TW202334414A - Compositions and methods for inhibiting melanocortin 2 receptor and cytochrome p450 11b1 expression - Google Patents

Abstract

Disclosed are oligonucleotides for inhibiting or reducing expression of MC2R or CYP11B1, and lipid conjugated oligonucleotides for targeting MC2R or CYP11B1 in the adrenal gland. Also disclosed are methods for treating, preventing, and alleviating diseases associated with MC2R and/or CYP11B1 expression, such as Cushing’s Disease, Cushing’s Syndrome, Familial Glucocorticoid Deficiency, hereditary adrenocortical unresponsiveness to ACTH, Congenital Adrenal Hypoplasia, familial Addison’s Disease, or other cortisol synthesis or signaling pathway associated conditions or complications, comprising administering an effective amount of the oligonucleotides, alone or in combination to a patient.

Description

Compositions and methods for inhibiting the expression of melanocortin 2 receptors and cytochrome P450-11B1

The present disclosure relates to oligonucleotides or oligonucleotides linked to a targeting moiety, which oligonucleotides can be used, for example, to inhibit, alleviate and/or control cortisol synthesis or signaling pathways in patients. disease or condition, such as Cushing's syndrome. In some embodiments, the present disclosure relates to methods of administering to a subject in need thereof a therapeutically effective amount of one or more RNAi oligonucleotides or one or more RNAi molecules that inhibit The subject's melanocortin receptor 2 ("MC2R") expression and/or 11β-hydroxylase ("CYP11B1"). Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 63/252,396, filed on October 5, 2021, which is incorporated herein by reference in its entirety.

The adrenal glands are located on top of each kidney and produce a variety of hormones. The outer structure of the adrenal gland (adrenal cortex) is the largest part of the adrenal gland. It is divided into three separate areas: the glomerular area, the fascicular area, and the reticular area. Each area is responsible for producing specific hormones.

In the human adrenal gland, aldosterone is produced in the zone glomeruli (ZG). The CYP11B2 gene expressed in ZG encodes aldosterone synthase. The zone fasciculata (ZF) produces cortisol. Cortisol is important for maintaining blood pressure, regulating blood sugar, and reducing inflammation (niddk.nih.gov). The CYP11B1 gene expressed in ZF encodes 11β-hydroxylase. The reticular zone (ZR) produces adrenal androgens (DHEA, DHEA-s) (Wallace, Cunningham and Lindsay, Ann Clin Biochem. 2009 Sep; 46 (Pt 5): 351-67).

Cushing's syndrome (CS) is a multisystem disorder characterized by hypercortisolism. Cushing's syndrome with a pituitary-dependent branch (eg, caused by a pituitary tumor) is called Cushing's Disease (CD). Most cases of CS are caused by pituitary-dependent Cushing's syndrome (ie, Cushing's disease). (Pivonello et al ., Endocr Rev. 2015 Aug; 36(4): 385–486). The regulation of cortisol synthesis and release is controlled by the hypothalamus-pituitary-adrenal (HPA) axis. In response to physiological signals, hypothalamic neurons secrete corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to release adrenocorticotropic hormone (ACTH). ACTH promotes the secretion of cortisol from the adrenal cortex of the adrenal glands. Cortisol in turn inhibits the axis at the level of the pituitary gland and hypothalamus (negative feedback).

In the cortisol synthesis pathway, ACTH binds to the melanocortin 2 receptor (MC2R) and activates the production of cyclic adenylate monophosphate (cAMP) through adenylyl cyclase (Gallo-Payet & Battista, Compr Physiol . 2014 Jul;4(3):889-964). Functional ACTH receptors include MC2R and MC2R accessory protein (MRAP) (Roy et al ., Molecular Endocrinology, Volume 25, Issue 11, 1 November 2011, Pages 1961–1977). As cytosolic cAMP increases, the phosphorylation cascade of activated steroidogenic acute regulatory (StAR) protein is activated (Gallo-Payet & Battista, Compr Physiol. 2014 Jul;4(3): 889-964). StAR proteins control the transport of lipophilic cholesterol (a substrate for the initial steps of steroidogenesis) from the cytosol to the inner mitochondrial membrane, where it is converted to pregnenolone by CYP1A1. From that point on, steroidogenesis occurs by transporting steroid products out of the mitochondria and then back into the mitochondria. Cortisol is produced by CYP11B1 in the final step. Cortisol diffuses into the extracellular fluid and into the microvessels of the adrenal cortex (Raff & Carroll, J Physiol . 2015 Feb 1; 593(Pt 3): 493–506).

