Antisense oligomers targeting GFRAL and uses thereof

Abstract

The present invention relates to a GDNF family receptor alpha like (GFRAL)-specific antisense oligomer targeting the GFRAL gene and a pharmaceutical composition containing the same for preventing or treating obesity, diabetes, anorexia, or cachexia. It has been confirmed that the antisense oligomer according to the present invention effectively suppresses GFRAL expression in vivo and shows ameliorative effects in an animal model of cancer cachexia. Therefore, it is expected to be highly applicable as a therapeutic agent for obesity or cachexia through the suppression of GFRAL expression.

Description

[Technical Field]

[0001] The present invention relates to a GFRAL (GDNF family receptor alpha like)-specific antisense oligomer that targets the GFRAL gene and uses thereof, and more particularly to a GFRAL-specific antisense oligomer containing a specific base sequence and a pharmaceutical composition containing the same for preventing or treating diseases associated with GFRAL activation due to increased GDF15. [Background technology]

[0002] Cachexia is a complex metabolic disorder characterized by muscle loss, often accompanied by adipose tissue loss, and is an irreversible disease state that cannot be reversed even with adequate calorie replacement. Cachexia is accompanied by anorexia, weight loss, adipose tissue and muscle loss, and depression, resulting in a poor quality of life and reduced survival rates. Cachexia is commonly caused by chronic diseases (e.g., chronic respiratory disease, heart failure, renal failure, chronic infection) and cancer. Treatment of cachexia caused by chronic diseases, including cancer, involves correcting the underlying cause and is not reversed by the administration of appetite-stimulating drugs, exercise, or anti-inflammatory drugs (Baracos et al., Nat Rev Dis Primers, Jan 18;4:17105, 2018). Therefore, cachexia remains an unmet medical need, with no treatments available to slow its progression and reverse its progression.

[0003] Cachexia, which is particularly prevalent in cancer patients, can occur in all types of cancer, including lung cancer, pancreatic cancer, and gastroesophageal cancer (Baracos et al., Nat Rev Dis Primers, Jan 18;4:17105, 2018). Cachexia occurs in more than 50% of cancer patients, and in 20% of cancer patients, cachexia is the direct cause of death. The diagnostic criteria for cachexia in cancer patients are a weight loss of more than 5% for 6 months or a BMI of 20 kg / m 2 It is defined as a weight loss of 2% or more in patients with sarcopenia or a weight loss of 2% or more in patients with sarcopenia (Feaonetal et al., Lancet Oncol, May;12(5):489-95, 2011).

[0004] GDF15 (Growth Differentiating Factor-15) is elevated in the blood of patients with cachexia-associated chronic diseases (e.g., chronic respiratory disease, heart failure, renal failure, chronic infections) and cancer, and is involved in the development of cachexia. While normal individuals exhibit low serum concentrations of GDF15 (0.2-1.2 ng / dL), in patients with chronic diseases and cancer, GDF15 levels are elevated by 10- or even 1,000-fold or more, inducing symptoms of cachexia such as appetite suppression, nausea, vomiting, and fat and muscle loss. Therefore, serum GDF15 levels are diagnostically useful because they increase with the severity of cachexia-inducing diseases (Breit et al., Annu Rev Physiol. 2021 Feb 10;83:127-151). Because GDF15, which increases in cancer, is directly related to the development of cancer cachexia, a neutralizing antibody that inhibits GDF15 activity showed an ameliorative effect on cachexia in a mouse model of cancer cachexia by alleviating weight gain and loss of muscle and adipose tissue (Lerner et al., Journal of cachexia, sarcopenia and muscle 2016;7:467-482).

[0005] The receptor for GDF15 is GDNF family receptor alpha like (GFRAL), which is specifically expressed in the AP (Area Postrema) and NTS (Nucleus Tractus Solitaris) regions of the hindbrain. Activation of the GFRAL receptor by GDF15 induces cancer cachexia (Mullican et al., Nature 2017; Hsu et al., Nature 2017). After binding to GFRAL, GDF15 activates GFRAL neurons through Ret signaling, leading to appetite suppression and weight loss (Goldman A et al., Nature 2017).

[0006] Research into the signal transduction process of the GDF15 / GFRAL / Ret complex has been attracting attention as a way to alleviate and suppress the symptoms of cancer cachexia. GDF15 dimers bind to domain 2 of GFRAL and activate cell signaling through the CLD1, 2, and 3 binding sites of Ret. Ret is known to phosphorylate and activate downstream signaling pathways, such as PI3K / AKT, PLC / PKC, and MEK / ERK (Mullican et al., Trends Endocrinol Metab. 2018 Aug;29(8):560-570).

[0007] The development of effective treatments for correcting the causes of cachexia in cancer patients remains a challenge, and the development of effective treatments is urgently needed. Therefore, the present inventors investigated the role and effect of antisense oligonucleotides against GFRAL in the hindbrain, leading to the completion of the present invention.

[0008] The above information provided in this Background section is merely intended to enhance understanding of the background of the present invention, and therefore may not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an oligomer that inhibits the expression of the human GFRAL (GDNF family receptor alpha like) gene.

[0010] Another object of the present invention is to provide a pharmaceutical composition and method for preventing or treating diseases associated with activation of GFRAL due to increased GDF15, comprising the above oligomer.

[0011] Yet another object of the present invention is to provide a use of the above oligomer for the prevention or treatment of diseases associated with GFRAL activation due to increased GDF15, and a use of the above oligomer for the manufacture of a medicament for the prevention or treatment of diseases associated with GFRAL activation due to increased GDF15. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides an oligomer having a length of 13 to 35 nt that can hybridize with at least 13 consecutive nucleic acid bases in the entire pre-mRNA of the human GFRAL (GDNF family receptor alpha like) gene represented by SEQ ID NO: 1 through Watson-Crick pairing A:T or G:C or wobble pairing G:U, I:A, I:C, or I:U.

[0013] The present invention also provides a pharmaceutical composition containing the above oligomer for preventing or treating a disease associated with activation of GFRAL due to increased GDF15.

[0014] The present invention also provides a method for preventing or treating diseases associated with activation of GFRAL due to increased GDF15, using the oligomer.

