composition for use according to claim 36 or 37, wherein the disease, symptom or syndrome is associated with abnormal or excessive activation of GPR146 and / or overexpression, ectopic expression or localization or accumulation of GPR146.

39. The nucleic acid or composition for use according to any one of claims 36 to 38, wherein the disease, condition, or syndrome is selected from: Hyperlipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), autosomal recessive hypercholesterolemia, atherosclerosis and / or atherosclerotic cardiovascular disease, aortic stenosis, ischemic stroke, ischemic heart disease / coronary artery disease, peripheral artery disease, pulmonary hypertension, diabetes, diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy.

40. Use of the nucleic acid of any one of claims 1 to 32 or the composition of claim 33 or 34 in the preparation of a medicament for the prevention or treatment of a disease, symptom or syndrome.

41. A method for preventing or treating a disease, condition, or syndrome, comprising administering to an individual requiring treatment a pharmaceutically effective dose of any one of claims 1 to 32 of a nucleic acid or any one of claims 33 or 34 of a composition, optionally wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously, or orally, rectally, or intraperitoneally.

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