Cushing's syndrome is associated with many associated symptoms and morbidities. Circulating serum and urinary cortisol levels are elevated, and a cortisol circadian rhythm deficiency may occur. Other symptoms and complications include weight gain, fatigue, thinning of the skin, and easy bruising. Comorbidities commonly associated with cortisol-induced weight gain include hypertension, diabetes, dyslipidemia, osteoporosis, and depression. In men, sexual dysfunction is common, while in women, menstrual irregularities, acne, and hirsutism frequently occur. Infertility can occur in both men and women (Pivonello et al., Endocrinol Metab Clin North Am. 2008;37(1):135–49)(Newell-Price J. et al., Lancet. 2006;367(9522) :1605–17).

The first-line treatment for CD is pituitary surgery, with subsequent remission in approximately 78% of patients and relapse in approximately 13% of patients during the 10-year postoperative period, so that nearly one-third of patients (i.e., 22% who do not initially achieve remission) patients and 13% of patients with recurrence) experience long-term surgical failure and require additional second-line therapy. Patients with persistent or relapsing CD require additional treatments, including pituitary radiation, adrenal surgery, and/or medications. In recent years, drug therapy has played a more important role in the preoperative and postoperative treatment of CD. Current treatments target pituitary ACTH secretion, adrenal cortisol production, or the effects of glucocorticoids on peripheral tissues. Approved treatments include mifepristone (a glucocorticoid receptor antagonist and competitive inhibitor of cortisol at this receptor), osilodrostat , and metyrapone ( Metyrapone) (CYP11B1 inhibitor), and Pasireotide (reduces ACTH production in the pituitary gland, but has no effect on ectopic ACTH secretion). Many current treatments come with their own side effects and risks, and often do not result in complete relief of symptoms (Pivonello et al ., Endocr Rev. 2015 Aug; 36(4): 385–486). For these reasons, there remains a need for therapeutic agents for the treatment of diseases or conditions related to cortisol synthesis and signaling pathways.

The present disclosure is based in part on the discovery of oligonucleotides (eg, RNAi oligonucleotides) that reduce MC2R or CYP11B1 expression in vitro and in vivo. Furthermore, lipid-conjugated oligonucleotides showed reduced expression of MC2R or CYP11B1 in the adrenal gland in vivo. As shown here, the mouse model of Cushing's disease exhibits increased plasma corticosterone and thicker adrenal glands, as well as ZF vacuolization and ZG extrusion. Without being bound by theory, a thicker adrenal cortex is caused by increased corticosterone synthesis and subsequent cellular hypertrophy, and thus the present disclosure provides guidance for targeting genes involved in cortisol synthesis and signaling pathways (e.g., MC2R and CYP11B1 ) oligonucleotides, which are shown herein to reduce plasma corticosterone levels and reduce the thickness of the adrenal cortex. Specifically, as shown herein, oligonucleotides targeting MC2R (i) reduce plasma corticosterone levels over time to near normal levels and (ii) reduce adrenocortical thickness, indicating reduced cortisol synthesis ; and (iii) reverse ZF vacuolation and ZG extrusion. Likewise, oligonucleotides targeting CYP11B1 reduce plasma corticosterone levels. When used together, oligonucleotides targeting MC2R and CYP11B1 further reduced plasma corticosterone levels compared to when the oligonucleotides were used alone (i.e., comparable to the 40 to 60% reduction achieved by the individual oligonucleotides). Compared to merging, it was reduced by about 80%). Without wishing to be bound by theory, oligonucleotides targeting MC2R or CYP11B1 alone or in combination may be used to treat diseases or conditions related to cortisol synthesis and signaling, such as Cushing's disease.

Accordingly, in some aspects, the present disclosure provides an oligonucleotide for reducing the expression of melanocortin 2 receptor (MC2R), the oligonucleotide comprising 15 to 30 nucleotides in length. A sense strand and a sense strand of 15 to 40 nucleotides in length, wherein the sense strand and the antisense strand form a double-stranded helix region, wherein the antisense strand has the same structure as any of SEQ ID NOs: 201 to 212. One shows the complementary region of the MC2R target sequence. In some aspects, the complementary region is completely complementary to the MC2R target sequence. In some aspects, the antisense strand includes the sequence set forth in any of SEQ ID NOs: 37-48. In some aspects, the sense strands comprise the sequence set forth in any of SEQ ID NOs: 85-96.