[0015] The present invention also provides use of the above oligomer for the prevention or treatment of diseases associated with GFRAL activation due to increased GDF15, and use of the above oligomer for the manufacture of a medicament for the prevention or treatment of diseases associated with GFRAL activation due to increased GDF15. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the results of a test to confirm the possibility of suppressing GFRAL mRNA expression when HEK293 GFRAL and RET cDNA overexpressing cells are transfected with antisense oligonucleotide candidates according to the present invention at a concentration of 200 nM for 24 hours. [Figure 2] Figure 2a is a graph showing the results of a test to confirm the ability of 16 antisense oligonucleotide candidates according to the present invention to suppress GFRAL mRNA expression when transfected into HEK293 GFRAL and RET cDNA-overexpressing cells at a concentration of 100 nM for 24 hours, and Figure 2b shows the results of a test to confirm the ability of GFRAL protein expression when transfected into HEK293 GFRAL and RET cDNA-overexpressing cells for 48 hours. [Figure 3] Figure 3a is a graph showing the results of a test to confirm the ability of 8 antisense oligonucleotide candidates according to the present invention to suppress GFRAL mRNA expression when transfected into HEK293 GFRAL and RET cDNA-overexpressing cells at a concentration of 50 nM for 24 hours, and Figure 3b is a graph showing the results of a test to confirm cytotoxicity when 8 antisense oligonucleotide candidates according to the present invention to HEK293 GFRAL and RET cDNA-overexpressing cells when transfected into HEK293 GFRAL and RET cDNA-overexpressing cells at a concentration of 50 nM for 48 hours. [Figure 4] Figure 4a shows the results of a test to confirm the ability of antisense oligonucleotide candidates according to the present invention to suppress GFRAL protein expression when transfected into HEK293 GFRAL and RET cDNA-overexpressing cells at a concentration of 50 nM for 48 hours, and Figure 4b is a graph quantitating the GFRAL protein expression levels in Figure 4a. [Figure 5] Figure 5a is a schematic diagram of the in vivo toxicity confirmation test of antisense oligonucleotide candidates according to the present invention, and Figure 5b shows the survival rates up to 7 days after administration of the negative and positive toxicity control groups and four antisense oligonucleotide candidates to experimental animals. [Figure 6] This figure shows the results of staining GFRAL protein expression using purified antibodies one week after in vivo administration of antisense oligonucleotide candidates according to the present invention. The arrows in the figure for each administration group indicate GFRAL-positive neurons. Fluorescence intensity measurements in the area postrema (AP) and the number of GFRAL-positive neurons in the AP were confirmed and shown in a graph. [Figure 7] Figure 7a shows the results of ASO distribution in the brain using A427 probe staining two weeks after in vivo administration of antisense oligonucleotide candidates according to the present invention, and Figure 7b is a graph quantifying ASO distribution in the brain. [Figure 8] Figure 8a shows the results of staining for GFRAL gene expression two weeks after in vivo administration of the antisense oligonucleotide candidate according to the present invention, and Figure 8b shows the results of measuring the fluorescence intensity in the area postrema (AP) and the number of GFRAL-positive neurons in the AP (Area Postrema) and NTS (Nucleus Tractus Solitaris). [Figure 9] Figure 9a is a schematic diagram showing the animal experiment schedule for verifying the therapeutic effect of antisense oligonucleotides according to the present invention on cancer cachexia, including the experimental design for creating an animal model of cancer cachexia and administering antisense oligonucleotides, and Figure 9b is a graph showing the concentration of GDF15 in serum isolated from the above experimental animals. [Figure 10] Figure 10a shows the results of measuring the weight of muscle tissue in the animal model one week after antisense oligonucleotide administration, and Figure 10b shows the results of Western blot analysis of Atrogin-1 and MuRF1 protein expression in the tissues, along with a graph showing the quantification of the results. DETAILED DESCRIPTION OF THE INVENTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0018] As used herein, the terms "GFRAL," "GDNF family receptor alpha like," "GDNF family receptor alpha," "Growth Differentiation Factor 15 receptor," or "GFRAL protein," and similar terms, unless otherwise specified, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native GFRAL from vertebrate sources, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats), and in certain embodiments, includes related GFRAL polypeptides, including SNP variants thereof. GFRAL is also known in the art as "C6orf144," "Chromosome 6 Open Reading Frame 144," "BA360D14.1," "IVF19356," and "UNQ9356."

[0019] Antisense oligonucleotide (ASO) technology is a technique that regulates gene-to-protein information transfer by altering the intermediary metabolism of mRNA through a single-stranded RNA or DNA chain. Specifically, a sufficiently complementary, specific, and hybridizable base sequence is selected to achieve the desired suppression of target protein expression. ASOs bind specifically to the target gene, without affecting the expression of other genes. Therefore, ASO technology is not only a useful tool for analyzing the biological role of specific proteins, but also has potential applications in gene therapy for specific diseases (FASEBJ.9, 1288-1296, 1995).

[0020] Antisense DNA binds to target mRNA to form an RNA / DNA double helix, which is then attacked and degraded by RNase H (RNase H; a type of ribonuclease that specifically degrades mRNA that forms an RNA / DNA hybrid double helix), which is present in the body. Antisense RNA also forms an RNA / RNA double helix, which is attacked and degraded by RNase L, a ribonuclease that preferentially degrades single-stranded RNA around the double-stranded RNA (Pharmacol. Toxicol. 32, 329-376, 1992).

[0021] In one aspect, the present invention relates to an oligomer having a length of 13 to 35 nt that can hybridize with at least 13 consecutive nucleic acid bases in the entire pre-mRNA of the human GFRAL (GDNF family receptor alpha like) gene represented by SEQ ID NO: 1 through Watson-Crick pairing A:T or G:C or wobble pairing G:U, I:A, I:C or I:U.

[0022] The antisense oligomer (antisense oligonucleotide) of the present invention can suppress the expression of the gene encoding human GFRAL, specifically the mRNA (pre-mRNA or mature mRNA). The antisense oligomer (antisense oligonucleotide) of the present invention comprises a sequence complementary to the mRNA encoding human GFRAL.

[0023] "Antisense activity" refers to any detectable or measurable activity attributable to hybridization of an antisense compound to its target nucleic acid. In a specific embodiment, antisense activity is a reduction in the amount or expression of a target nucleic acid or a protein encoded by such a target nucleic acid. Antisense activity according to the present invention acts on a target nucleic acid encoding GFRAL to reduce the amount or expression of the encoded GFRAL protein.

[0024] "Targeting," "targeting," or "targeted" means to specifically hybridize to a target nucleic acid and induce a desired effect.

[0025] "Target nucleic acid," "target RNA," and "target RNA transcript" all refer to a nucleic acid that can be targeted by an antisense oligomer.

[0026] The present invention includes oligomers that are capable of hybridizing to a target nucleic acid through hydrogen bonding.

[0027] "Inhibition" refers to a decrease in target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to the target nucleic acid levels or target protein levels in the absence of the antisense oligomer.

[0028] An "antisense oligomer" comprises a single-stranded oligonucleotide having a nucleobase sequence that enables it to hybridize to a corresponding site or segment of a target nucleic acid.

[0029] In the present invention, the oligomer can be characterized by being capable of hybridizing with at least 13 consecutive nucleic acid bases in the whole pre-mRNA of the human GFRAL gene and having a length of 13 to 35 nt.

[0030] The entire pre-mRNA of the human GFRAL gene can be characterized by being represented by the nucleic acid sequence of SEQ ID NO: 1, which is GenBank Homo sapiens chromosome 6, GRCh38.p14 Primary Assembly NC_000006.12 REGION:55327469..55402493.

[0031] Oligomers according to the present invention are capable of hybridizing to at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 consecutive nucleobases of the above nucleic acid sequences.

[0032] The conditions used to achieve a particular level of stringency in hybridization vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid segment being hybridized, the degree of affinity, the nucleotide sequence composition (e.g., GC / AT composition ratio), and the type of nucleic acid (e.g., RNA, DNA) are taken into consideration when selecting hybridization conditions. An additional consideration is whether the nucleic acid is immobilized, for example, on a filter.

[0033] Examples of highly stringent conditions include: 2x SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2x SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2x SSC / 0.1% SDS at 42°C (normal stringency conditions); and 0.1x SSC at 68°C (high stringency conditions). Washing can be performed using one of these conditions, e.g., high stringency conditions, or each of the above conditions, in the order listed above, for 10-15 minutes each, with all or some of the above conditions repeated. However, as noted above, optimal conditions will vary depending on the particular hybridization reaction involved and can be determined empirically. Generally, high stringency conditions are used for hybridization of the probe of interest.

[0034] The hybridizing base pairs may include at least one wobble pair G:U, I:A, I:C, or I:U.

[0035] In the present invention, the oligomer can be characterized by being capable of hybridizing with at least 13 consecutive nucleic acid bases contained in any one nucleic acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 18, and having a length of 13 to 35 nt.

[0036] In one embodiment of the present invention, the oligomer is an antisense oligomer comprising 15 to 25 linked nucleosides, and can hybridize to at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 consecutive nucleic acid bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 18, and can be characterized as an oligomer having a length of 13 to 35 nt.

[0037] Specifically, it may include an oligomer having a length of 13 to 35 nt that can hybridize with 19 or more to 20 consecutive nucleic acid bases selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 18.

[0038] In the present invention, the oligomer may be characterized by being capable of hybridizing with at least 13 consecutive nucleic acid bases in one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11, 14, 17, and 18, and having a length of 13 to 35 nt.

[0039] In the present invention, the oligomer may include an oligomer having a length of 13 to 35 nt that can hybridize with at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 consecutive nucleic acid bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 11.

[0040] In the present invention, the oligomer may have a length of 13 to 35 nt or 16 to 25 nt, but is not limited thereto, and specifically may have a length of 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt.

[0041] The hybridizing base pairs may include at least one wobble pair G:U, I:A, I:C, or I:U.

[0042] In the present invention, the oligomer may be characterized by being capable of hybridizing with at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 consecutive nucleic acid bases in the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11 and having a length of 13 to 35 nt, and at least one of the hybridizing base pairs may include a wobble pair G:U, I:A, I:C, or I:U.

[0043] "Contiguous nucleobases" means nucleobases immediately adjacent to each other. "Internucleoside linkage" refers to the chemical bond between nucleosides. "Linked nucleosides" means adjacent nucleosides joined to each other.

[0044] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering RNA (siRNA), and microRNAs (miRNA). Nucleic acids can also include combinations of these elements within a single molecule.

[0045] "Nucleobase" means a heterocyclic moiety capable of binding to a base of another nucleic acid.

[0046] "Nucleobase sequence" means the order of contiguous nucleobases without regard to any sugar, linkage, or nucleobase modification.

[0047] "Nucleoside" means a nucleobase linked to a sugar, and "nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0048] "Oligomer" means a polymer of linked monomeric subunits that are capable of hybridizing to a region of a nucleic acid molecule.

[0049] "Oligonucleotide" means a polymer of linked nucleosides, each of which can be modified or unmodified, independently of the other.

[0050] "Single-stranded oligonucleotide" means an oligonucleotide that is not hybridized to a complementary strand.

[0051] At least one of the hybridizing base pairs may include a wobble pair G:U, I:A, I:C, or I:U. The antisense oligomer according to the present invention may include, for example, a sequence having 90% or more sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 19 to 34.

[0052] Specifically, it may include a sequence that shows 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence homology with a sequence selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 34.

[0053] The antisense oligomer according to the present invention may include, for example, a sequence having 90% or more affinity with one or more sequences selected from the group consisting of SEQ ID NOs: 24 to 27, 30, 33, and 34.

[0054] Specifically, it may include sequences that exhibit 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with one or more sequences selected from the group consisting of SEQ ID NOs: 24 to 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 34. The antisense oligomer according to the present invention may include one of the sequences selected from the group consisting of SEQ ID NOs: 24 to 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 34.

[0055] Antisense oligomers according to the present invention can include, for example, a sequence having 90% or greater sequence homology with the sequence of SEQ ID NO:24 or SEQ ID NO:27.

[0056] Specifically, it may include a sequence that shows 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the sequence of SEQ ID NO: 24 or SEQ ID NO: 27. The antisense oligomer according to the present invention may, for example, include the sequence of SEQ ID NO: 24 or SEQ ID NO: 27.

[0057] As used herein, "homology" refers to the percent identity between polynucleotide moieties. "Identity" refers to the degree to which sequences are functionally or structurally identical based on polynucleotide sequences over a comparison window. Sequence identity can be determined by comparing sequences using standard software, such as programs called BLASTN and BLASTX, which were developed based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993).

[0058] In an embodiment of the present invention, an antisense oligomer according to the present invention may comprise 20 linked nucleosides.

[0059] As used herein, the term "complementary sequence" encompasses partial base deletions and incomplete complementarity to the extent that expression of mRNA encoding GFRAL can be inhibited.

[0060] In one embodiment of the present invention, the antisense oligonucleotide is prepared by selecting one or more target sites in the nucleic acid base sequence of the gene encoding GFRAL, and selecting an oligonucleotide that is sufficiently complementary to the target site so that it can hybridize with sufficient specificity to the target site to achieve the desired effect on regulating GFRAL expression.

[0061] As used herein, "hybridization" refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0062] As used herein, the terms "hybridizable" or "complementary" or "substantially complementary" mean that a nucleic acid (e.g., RNA, DNA) comprises a base sequence of nucleotides that can non-covalently bind, i.e., form adenine (A) and thymine (T) pairing, adenine (A) and uracil (U) pairing, and guanine (G) and cytosine (C) pairing, "anneal," or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength.

[0063] In one embodiment of the invention, the hybridization occurs between the antisense oligonucleotides disclosed herein and the nucleobase sequence of the gene encoding GFRAL. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleobases of nucleic acid molecules.

[0064] Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecule being hybridized. Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art.

[0065] As used herein, the term "complementary" refers to the property of two nucleotides being capable of precise pairing. For example, when the base sequences of two different nucleic acids or oligonucleotides are written in the 5' to 3' direction, if the base sequence of a certain portion of one nucleic acid or oligonucleotide is aligned in the opposite direction and non-covalently binds to a certain portion of the other nucleic acid or oligonucleotide, i.e., adenine (A) and thymine (T) pairing, adenine (A) and uracil (U) pairing, and guanine (G) and cytosine (C) pairing, the two nucleic acids or oligonucleotides are said to be complementary.

[0066] Thus, "specifically hybridizable" and "complementary" can be interpreted as terms used to indicate a sufficient degree of complementarity or precise pairing to allow stable, specific binding to occur between an oligonucleotide and a DNA or RNA target. It is known in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to the sequence of the target nucleic acid to which it is specifically hybridized.

[0067] The antisense oligonucleotides described above are interpreted as being capable of specifically hybridizing with target DNA or RNA and inhibiting the normal function of the target DNA or RNA, and as having a sufficient degree of complementarity to prevent nonspecific binding of the antisense oligonucleotide to non-target sequences under conditions where specific binding is desirable, i.e., under physiological conditions in the case of in vivo analysis or therapy, or under analytical conditions in the case of in vitro analysis.

[0068] That is, as long as the antisense oligonucleotide can specifically hybridize with the target nucleic acid, the antisense oligonucleotide can tolerate non-complementary nucleobases between the target nucleic acid and the antisense oligonucleotide. Furthermore, the antisense oligonucleotide can hybridize with one or more nucleic acid moieties such that intervening or adjacent moieties are not involved in hybridization (e.g., a loop structure, mismatch, or hairpin structure).

[0069] As used herein, "fully complementary" means that each nucleobase of an antisense oligonucleotide is capable of precise base pairing with the corresponding base of a target nucleic acid.

[0070] In one embodiment of the present invention, the non-complementary nucleobase position can be located at the 5'-end or 3'-end of the antisense oligonucleotide. Alternatively, the non-complementary nucleobase or nucleobases can be located internally in the antisense oligonucleotide. When two or more non-complementary nucleobases are present, they can be adjacent (i.e., linked) or non-adjacent. In one embodiment of the present invention, the non-complementary nucleobase can be located in the wing portion of a gapmer antisense oligonucleotide.

[0071] In one embodiment of the present invention, the antisense oligonucleotides of the present invention can include those complementary to a portion of the nucleobase sequence of a gene encoding GFRAL. As used herein, "portion" refers to a region of a target nucleic acid but a predetermined number of contiguous nucleobases within the portion. The portion can also refer to a specific number of contiguous nucleobases in the antisense oligonucleotide. According to one embodiment, the antisense oligonucleotide can be complementary to at least an 8 nucleobase portion of the target portion, at least a 12 nucleobase portion, or at least a 15 nucleobase portion. Antisense oligonucleotides complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portions of the target portion, but within a range defined by any two of these values, are also construed as being within the above range.