In some aspects, the present disclosure provides an oligonucleotide for reducing melanocortin 2 receptor (MC2R) expression, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and A sense strand 15 to 40 nucleotides long, wherein the sense strand and the antisense strand form a double-stranded helix region, wherein the antisense strand has the same structure as any one of SEQ ID NOs: 229 to 420 The complementary region of the MC2R target sequence is shown.

In some aspects, the present disclosure provides an oligonucleotide for reducing melanocortin 2 receptor (MC2R) expression, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and A sense strand of 15 to 40 nucleotides long, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has the same properties as in SEQ ID NOs: 201 to 212 and 229 to 240 The complementary region of the target sequence of MC2R shown in either.

In some aspects, the antisense strand includes a sequence set forth in any of SEQ ID NOs: 805-996. In some aspects, the sense shares comprise the sequence set forth in any of SEQ ID NOs: 613-804.

In some aspects, the present disclosure provides an oligonucleotide for reducing the expression of cytochrome P450 family 11 subfamily B member 1 (CYP11B1), the oligonucleotide comprising 15 to 30 nucleotides in length. An antisense strand and a sense strand of 15 to 40 nucleotides in length, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has a complementary region to the target sequence of CYP11B1, wherein the The target sequence is 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and wherein the complementary region is at least 15 contiguous nucleotides long. In some aspects, the complementary region is completely complementary to the target sequence of CYP11B1.

In any of the foregoing or related aspects, the antisense strand is 19 to 27 nucleotides long. In some aspects, the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long. In any of the foregoing or related aspects, the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long. In some aspects, the double helix region is at least 19 nucleotides long. In some aspects, the double helix region is at least 20 nucleotides long, optionally wherein the double helix region is 21 nucleotides long.

In any of the foregoing or related aspects, the complementary region is at least 19 contiguous nucleotides long. In some aspects, the complementary region is at least 21 contiguous nucleotides long.

In any of the foregoing or related aspects, the sense strand includes at its 3' end a stem-loop as follows: S1-L-S2, where S1 is complementary to S2, and where L is between S1 and S2 A loop of 3 to 5 nucleotides in length is formed between them.

In other aspects, the present disclosure provides an oligonucleotide for reducing MC2R performance, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and having a complementary region to the target sequence of MC2R as set forth in any one of SEQ ID NOs: 201 to 212, wherein the sense strand contains at its 3' end a stem-loop as shown below: S1- L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a loop that is at least 19 nucleotides long. The double helix region. In some aspects, the complementary region is at least 19 nucleotides long and is completely complementary to the target sequence.

In some aspects, the present disclosure provides an oligonucleotide for reducing MC2R performance, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and having a complementary region to the target sequence of MC2R as set forth in any one of SEQ ID NOs: 229 to 420, wherein the sense strand contains at its 3' end a stem-loop as shown below: S1- L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a loop that is at least 19 nucleotides long. The double helix region.

In some aspects, the invention provides an oligonucleotide for reducing MC2R expression, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long , and having a complementary region to the target sequence of MC2R as set forth in any one of SEQ ID NOs: 201 to 212 and 229 to 420, wherein the sense strand includes at its 3' end a stem loop as shown below :S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a 3 to 5 nucleotide long loop between S1 and S2, and wherein the antisense strand and the sense strand form at least 19 nuclei A long double helix region of nucleotides.

In some aspects, the invention provides an oligonucleotide for reducing CYP11B1 expression, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long , and has a complementary region to the target sequence of CYP11B1, wherein the target sequence is 15 to 30 contiguous nucleotides of SEQ ID NO: 226, wherein the sense strand includes the stem shown below at its 3' end Loop: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a 3 to 5 nucleotide long loop between S1 and S2, and wherein the antisense strand and the sense strand form at least 19 The long double helix structure of nucleotides. In some aspects, the complementary region is 19 contiguous nucleotides in length and is completely complementary to the target sequence.

In any of the foregoing or related aspects, L is four rings. In some aspects, L is 4 nucleotides long. In some aspects, L includes the sequence represented by GAAA.

In any of the foregoing or related aspects, (i) the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long, or (ii) the antisense strand is 22 nucleosides The acid and sense strand is 36 nucleotides long. In some aspects, the antisense and sense strands form a double helix region that is 25 nucleotides long or 20 nucleotides long.