[0072] As used herein, the term "nucleotide" refers to a unit molecule constituting a nucleic acid, which is composed of a nucleobase, a sugar moiety, and a phosphate group. The term "nucleotide" may be construed as including all unmodified and modified nucleobases, sugar moieties, and / or phosphate groups, such as nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides.

[0073] As used herein, the term "nucleoside" refers to a glycosylamine, which is considered to be the portion of a nucleotide excluding the phosphate group, and refers to a unit molecule composed of a nucleobase and a sugar moiety. The term "nucleoside" can be interpreted as including all nucleosides, whether or not the nucleobase "G" or sugar moiety is modified, as well as nucleotides.

[0074] As used herein, the term "oligonucleotide" refers to an oligonucleotide or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or their analogs. The term "oligonucleotide" refers not only to oligonucleotides typically found in living organisms, which are composed of nucleobases, sugars, and internucleoside (backbone) covalent bonds, but also to modified or substituted oligonucleotides composed of nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides that function similarly to these. These modified or substituted oligonucleotides have properties such as enhanced cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases compared to unmodified or unsubstituted oligonucleotides.

[0075] As used herein, the term "antisense oligonucleotide (ASO)" is intended to include oligonucleotides capable of hybridizing with a target nucleic acid sequence through hydrogen bonding. Antisense oligonucleotides include, but are not limited to, oligonucleotides, oligonucleotide analogs, oligonucleotide mimetics, siRNA, single-stranded siRNA (sssiRNA), short hairpin RNA (shRNA), microRNA mimics, ribozymes, external guide sequence oligonucleotides, and other oligonucleotides that hybridize with a target nucleic acid sequence to regulate its expression. The term "antisense oligonucleotide" is intended to encompass both single-stranded and double-stranded oligonucleotides.

[0076] In one embodiment of the present invention, the antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion of the target nucleic acid sequence to be targeted. Preferably, the antisense oligonucleotide can bind complementarily to the nucleobase sequence of a gene encoding GFRAL. The gene encoding GFRAL can be selected from mRNA and pre-mRNA including introns, exons, and untranslated regions as the nucleic acid targeted by the antisense oligonucleotide.

[0077] In the present invention, the oligomers can be characterized as including modified internucleoside linking groups, modified nucleobases, or modified nucleoside chemical modifications.

[0078] Specifically, the modified nucleobase can be characterized as being 5'-methyl-cytosine.

[0079] Specifically, the internucleoside linkages can be phosphorothioate, boranophosphate, or methyl phosphonate linkages.

[0080] Internucleoside linkages according to the present invention can be SOOOSSSSSSSSSSSOOOS linkages, where S can be a phosphorothioate internucleoside linkage and O can be a phosphodiester internucleoside linkage.

[0081] The nucleosides may include the following modified sugars: Variations in the substitution at the 2'-carbon position of the sugar structure include 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-methoxyethyl-5'-methyl, 2'-O-aminoethyl, 5'-methyl, 2'-O-propyl, 2'-methylthioethyl, or 2'-fluoro.

[0082] The above nucleosides can include variations, for example, where the internucleoside linkage is a phosphorothioate, boranophosphate, or methyl phosphonate linkage; and the substitution at the 2' carbon position of the sugar structure is 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-methoxyethyl-5'methyl, 2'-O-aminoethyl, 5'-methyl, 2'-O-propyl, 2'-methylthioethyl, or 2'-fluoro.

[0083] The nucleoside can be characterized by including a modification selected from the group consisting of: a modification at the 2' carbon position of the sugar structure within the nucleotide, specifically to 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-methoxyethyl-5' methyl, 2'-O-aminoethyl, 5'-methyl, 2'-O-propyl, 2'-methylthioethyl, or 2'-fluoro; a modification of the nucleotide bond to phosphorothioate, boranophosphate, or methyl phosphonate; a modification to a peptide nucleic acid (PNA), locked nucleic acid (LNA), or unlocked nucleic acid (UNA) form; and a phosphate group.

[0084] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring.

[0085] "2'-O-methoxyethyl nucleotide" means a nucleotide containing a sugar moiety that has been modified with a 2'-O-methoxyethyl.

[0086] "Modified sugars" refer to substitutions or variations from natural sugars.

[0087] "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0088] "Modified internucleoside linkage" means a substitution or any change from a naturally occurring internucleoside bond.

[0089] "Modified nucleobase" means any nucleobase that is not adenine, cytosine, guanine, thymidine, or uracil. "Modified nucleobase" means the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0090] "Modified nucleotide" means, independently, a nucleotide having a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase. "Modified nucleoside" means, independently, a nucleotide having a modified sugar moiety or a modified nucleobase.

[0091] "Modified oligonucleotide" means an oligomer containing at least one modified nucleotide.

[0092] Antisense oligomers according to the invention may contain modifications to 2'-O-methoxyethyl (2'MOE), 2'-O-methoxyethyl-5'methyl, 5'methyl, or phosphorothioate linkages.

[0093] Deformation of the sugar moiety In one embodiment of the present invention, the modified nucleosides can be modified nucleosides containing non-bicyclic modified sugar moieties and / or bicyclic or tricyclic sugar moieties, and / or sugar moieties modified with sugar surrogates or sugar mimetics.

[0094] In the present invention, the modified nucleosides include, for example, 2'-O-alkyl such as 2'-O-methyl, 2'-O-alkoxy such as 2'-O-methoxy, 2'-O-alkoxyalkyl such as 2'-O-methoxyethyl, 2'-amino, 2'-allyl, 2'-fluoro, 2'-arabino-fluoro, and 2'-arabino-fluoro. The sugar moiety may be, but is not limited to, a sugar moiety having one or more substituents introduced therein, selected from the group consisting of fluoro, 2'-ON-substituted acetamide such as 2'-OCH2C(=O)-NHCH3 (NMA), 2'-O-benzyl and 2'-O-methyl-4-pyridine, 4'-O-methyl, 5'-methyl, 5'-vinyl, and 5'-methoxy.

[0095] Antisense oligomers of the present invention can include one or more modified nucleosides with selectively substituted or modified sugar moieties. Modifications to the sugar moiety can confer nuclease stability, binding affinity, or other advantageous biological properties to the oligomer. The (pento)furanosyl sugar ring of the natural nucleosides can be modified in a variety of ways, including, but not limited to, the addition of substituents (especially at the 2'-position); bridging of two different ring atoms to form bicyclic nucleic acids (BNAs); and substitution of atoms or groups such as -S-, -N(R)-, or -C(R1)(R2) at the 4'-position ring oxygen. Modified sugar moieties can include substituted sugars, particularly 2'-substituted sugars with 2'-F, 2'-OCH2 (2'-OMe), or 2'-O(CH2)2-OCH3 (2'-O-methoxyethyl or 2'-MOE) substituents; and 4'-(CH2) n Modified sugars include, but are not limited to, bicyclic modified sugars (BNAs) with an -O-2' (n=1 or n=2) bridge. Methods for preparing such modified sugars are known in the art. In nucleosides containing modified sugar moieties, the base moiety can be maintained so as to hybridize with a target nucleic acid.

[0096] In the present invention, the modified nucleosides include one of the following at the 2' position: F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-alkyl-O-alkyl; O-alkyl-O-alkyl-N(dialkyl); or O-alkyl-carboxylamido, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 alkenyl and alkynyl). O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, O(CH2) n O(CH2) n N[(CH2) mCH3]2, O(CH2) n C(=O)-NHCH3 and O(CH2) n ON[(CH2) m CH3]2 (where n and m are 0 to about 10) is particularly preferred.

[0097] Preferably, the variants include 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chem. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group; the variants include 2'-dimethylaminooxyethoxy (i.e., the (CH2)2ON(CH3)2 group, also known as 2'-DMAOE), and 2'-dimethylaminoethoxyethoxy (i.e., the 2'-O(CH2)2O(CH2)2-N(CH3)2 group, also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE).

[0098] The bicyclic or tricyclic sugar moiety may be selected from the group consisting of, but not limited to, locked nucleic acid (LNA), constrained ethyl bicyclic nucleic acid (cEt), 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA), and tricyclo-DNA.

[0099] In one embodiment, the modified nucleoside can include a sugar substitute having a hexacyclic or acyclic moiety. The sugar substitute can be selected from the group consisting of, but not limited to, a morpholino ring, such as a phosphorodiamidate morpholino oligomer (PMO), a cyclohexyl ring, or a tetrahydropyranyl ring, such as hexitol, anethole, mannitol, or fluorohexitol. Various other bicyclic and tricyclic sugar-substituted ring systems that can be used to modify the nucleosides incorporated into the antisense oligomers of the present invention are known in the art. The activity of such ring systems can be enhanced through various substitution processes.

[0100] The sugar substitute can also be an acyclic moiety such as, but not limited to, an unlocked nucleic acid (USA) or a peptide nucleic acid (PNA).

[0101] Peptide nucleic acid (PNA) is a type of nucleic acid analogue in which nucleobases are linked by peptide bonds instead of phosphate bonds, and contains nucleobases such as adenine, thymine, guanine, and cytosine, allowing it to specifically hybridize with nucleic acids. PNA is not found in nature but is artificially synthesized by chemical methods and can form double-stranded chains through hybridization with nucleic acids of complementary base sequences. Furthermore, PNA is not only chemically stable due to its electrically neutrality, but also biologically stable, as it is resistant to degradation by nucleases and proteases. PNAs with an N-aminoethylglycine backbone are most commonly used, but PNAs with modified backbones can also be used as described in the art (P.E. Nielsen and M. Egholm, "An Introduction to PNA," in P.E. Nielsen (Ed.), "Peptide nucleic acids: Protocols and Applications," 2nd Ed., p. 9 (Horizon Bioscience, 2004)).

[0102] Unlocked nucleic acid (USA) is a modified nucleoside that does not have the C2'-C3' bond of ribose. Its open-chain structure allows for unrestricted steric conformation, allowing for the flexibility of the oligonucleotide to be adjusted. When UNA is included in an oligomer, it is known to reduce the Tm value by 5-10°C, thereby reducing off-target effects.

[0103] Nucleobase transformation In the present invention, the modified nucleosides include pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2-deoxyuridine, N-ethylpiperidine 7'-EAA triazol modified adenine, and N-ethylpiperidine 6'-triazol modified adenine. The modified nucleoside may be characterized as a modified nucleoside containing one or more modified nucleobases selected from the group consisting of 2',4'-difluorotoluyl ribonucleoside, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluyl ribonuleoside, and 5'-nitroindole, but is not limited thereto.

[0104] Unmodified or natural nucleobases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0105] The modified nucleosides may also contain nucleobase modifications or substitutions. Nucleobase modifications or substitutions are structurally distinct but may be functionally interchangeable with naturally occurring, but not synthetically modified, nucleobases. Naturally occurring nucleobases and modified nucleobases can participate in hydrogen bonds. Such nucleobase modifications may impart nuclease stability, binding affinity, or other advantageous biological properties to the antisense oligomer. For example, certain nucleobase substitutions, such as 5-methylcytosine substitutions, are known to increase nucleic acid duplex stability by 0.6-1.2°C and may be particularly useful for enhancing the binding affinity of antisense oligomers to target nucleic acids.

[0106] For example, such modified nucleobases include 5'-hydroxymethylcytosine, xanthine, hypoxanthine, 2'-aminoadenine, 6'-methyl and other alkyl derivatives of adenine and guanine, 2'-propyl and other alkyl derivatives of adenine and guanine, 2'-thiouracil, 2'-thiothymine and 2'-thiocytosine, 5'-halouracil and cytosine, 5'-propynyl (-C≡C-CH3)uracil and cytosine, and and pyrimidine bases, as well as alkynyl derivatives, 6'-azouracil, cytosine and thymine, 5'-uracil (analogous uracil), 4'-thiouracil, 8'-halo, 8'-amino, 8'-thiol, 8'-thioalkyl, 8'-hydroxy and other 8'-substituted adenines and guanines, 5'-halo (especially 5'-bromo), 5'-trifluoromethyl and other 5'-substituted uracils and cytosines, 7'-methylguanine and 7' -methyladenine, 2'-F-adenine, 2'-amino-adenine, 8'-azaguanine and 8'-azaadenine, 7'-diazaguanine and 7'-diazaadenine and 3'-diazaguanine and 3-diazaadenine, phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one) G-clamps include, but are not limited to, tricyclic pyrimidines such as 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), substituted phenothiazine cytidines such as carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrole[2,3-d]pyrimidin-2-one).

[0107] The modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993.

[0108] Modified internucleoside linkers In the present invention, the modified internucleoside linking group in the antisense oligomer may be one or more modified internucleoside linking groups selected from the group consisting of phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, mesylphosphoramidate, methylphosphonate, methoxypropyl-phosphonate, and boranophosphate.

[0109] As known in the art, a nucleoside is a combination of a nucleobase and a sugar moiety. Nucleotides further contain a phosphate group covalently linked to the sugar moiety of the nucleoside. In nucleotides containing pentoprasinol sugars, the phosphate group can be linked to the 2', 3', or 5' hydroxyl group of the linked sugar. In oligonucleotide formation, the phosphate groups covalently link adjacent nucleosides to form a linear polymeric compound. In turn, the ends of the linear polymeric structure can also be linked to form a circular structure, although open linear structures are generally preferred. In oligonucleotide structures, the phosphate groups typically form the internucleoside backbone of the oligonucleotide; the naturally occurring linkages and backbones of RNA and DNA are 3' to 5' phosphodiester linkages. In one embodiment, an antisense oligomer may contain one or more modified internucleoside linkages in addition to naturally occurring internucleoside linkages, which are preferred over antisense oligomers containing naturally occurring internucleoside linkages due to properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0110] One specific example of a preferred antisense oligomer that can be used in the present invention is an oligonucleotide containing a modified backbone or an unnatural internucleoside linkage. As defined above, oligonucleotides with modified backbones include nucleotides that contain a phosphorus atom in the backbone and nucleotides that do not contain a phosphorus atom in the backbone. Furthermore, as used in the art, modified oligonucleotides that do not contain a phosphorus atom in the internucleoside backbone are also construed herein as oligonucleotides.

[0111] In an embodiment of the antisense oligomer, modified internucleoside linkages can include internucleoside linkages that do not contain a phosphate, as well as internucleoside linkages that contain a phosphate. Representative phosphate-containing internucleoside linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates, phosphinates, 3'-amino phosphoramidates and aminoalkyl phosphoramidates with their normal 3'-5' and 2'-5' linked analogs, mesyl phosphoramidate, thionophosphoamidate, thionoalkylphosphonates, thionoalkylphosphorotriesters, phosphoamidates including selenophosphates and boranophosphates, and compounds of opposite polarity in which one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linked.

[0112] In the present invention, the modified oligonucleotide comprises a gap segment composed of linked deoxynucleosides, a 5' wing segment composed of linked nucleosides, and a 3' wing segment composed of linked nucleosides, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment may comprise, but are not limited to, a modified sugar moiety or sugar substitute.

[0113] In one embodiment of the present invention, the modified oligonucleotide may be characterized in that it comprises a gap segment consisting of 8 to 10 linked deoxynucleosides; a 5' wing segment consisting of 3 to 5 linked nucleosides; and a 3' wing segment consisting of 3 to 5 linked nucleosides, each nucleoside of each wing segment comprising a modified sugar moiety, but is not limited thereto.