In any of the foregoing or related aspects, the antisense strand comprises a 3' overhang sequence of one or more nucleotides in length. In some aspects, the 3' overhang sequence is 2 nucleotides long. In some aspects, the 3' overhang contains two purine nucleotides. In some aspects, the 3' overhang is GG, AA, GA, or AG. In some aspects, the 3' overhang sequence is GG.

In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide includes a 2'-modification. In some aspects, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy -Modification of 2'-fluoro-β-d-arabinic acid. In some aspects, about 10 to 15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise 2'-fluorine modifications. In some aspects, the antisense strand is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% contained 2'-fluoro modifications. In some aspects, the oligonucleotide is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% containing 2'-fluoro modifications. In some aspects, the sense strand includes 36 nucleotides from 5' to 3 at positions 1 to 36, wherein positions 8 to 11 include a 2'-fluoro modification. In some aspects, positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications. In some aspects, the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, and wherein positions 2, 3, 4, 5, 7, 10, and 14 include 2'- Fluorine modification. In some aspects, the antisense strand contains 2'-O-methyl modifications at positions 1, 6, 8, 9, 11 to 13, and 15 to 22. In some aspects, the remaining nucleotides contain 2'-O-methyl modifications. In some aspects, all nucleotides of the oligonucleotide are modified.

In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide linkage. In some aspects, at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand includes a phosphorothioate linkage between positions 1 and 2 of the sense strand. In some aspects, the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand is included at positions 1 and 2, 2 and 3, 20 and 21, and Phosphorothioate linkage between 21 and 22. In some aspects, the sense strand includes a phosphorothioate linkage between positions 1 and 2 of the sense strand. In some aspects, the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand is included at positions 1 and 2, 2 and 3, 3 and 4, Phosphorothioate linkages between 20 and 21 and 21 and 22.

In any of the foregoing or related aspects, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analogue. In some aspects, the phosphate analog is methoxyphosphonate, vinyl phosphonate, or malonyl phosphonate.

In any of the foregoing or related aspects, at least one nucleotide of the oligonucleotide is coupled to one or more targeting ligands. In some aspects, a nucleotide is bound to more than one target ligand, wherein the target ligands are the same or different. In some aspects, one or more targeting ligands are selected from carbohydrates, amino sugars, cholesterol, polypeptides, or lipids.

In some aspects, one or more targeting ligands are saturated or unsaturated fatty acid moieties. In some aspects, the target ligand is a long saturated fatty acid moiety ranging in size from C10 to C24. In some aspects, the target ligand is a C16 saturated fatty acid moiety. In some aspects, the target ligand is a C18 saturated fatty acid moiety. In some aspects, the target ligand is a C22 saturated fatty acid moiety.

In some aspects, one or more targeting ligands comprise an N-acetylgalactosamine (GalNAc) moiety. In some aspects, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some aspects, up to four nucleotides of L of the stem loop are each joined to a monovalent GalNAc moiety.

In any of the foregoing or related aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively.

In any of the foregoing or related aspects, the sense strand and antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; (l) SEQ ID NO: 48 and 96 respectively; (m) are SEQ ID NO: 632 and 824 respectively; (n) SEQ ID NO: 765 and 957 respectively; (o) SEQ ID NO: 770 and 962 respectively; and (p) are SEQ ID NO: 38 and 86 respectively.

In any of the foregoing or related aspects, the sense strand and antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) SEQ ID NO: 38 and 86 respectively.

In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:37 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:85. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:38 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:86.

In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:248 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:440. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:381 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:573. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:386 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:578. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:203 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:215.

In some aspects, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 176 to 187. In some aspects, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 174-175. In some aspects, the MC2R oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 188 to 199.

In some aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 186 and 188 respectively; (b) SEQ ID NO: 187 and 189 respectively; (c) SEQ ID NO: 176 and 190 respectively; (d) SEQ ID NO: 177 and 191 respectively; (e) SEQ ID NO: 178 and 192 respectively; (f) SEQ ID NO: 179 and 193 respectively; (g) SEQ ID NO: 180 and 194 respectively; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 184 and 198 respectively; and (l) SEQ ID NO: 185 and 199 respectively.

In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:186 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:188. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:187 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:189.

In some aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 174 and 188 respectively; and (b) SEQ ID NO: 175 and 189 respectively.

In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:174 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:188. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:175 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:189.