[0114] In a gapmer, an internal region containing multiple nucleotides that support RNase H cleavage is located between an external region containing multiple nucleosides that are chemically different from the nucleosides in the internal region. In antisense oligonucleotides with a gapmer motif, the gap segment supports cleavage of the target nucleic acid, while the wing segments can comprise modified oligonucleotides containing modified nucleosides to improve stability, affinity, and exonuclease resistance.

[0115] If desired, the gap segment may also comprise a modified oligonucleotide, which may comprise one or more modifications selected from one or more modified internucleoside linking groups, one or more modified nucleosides containing modified sugar moieties, and one or more modified nucleosides containing modified nucleobases, each of which is as described above.

[0116] In one embodiment of the present invention, the antisense oligomer preferably comprises the base sequences shown in SEQ ID NO: 19 to SEQ ID NO: 34 and may be characterized by including the chemical modifications shown in Table 2, but is not limited thereto.

[0117] In the present invention, the oligomer can be most preferably characterized by comprising a structure of formula (I) or formula (II): G*GTATA*8*8*8*8*7*5*8*5*7*5*AGAAC*C Chemical formula (I); C*ACAA*5*5*5*7*6*6*5*5*6*5*TCTG*G Formula (II), Here, A means 2'MOE-A, C means 2'MOE-5'-methyl-C, G means 2'MOE-G, T means 2'MOE-T, 5 means DNA-A, 6 means DNA-5'-methyl-C, 7 means DNA-G, 8 means DNA-T, * means PS (phosphorothioate), and 2'MOE means 2'-O-methoxyethyl.

[0118] To facilitate the use of oligomers in the present invention, various dosage forms have been developed that can deliver oligonucleotides to a subject or cellular environment, for example, using dosage forms that minimize degradation, facilitate delivery and / or absorption, or provide other beneficial properties to the oligonucleotide in the dosage form.

[0119] In the present invention, antisense oligomers for reducing GFRAL expression can be formulated so that when administered to a subject in the presence of target cells or systemically, a sufficient portion of the oligomer enters the cells and reduces GFRAL expression. According to one embodiment, the antisense oligomer can be formulated in a buffer solution, such as phosphate buffered saline, liposomes, micellar structures, or capsids. It can also be formulated in water or an aqueous solution (e.g., pH-adjusted water) or a basic buffered aqueous solution (e.g., PBS).

[0120] In the present invention, oligomer formulations containing cationic lipids can be used to facilitate the introduction of oligomers into cells. For example, cationic lipids, such as lipofection, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine), can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc.), or FuGene6 (Roche), all of which can be used according to the manufacturer's protocol. Such formulations can include lipid nanoparticles.

[0121] The dosage form may also include an excipient. The excipient may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to cells, tissues, organs, or the body of a subject in need thereof (see, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013). The excipient may provide the composition with improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. The excipient may also be a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0122] In one embodiment, the oligomer can be lyophilized to extend its shelf life and then reconstituted into a solution prior to use. Thus, the excipient in a composition containing an oligomer according to the present invention can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0123] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers can include physiological saline, bacteriostatic water, Cremophor EL™ (BASF), or phosphate-buffered saline (PBS). The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and compatible mixtures thereof. In various cases, it may be preferable to include isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide in the selected solvent, optionally with one or a combination of the above-listed ingredients, followed by sterile filtration. The pharmaceutical composition may contain at least about 0.1% of a therapeutic agent (e.g., an antisense oligonucleotide for reducing WFDC2 expression) or more, but the percentage of active ingredient is preferably about 1% to about 80% by weight or volume of the total composition. This is because factors such as solubility, bioavailability, biological half-life, route of administration, and product shelf life, as well as other pharmaceutical considerations, must be taken into account in formulating the dosage form.

[0124] In another aspect, the present invention relates to a pharmaceutical composition containing the above oligomer for preventing or treating a disease associated with activation of GFRAL due to an increase in GDF15.

[0125] In still another aspect, the present invention relates to a method for preventing or treating a disease associated with activation of GFRAL due to an increase in GDF15, comprising administering the oligomer to a subject.

[0126] In another aspect, the present invention relates to the use of the oligomer for the prevention or treatment of diseases associated with activation of GFRAL due to increased GDF15.

[0127] In another aspect, the present invention relates to use of the above oligomer for producing a drug for preventing or treating a disease associated with activation of GFRAL due to increased GDF15.

[0128] As used herein, the term "prevention" refers to any action of administering a composition of the present invention to suppress or delay the progression of a disease associated with GFRAL activation due to increased GDF15, and "treatment" refers to the suppression of the development of a disease associated with GFRAL activation due to increased GDF15, or the alleviation or elimination of symptoms.

[0129] In the present invention, the diseases associated with activation of GFRAL due to increased GDF15 may be characterized as obesity, diabetes, anorexia, or cachexia, but are not limited thereto.

[0130] Specifically, the disease can be cancer cachexia.

[0131] Cachexia is a debilitating metabolic syndrome associated with numerous diseases, including cancer, AIDS, chronic heart failure (also known as congestive heart failure), chronic obstructive pulmonary disease (COPD), chronic kidney disease, tuberculosis, sepsis, and other forms of systemic inflammation. In particular, cachexia is a wasting disorder associated with involuntary weight loss and can be associated with systemic inflammation and / or acute inflammatory responses. Loss of muscle mass, as well as loss of fat mass, is often the primary clinical feature of cachexia. Sarcopenia due to loss of muscle mass can cause functional impairments, including loss of strength, increased tendency to collapse, and loss of autonomy. Respiratory function can also be impaired, resulting in reduced vital capacity. While sarcopenia is largely a geriatric disease, its development can also be associated with muscle disuse and nutritional deficiencies, and can occur simultaneously with cachexia. Cachexia can progress through stages designated as pre-cachexia, cachexia, and refractory cachexia.

[0132] GDF15 is expressed at elevated levels in serum in many malignant cancers, particularly invasive brain cancer, melanoma, lung cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer, and breast cancer. Similarly, chemoresistance can be correlated with high GDF15 expression, and a correlation between high GDF15 expression levels and cancer prognosis has also been reported. GDF15, a member of the TGF-β superfamily, has been reported to mediate cachexia induced by various diseases. Because GFRAL is a high-affinity receptor for GDF15, the antisense oligomers of the present invention can be used to prevent or treat GDF15-induced cancer cachexia.

[0133] In the present invention, "cancer" and "tumor" are used interchangeably to refer to or denote the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0134] In the present invention, the pharmaceutical composition can be characterized by containing a therapeutically effective amount of an antisense oligomer.

[0135] The "pharmaceutically acceptable carrier" refers to a substance that can be added to an active ingredient to help formulate or stabilize the formulation, without causing significant adverse toxic effects to the patient. Pharmaceutically acceptable carriers are those commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0136] In addition to the above ingredients, the pharmaceutical composition may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0137] The term "administration" as used herein means introducing the pharmaceutical composition of the present invention into a patient by any suitable method. The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, by infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0138] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, and an ordinarily skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat obesity, diabetes, anorexia or cachexia.

[0139] The pharmaceutical composition according to the present invention can be used in combination with a conventional therapeutic agent, meaning that the antisense oligomer according to the present invention and a pharmaceutical composition containing the same can be administered simultaneously with a conventional therapeutic agent such as an anticancer agent, or administered sequentially or in reverse order, and can be administered in an appropriate effective amount combination within the scope of a person skilled in the art. [Example]

[0140] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0141] Example 1. Development of GFRAL-targeted antisense oligonucleotides Taking into consideration target specificity and stability, 16 GFRAL mRNA target sequences (SEQ ID NOs: 3 to 18) were developed from the GFRAL gene (pre-mRNA sequence) and GFRAL mature-RNA sequence (SEQ ID NOs: 1 to 2), and 16 antisense sequences (SEQ ID NOs: 19 to 34) were developed against these sequences (Table 1).

[0142] In addition, we developed 16 antisense oligonucleotides by chemically modifying the 16 complementary sequences listed in Table 1 (Table 2). The antisense oligonucleotide structure used was a "gapmer," which includes a 10-nt internal "gap" region and 4-6-nt "wing" regions with 2'MOE (2'-Methoxyethyl) modified backbones at the 5' and 3' ends. In the gapmer, phosphorothioate (PS) bonds were used instead of the usual phosphodiester (PO) bonds in the region connecting the two DNA nucleosides and in the bond connecting the DNA nucleoside to the 2'MOE nucleoside. PS bonds were used only at the final ends of the 2'MOE nucleotide-based wing region, while PO bonds were used for the remaining regions. Meanwhile, all cytosines (C) are replaced with methylated cytosines, and all uracils (U) in the RNA portion are replaced with thymines (T), which further enhances the stability of the antisense oligonucleotide and reduces the host immune response.

[0143] For example, A427 has the sequence and chemical variant G*GTATA*8*8*8*8*7*5*8*5*7*5*AGAAC*C (A=2'MOE-A, C=2'MOE-5'-methyl-C, G=2'MOE-G, T=2'MOE-T, 5=DNA-A, 6=DNA-5'-methyl-C, 7=DNA-G, 8=DNA-T, *=PS).

[0144] Sequence number 1 (GenBank Homo sapiens chromosome 6, GRCh38.p14 Primary Assembly NC_000006.12 REGION: 55327469..55402493)

[0145] Sequence number 2 (GenBank Homo sapiens GFRAL mRNA, NM_207410.2)

[0146] [Table 1]

[0147] [Table 2]

[0148] Experimental example 1: Primary screening Antisense oligonucleotide candidates targeting GFRAL mRNA were derived as listed in Table 1 and then manufactured by Chemgenes (USA). HEK293 cells overexpressing GFRAL and RET cDNAs were transfected with the antisense oligonucleotides at a concentration of 200 nM using Lipofectamine (Invitrogen, USA) reagent. After 24 hours, RNA was purified using an RNA extraction kit (Nioneer, Korea) and DNase (NEB, USA). Reverse transcription was then performed using RT-premix (Applied Biosystems, USA). qRT-PCR was then performed to measure GFRAL gene expression levels. The following primer and probe sequences were used for qRT-PCR: GFRAL:predesigned qRT Assay (Hs.PT.58.21179605, IDT, USA) GAPDH: Forward: 5'-GGTGTGAACCATGAGAAGTATGA-3' (SEQ ID NO: 35) reverse: 5'-GAGTCCTTCCACGATACCAAAG-3' (SEQ ID NO: 36) probe: 5'-AGATCATCAGCAATGCCTCCTGCA-3' (SEQ ID NO: 37)

[0149] As a result, as can be seen in Figure 1, it was confirmed through primary screening that the expression of the GFRAL gene was suppressed by antisense oligonucleotides.

[0150] Experimental example 2: Secondary screening We tested 16 antisense oligonucleotides in HEK293 cells expressing GFRAL and RET cDNAs by administering 100 nM ASOs using Lipofectamine (Invitrogen, USA) reagent to repeatedly examine whether GFRAL gene knockdown was possible. After transfection with the ASOs for 24 hours, cells were lysed using the lysis and Rt kits and reverse transcription was performed. Next, qRT-PCR was performed using Taqman probes and qRT-PCR premix (Applied Biosystems, USA) to measure GFRAL gene expression levels. As shown in Figure 2a, eight antisense oligonucleotides demonstrated knockdown effects at a concentration of 100 nM.

[0151] Additionally, after transfection with the ASO for 48 hours, Western blot assays were performed to confirm GFRAL gene protein expression. As shown in Figure 2b, eight antisense oligonucleotide candidates were found to reduce protein expression. Based on the results of Figures 1 and 2, eight candidates were selected: A426, A427, A428, A429, A430, A433, A436, and A437.

[0152] Experimental example 3: Third screening Tertiary screening was performed on the eight antisense oligonucleotides selected in Experiments 1 and 2, administering 50 nM ASO using the same method as in Experiments 1 and 2. As shown in Figure 3a, all eight antisense oligonucleotides showed excellent knockdown efficiency at a concentration of 50 nM. After transfection with the ASO for 48 hours, cytotoxicity was measured using the Quantimax WST-8 cell viability assay kit (Abcam, UK), and GFRAL gene expression was confirmed by Western blot assay under the same conditions. As shown in Figure 3b, all eight antisense oligonucleotide candidates showed no decrease in cell viability.

[0153] Furthermore, as shown in Figures 4a and 4b, protein expression inhibition was repeatedly confirmed with A427, A428, A429, and A430. Based on the results of Figures 1, 2, 3, and 4, A427, A428, A429, and A430 were selected as the final candidate substances.

[0154] Experimental Example 4: In vivo toxicity experiment Based on the results of the cell experiments in Experimental Examples 1, 2 and 3, animal toxicity experiments were carried out three times to verify the stability of the four antisense oligonucleotide candidates.

[0155] The antisense oligonucleotide of the present invention was administered intracerebroventricularly (ICV) to the left hemisphere of the brain of 0-2 day old C57BL / 6J wild type mice at 2 μl (20 μg), and the survival rate was measured after 7 days (FIG. 5a).

[0156] As a result, as shown in Figure 5b, none of the 12 animals administered PBS or A114 in the negative control group died, while 11 of the 12 animals administered A386 in the positive toxicity control group died within 6 hours, confirming its significant toxicity. One of the seven animals administered A427 in the experimental group died, while all animals administered A428, A429, and A430 survived, confirming that these antisense oligonucleotide candidate substances are relatively safe for in vivo administration.

[0157] Experimental Example 5: In vivo GFRAL protein expression regulation analysis The following animal experiments were carried out to verify the pharmacological effects of the antisense oligonucleotide candidates.

[0158] Eight-week-old male C57BL / 6N wild-type mice were intracerebroventricularly (ICV) injected with 8.5 μl (500 μg) of antisense oligonucleotides (A114, A427). One week later, the brain tissue was fixed by sequentially infusing saline (sodium chloride, perfusate; JW Pharmaceutical Corporation, Korea) and 4% paraformaldehyde (FUJIFILM, Japan) through the aorta. The brain tissue was then removed and fixed in the same fixative for 24 hours. After washing twice with phosphate buffered saline containing 30% sucrose at 4°C, the tissue was frozen and sectioned at 30 μm using a cryostat. The sections were blocked for 1 hour with 0.1% PBST (triton-X100) containing 5% donkey serum (Jackson Immunoresearch, USA) and then incubated with purified primary antibodies for 24 hours. After incubation with a secondary antibody for 2 hours, changes in the expression level of the GFRAL gene protein were confirmed using immunohistochemistry (IHC) fluorescent staining techniques.

[0159] As a result, as shown in Figure 6, when A427 antisense oligonucleotide was administered, the fluorescent signal intensity of the GFRAL gene protein decreased, and the proportion of GFRAL gene-expressing neurons among all neurons decreased.

[0160] Experimental Example 6: In vivo ASO distribution analysis The following animal experiments were carried out to verify the pharmacological effects of the antisense oligonucleotide candidates.

[0161] Eight-week-old male C57BL / 6N wild-type mice were administered 8.5 μl (500 μg) of antisense oligonucleotides (A114, A427, A428) intracerebroventricularly (ICV). Two weeks later, the brain tissue was primarily fixed by infusing saline (sodium chloride, perfusate) and 4% paraformaldehyde through the aorta in that order. The brains were then removed and postfixed in the same fixative for 24 hours. After washing twice in phosphate buffered saline containing 30% sucrose at 4°C, the brains were frozen and sectioned at 10 μm using a cryostat. RNAscope TM After staining the ASO probe with Plus smRNA-RNA HD Reagent kit (ACD bio, USA), the biodistribution of the ASO probe was observed using fluorescence in situ hybridization (FISH) technique.