In some aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 186 and 188 respectively; (b) SEQ ID NO: 187 and 189 respectively; (c) SEQ ID NO: 176 and 190 respectively; (d) SEQ ID NO: 177 and 191 respectively; (e) SEQ ID NO: 178 and 192 respectively; (f) SEQ ID NO: 179 and 193 respectively; (g) SEQ ID NO: 180 and 194 respectively; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 184 and 198 respectively; (l) SEQ ID NO: 185 and 199 respectively; (m) are SEQ ID NO: 997 and 1001 respectively; (n) SEQ ID NO: 998 and 1002 respectively; (o) SEQ ID NO: 999 and 1003 respectively; and (p) are SEQ ID NO: 1000 and 1004 respectively.

In some aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 997 and 1001 respectively; (b) SEQ ID NO: 998 and 1002 respectively; (c) SEQ ID NO: 999 and 1003 respectively; and (d) SEQ ID NO: 1000 and 1004 respectively.

In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:997 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1001. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:998 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1002. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:999 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1003. In some aspects, MC2R oligonucleotides comprise a sense strand comprising the nucleotide sequence of SEQ ID NO:1000 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1004.

In some aspects, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 1005 and 1001 respectively; (b) SEQ ID NO: 1006 and 1002 respectively; (c) SEQ ID NO: 1007 and 1003 respectively; and (d) SEQ ID NO: 1008 and 189 respectively.

In some aspects, MC2R oligonucleotides comprise a sense strand comprising the nucleotide sequence of SEQ ID NO:1005 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1001. In some aspects, MC2R oligonucleotides comprise a sense strand comprising the nucleotide sequence of SEQ ID NO:1006 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1002. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:1007 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:1003. In some aspects, the MC2R oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO:1008 and an antisense strand comprising the nucleotide sequence of SEQ ID NO:189.

In some aspects, the present disclosure provides an oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising A region complementary to the target sequence in MC2R mRNA and (ii) one or more targeting ligands, wherein the oligonucleotide-ligand conjugate is represented by Formula Ia: Or its medically acceptable salt, wherein: B is a nucleobase; R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R , -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or R ¹ and R ² on the same carbon together with their intervening atoms form 0 to 3 independently selected from nitrogen, oxygen and a 3 to 7-membered saturated or partially unsaturated ring of sulfur heteroatoms; each R ^A is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, 4 to 7 membered saturated or partially unsaturated heterocycles having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur to a 6-membered heteroaromatic ring; each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaryls having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur Ring; or two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaryl having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur ring; each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain 0 to 10 methylene units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O- , -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR- substitution; Each -Cy- is independently a bivalent ring that is optionally substituted, The divalent ring system is selected from the group consisting of a phenylene group, an 8- to 10-membered bicyclic aryl group, a 4- to 7-membered saturated or partially unsaturated carbocyclylenyl group, and a 4- to 11-membered saturated or partially unsaturated spirocyclic ring. Carbocyclyl, 8 to 10-membered bicyclic saturated or partially unsaturated carbocyclyl, 4 to 7-membered saturated or partially unsaturated heteroatoms with 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. Cyclocyclyl, a 4 to 11-membered saturated or partially unsaturated spirocyclic heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, having 1 to 2 heteroatoms independently selected from nitrogen, 8 to 10 membered bicyclic saturated or partially unsaturated heterocyclyl groups of oxygen and sulfur heteroatoms, 5 to 6 membered heteroaryl groups having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or An 8- to 10-membered bicyclic heteroaryl group having 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or Branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-, -V ¹ CR ² W ¹ -or Substitution; m is 1 to 50; X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-; Y is hydrogen , Suitable hydroxyl protecting group, or ; R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from X ² is ^{O ,} S or _NR ; The linking group; Y ² is hydrogen, a suitable protecting group, a phosphoramidite analogue, an internucleotide linking group attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a solid support attached and Z is -O-, -S-, -NR- or -CR ₂ -.

In other aspects, the present disclosure provides an oligonucleotide-ligand conjugate for reducing CYP11B1 mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising A region complementary to the target sequence in CYP11B1 mRNA and (ii) one or more target ligands, wherein the oligonucleotide-ligand conjugate is represented by Formula Ia : Or its medically acceptable salt, wherein: B is a nucleobase; R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R , -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or R ¹ and R ² on the same carbon together with their intervening atoms form 0 to 3 independently selected from nitrogen, oxygen and a 3 to 7-membered saturated or partially unsaturated ring of sulfur heteroatoms; each R ^A is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, 4 to 7 membered saturated or partially unsaturated heterocycles having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur to a 6-membered heteroaromatic ring; each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaryls having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur Ring; or two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaryl having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur ring; each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain 0 to 10 methylene units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O- , -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR- substitution; Each -Cy- is independently a bivalent ring that is optionally substituted, The divalent ring system is selected from the group consisting of a phenylene group, an 8- to 10-membered bicyclic aryl group, a 4- to 7-membered saturated or partially unsaturated carbocyclic group, and a 4 to 11-membered saturated or partially unsaturated spirocyclic carbocyclic group. radical, an 8- to 10-membered bicyclic saturated or partially unsaturated carbocyclyl group, a 4 to 7-membered saturated or partially unsaturated heterocyclyl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. , having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4 to 11 membered saturated or partially unsaturated spirocyclic heterocyclyl group, having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur An 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic aryl group having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 5 8- to 10-membered bicyclic heteroarylene groups independently selected from nitrogen, oxygen or sulfur heteroatoms; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched chain C _1-50 hydrocarbon chains, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C( O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-, -V ¹ CR ² W ¹ -or Substitution; m is 1 to 50; X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-; Y is hydrogen , Suitable hydroxyl protecting group, or ; R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from X ² is ^{O ,} S or _NR ; The linking group; Y ² is hydrogen, a suitable protecting group, a phosphoramidite analogue, an internucleotide linking group attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a solid support attached and Z is -O-, -S-, -NR- or -CR ₂ -.

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate is represented by Formula II-b or II-c : Or a medically acceptable salt thereof, wherein: L ¹ is a covalent bond, a monovalent or divalent saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene The base units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O) -, -S(O) ₂ -, -P(O)OR-, -P(S)OR-or Replacement; R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene units independently -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O ) ₂ -, -P(O)OR- or -P(S)OR substitution.

In some aspects, the present disclosure provides an oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising A region complementary to the target sequence in MC2R mRNA and (ii) one or more targeting ligands, wherein the oligonucleotide-ligand conjugate is represented by formula II-Ib or II-Ic : or its medically acceptable salt; wherein B is a nucleobase; m is 1 to 50; X ¹ is -O- or -S-; Y is hydrogen, or ; ^{R 3} is hydrogen or a suitable protecting group; ^{X 2} is O or S; A linker at the 2' or 3'end; Y ² is hydrogen, a phosphoramidite analog, an inter-nucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or an attached solid The linking group of the support; R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, in which the 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C ( O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S)OR substitution; and R is hydrogen, a suitable protecting group or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently 4 to 7 membered saturated or partially unsaturated heterocyclic rings having heteroatoms selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

In other aspects, the present disclosure provides an oligonucleotide-ligand conjugate for reducing CYP11B1 mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising Complementary region to the target sequence in CYP11B1 mRNA and (ii) one or more target ligands, wherein the oligonucleotide-ligand conjugate is represented by formula II-Ib or II-Ic: or its medically acceptable salt; wherein B is a nucleobase; m is 1 to 50; X ¹ is -O- or -S-; Y is hydrogen, or ; ^{R 3} is hydrogen or a suitable protecting group; ^{X 2} is O or S; A linker at the 2' or 3'end; Y ² is hydrogen, a phosphoramidite analog, an inter-nucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or an attached solid The linking group of the support; R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, in which the 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C ( O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S)OR substitution; and R is hydrogen, a suitable protecting group or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently 4 to 7 membered saturated or partially unsaturated heterocyclic rings having heteroatoms selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

In any of the foregoing or related aspects, ^R5 is selected from

In any of the foregoing or related aspects, ^R5 is selected from and

In some guises, the R ⁵ Series .

In any of the foregoing or related aspects, the nucleotide sequence includes 1 to 10 targeting ligands. In some aspects, the nucleotide sequence contains 1, 2, or 3 targeting ligands.

In any of the foregoing or related aspects, the cells in the adrenal cortex or adrenal gland are epithelial cells.

In any of the foregoing or related aspects, the oligonucleotide is single-stranded. In some aspects, single-stranded oligonucleotides are 15 to 30 nucleotides long. In other aspects, the oligonucleotide is double-stranded.