[0162] As a result, as seen in Figures 7a and 7b, A427 was found to be distributed in the hindbrain, including the AP (Area postrema) and NTS (Nucleus tractus solitaris).

[0163] Experimental Example 7: In vivo GFRAL RNA expression regulation analysis The following animal experiments were carried out to verify the pharmacological effects of the antisense oligonucleotide candidates.

[0164] Eight-week-old male C57BL / 6N wild-type mice were administered 8.5 μl (500 μg) of antisense oligonucleotides (A114, A427, A428, A429, A430) intracerebroventricularly (ICV). Two weeks later, the brain tissue was primarily fixed by infusing saline (sodium chloride, perfusate) and 4% paraformaldehyde through the aorta in that order. The brain tissue was then excised and postfixed in the same fixative for 24 hours. After washing twice in phosphate buffered saline containing 30% sucrose at 4°C, the tissue was frozen and sectioned at 10 μm using a cryostat. RNAscope TM After staining GFRAL RNA with Multiplex Fluorescent Reagent kit v2 kit (ACD bio, USA), changes in GFRAL RNA expression were confirmed using fluorescence in situ hybridization (FISH) technique.

[0165] As a result, as shown in Figures 8a and 8b, when A427, A428, A429, and A430 antisense oligonucleotides were administered, the fluorescent signal intensity of GFRAL RNA decreased, and the proportion of GFRAL gene-expressing neurons among all neurons decreased.

[0166] Experimental Example 8: Confirmation of the efficacy of ASO in a cancer cachexia model Colon cancer cells (MC38) were injected intraperitoneally (IP) into 8-week-old male C57BL / 6J wild-type mice. Two weeks later, 500 μg of antisense oligonucleotides (A427 and A430) were administered intracerebroventricularly. One week after antisense oligonucleotide administration, serum GDF15 levels, which are useful for diagnosing cancer cachexia, were measured using a Quantikine ELSIA kit (R&D Systems, USA) (Figure 9a).

[0167] As seen in Figure 9b, serum GDF15 levels increased more than 1000-fold in the MC38-treated group, suggesting that the cancer cachexia model was well established.

[0168] As shown in Figure 10a, in the cancer cachexia animal model, the A427-treated group exhibited increased gastrocnemius weight compared to the negative control group (PBS). Furthermore, the expression of Atrogin-1 and MuRF1 proteins, key molecular markers of muscle atrophy, was measured in gastrocnemius muscle tissue by Western blot assay. It was confirmed that Atrogin-1 protein expression was significantly reduced by the ASO (Figure 10b). [Industrial Applicability]

[0169] In the present invention, we have discovered candidate antisense oligonucleotides that regulate GFRAL expression and confirmed that these candidate compounds effectively suppress GFRAL expression in vivo and show ameliorative effects in animal models of cancer cachexia. Therefore, the antisense oligonucleotides of the present invention are expected to be highly applicable as therapeutic agents for obesity or cancer cachexia through their inhibition of GFRAL expression.

[0170] While the present invention has been described in detail above, it is obvious to those skilled in the art that the specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0171] Sequence Catalog Free Text Electronic file attached.

Claims

1. An oligomer having a length of 13 to 35 nt that can hybridize with at least 13 consecutive nucleic acid bases in the entire pre-mRNA of the human GFRAL (GDNF family receptor alpha like) gene represented by SEQ ID NO: 1 through Watson-Crick base pairing A:T or G:C or wobble base pairing G:U, I:A, I:C or I:U.

2. The oligomer of claim 1, which is capable of hybridizing with at least 13 consecutive nucleic acid bases contained in any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 to 18 via Watson-Crick base pairing A:T or G:C or wobble base pairing G:U, I:A, I:C, or I:U, and has a length of 13 to 35 nt.

3. The oligomer of claim 1, which is capable of hybridizing with at least 13 consecutive nucleic acid bases contained in any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11, 14, 17, and 18 via Watson-Crick base pairing A:T or G:C or wobble base pairing G:U, I:A, I:C, or I:U, and has a length of 13 to 35 nt.

4. The oligomer of claim 1, which is capable of hybridizing with at least 13 consecutive nucleic acid bases contained in any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11 via Watson-Crick base pairing (A:T or G:C) or wobble base pairing (G:U, I:A, I:C, or I:U) and has a length of 13 to 35 nt.

5. The oligomer according to claim 1, which is capable of hybridizing with at least 13 consecutive nucleic acid bases contained in the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11 via Watson-Crick base pairing (A:T or G:C) or wobble base pairing (G:U, I:A, I:C, or I:U) and has a length of 13 to 35 nt.

6. 10. The oligomer of claim 1, wherein the oligomer comprises a modified internucleoside linking group, a modified nucleobase, or a chemical modification of a modified nucleoside.

7. 7. The oligomer of claim 6, wherein the modified nucleobase is 5'-methyl-cytosine.

8. The modified nucleosides include 2'-O-methyl, 2'-O-methoxy, 2'-O-methoxyethyl, 2'-amino, 2'-allyl, 2'-fluoro, 2'-arabino-fluoro, and 2'-OCH ２ C(=O)-NHCH ３ 7. The oligomer according to claim 6, which is a nucleoside having a sugar moiety into which one or more substituents selected from the group consisting of NMA, 2'-O-benzyl, 2'-O-methyl-4-pyridine, 4'-O-methyl, 5'-methyl, 5'-vinyl, and 5'-methoxy have been introduced; or a bicyclic nucleoside having one or more modified nucleosides in LNA (locked nucleic acid) or cEt (constrained ethyl).

9. 7. The oligomer of claim 6, wherein the modified internucleoside linking group is at least one selected from the group consisting of phosphorothioate, phosphorodithiate, phosphotriester, phosphoramidate, mesylphosphoramidate, methylphosphonate, methoxypropylphosphonate, and boranophosphate.

10. the oligomer comprises a gap segment comprised of linked deoxynucleosides, a 5' wing segment comprised of linked nucleosides, and a 3' wing segment comprised of linked nucleosides; 7. The oligomer of claim 6, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and wherein the nucleosides of each wing segment comprise a modified sugar moiety or sugar surrogate.

11. 11. The oligomer of claim 10, wherein the oligomer comprises a gap segment of 8 to 10 linked deoxynucleosides; a 5' wing segment of 3 to 7 linked nucleosides; and a 3' wing segment of 3 to 7 linked nucleosides, wherein each nucleoside in each wing segment comprises a modified sugar moiety or sugar surrogate.

12. The oligomer of claim 1, wherein the oligomer comprises a structure of the following formula (I) or (II): G*GTATA*8*8*8*8*7*5*8*5*7*5*AGAAC*C Chemical formula (I); C*ACAA*5*5*5*7*6*6*5*5*6*5*TCTG*G Chemical formula (II), Here, A means 2'MOE-A, C means 2'MOE-5'-methyl-C, G means 2'MOE-G, T means 2'MOE-T, 5 means DNA-A, 6 means DNA-5'-methyl-C, 7 means DNA-G, 8 means DNA-T, * means PS (phosphorothioate), and 2'MOE means 2'-O-methylethyl.

13. A pharmaceutical composition for preventing or treating a disease associated with GFRAL activation due to increased GDF15, comprising the oligomer according to any one of claims 1 to 12.

14. The pharmaceutical composition according to claim 13, wherein the disease associated with GFRAL activation due to increased GDF15 is obesity, diabetes, anorexia, or cachexia.

15. The pharmaceutical composition according to claim 13, wherein the disease associated with GFRAL activation due to increased GDF15 is cancer cachexia.

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