In some aspects, a double-stranded oligonucleotide includes an antisense strand that is 15 to 30 nucleotides long and a sense strand that is 15 to 40 nucleotides long, wherein the sense strand and the antisense strand form A double helix region, wherein the antisense strand comprises a region of complementarity to the target sequence, and wherein the region of complementarity is at least 15 contiguous nucleotides in length. In some aspects, the antisense strand is 19 to 27 nucleotides long. In some aspects, the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long. In some aspects, the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long. In some aspects, the double helix region is at least 19 nucleotides long. In some aspects, the double helix region is at least 20 nucleotides long, optionally wherein the double helix region is 21 nucleotides long. In some aspects, the complementary region is at least 19 contiguous nucleotides long. In some aspects, the complementary region is at least 21 contiguous nucleotides long.

In any of the foregoing or related aspects, the sense strand of the double-stranded oligonucleotide includes at its 3' end a stem loop as shown below: S1-L-S2, wherein S1 is complementary to S2, and Where L forms a 3 to 5 nucleotide long loop between S1 and S2. In some forms, L is four rings. In some aspects, L includes the sequence GAAA. In some aspects, one or more targeting ligands are associated with the stem loop. In some aspects, one or more targeting ligands are coupled to one or more nucleotides of L. In some aspects, L includes four nucleotides at positions numbered 1 to 4 from 5' to 3', with one or more targeting ligands bound to position 2.

In any of the foregoing or related aspects, the antisense strand of the double-stranded oligonucleotide comprises one or more nucleotide-long 3' overhang sequences. In some aspects, the 3' overhang sequence is 2 nucleotides long. In some aspects, the 3' overhang sequence is 2 purine nucleotides. In some aspects, the 3' overhang sequence is GG, AA, GA, or AG. In some aspects, the 3' overhang sequence is GG.

In any of the foregoing or related aspects, the oligonucleotide of the oligonucleotide-ligand conjugate comprises at least one modified nucleotide. In some aspects, the modified nucleotide includes a 2'-modification. In some aspects, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy -Modification of 2'-fluoro-β-d-arabinic acid. In some aspects, about 10 to 15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise 2'-fluorine modifications. In some aspects, the antisense strand is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% contained 2'-fluoro modifications. In some aspects, the oligonucleotide is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% containing 2'-fluoro modifications. In some aspects, the sense strand includes 36 nucleotides from 5' to 3' at positions 1 to 36, wherein positions 8 to 11 include a 2'-fluoro modification. In some aspects, positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications. In some aspects, the antisense strand includes 22 nucleotides from 5' to 3' at positions 1 to 22, and wherein positions 2, 3, 4, 5, 7, 10, and 14 include 2'- Fluorine modification. In some aspects, the antisense strand contains 2'-O-methyl modifications at positions 1, 6, 8, 9, 11 to 13, and 15 to 22. In some aspects, the remaining nucleotides contain 2'-O-methyl modifications. In some aspects, all nucleotides of the oligonucleotide are modified.

In any of the foregoing or related aspects, the oligonucleotide of the oligonucleotide-ligand conjugate comprises at least one modified internucleotide linkage. In some aspects, at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand includes a phosphorothioate linkage between positions 1 and 2 of the sense strand. In some aspects, the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand is included at positions 1 and 2, 2 and 3, 20 and 21, and Phosphorothioate linkage between 21 and 22.

In any of the foregoing or related aspects, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand of the oligonucleotide-ligand conjugate comprises a phosphate analogue. In some aspects, the phosphate analog is methoxyphosphonate, vinyl phosphonate, or malonyl phosphonate.

In any of the foregoing or related aspects, R ⁵ is . In some guises, the R ⁵ Series . In some guises, the R ⁵ Series .

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a component selected from the group consisting of: The nucleotide sequence of the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively.

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a component selected from the group consisting of: The nucleotide sequence of the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; (l) SEQ ID NO: 48 and 96 respectively; (m) SEQ ID NO: 632 and 824 respectively; (n) SEQ ID NO: 765 and 957 respectively; (o) SEQ ID NOs: 770 and 962 respectively; and (p) SEQ ID NO: 38 and 86 respectively.

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a component selected from the group consisting of: The nucleotide sequence of the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) SEQ ID NO: 38 and 86 respectively.

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 174 and a sense strand comprising SEQ ID NO: 188 The antisense nucleotide sequence. In some aspects, an oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 175 and a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 189. Antisense stocks.

In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1005 and a sense strand comprising SEQ ID NO: 1001 The antisense nucleotide sequence. In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1006 and a sense strand comprising SEQ ID NO: 1002 The antisense nucleotide sequence. In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1007 and a sense strand comprising SEQ ID NO: 1003 The antisense nucleotide sequence. In any of the foregoing or related aspects, the oligonucleotide-ligand conjugate targeting MC2R comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1008 and a sense strand comprising SEQ ID NO: 189 The antisense nucleotide sequence.

In some aspects, the present disclosure provides a pharmaceutical composition comprising an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, and a medically acceptable carrier. agents, delivery agents or excipients.

In other aspects, the present disclosure provides a kit comprising an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, optionally a medically acceptable A vehicle and instructions containing instructions for administration to a subject suffering from a disease, disorder or condition associated with expression of MC2R. In some aspects, the kit includes instructions for administering an oligonucleotide or oligonucleotide-ligand conjugate targeting MC2R to a subject that has received or is receiving the inhibitor CYP11B1.

In additional aspects, the present disclosure provides a method for treating a subject suffering from a disease, disorder, or condition associated with expression of MC2R, the method comprising administering to the subject a therapeutically effective amount of a method described herein MC2R-targeting oligonucleotides or oligonucleotide-ligand conjugates or pharmaceutical compositions thereof. In some aspects, the disease, disorder, or condition associated with manifestation of MC2R is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital Adrenal agenesis and familial Addison's disease group. In some aspects, the disease, disorder, or condition associated with manifestations of MC2R is Cushing's syndrome. In some aspects, an oligonucleotide or oligonucleotide-ligand conjugate targeting MC2R is used together with a second composition or therapeutic agent. In some aspects, the second composition or therapeutic agent is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production, or glucocorticoid effects on surrounding tissue, surgical procedures, radiation Procedures, gene therapy, mifepristone, glucocorticoid receptor antagonists and competitive inhibitors of cortisol at this receptor, osilodrostat, metyrapone, CYP11B1 inhibitors, pasireotide or combination thereof. In some aspects, the second composition or therapeutic agent targets CYP11B1. In some aspects, an oligonucleotide or oligonucleotide-ligand conjugate targeting MC2R is administered with at least 2 additional therapeutic agents.

In other aspects, the present disclosure provides a method of treating Cushing's syndrome in a subject, comprising administering to the subject an oligonucleotide or oligonucleotide-ligand targeting MC2R as described herein A conjugate or a pharmaceutical composition thereof, wherein the cells in the adrenal gland or adrenal cortex express MC2R.

In additional aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, wherein the The oligonucleotide or oligonucleotide-ligand conjugate reduces the expression, production or activity of MC2R.

In some aspects, the present disclosure provides use of an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, or a pharmaceutical composition thereof, to reduce MC2R expression in the adrenal gland. .

In other aspects, the present disclosure provides an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein or a pharmaceutical composition thereof for reducing the risk of disease associated with MC2R manifestations. Use of MC2R representations of diseases, disorders or conditions. In some aspects, the disease, disorder, or condition associated with manifestation of MC2R is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital Adrenal agenesis and familial Addison's disease group.

In some aspects, the present disclosure provides a pharmaceutical composition comprising a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, and a medically acceptable carrier. agents, delivery agents or excipients.

In other aspects, the present disclosure provides a kit comprising a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, optionally a medically acceptable A vehicle and instructions containing instructions for administration to a subject suffering from a disease, disorder or condition associated with expression of CYP11B1. In some aspects, the kit includes instructions for administering an oligonucleotide or oligonucleotide-ligand conjugate targeting CYP11B1 to a subject that has received or is receiving an inhibitor MC2R.

In additional aspects, the present disclosure provides a method for treating a subject suffering from a disease, disorder, or condition associated with expression of CYP11B1, the method comprising administering to the subject a therapeutically effective amount of a drug described herein. Oligonucleotides or oligonucleotide-ligand conjugates targeting CYP11B1 or pharmaceutical compositions thereof. In some aspects, the disease, disorder, or condition associated with expression of CYP11B1 is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital Adrenal agenesis and familial Addison's disease group. In some aspects, the disease, disorder, or condition associated with expression of CYP11B1 is Cushing's syndrome. In some aspects, an oligonucleotide or oligonucleotide-ligand conjugate targeting CYP11B1 is administered with a second composition or therapeutic agent. In some aspects, the second composition or therapeutic agent is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production, or glucocorticoid effects on surrounding tissue, surgical procedures, radiation Procedures, gene therapy, mifepristone, glucocorticoid receptor antagonists and competitive inhibitors of cortisol at this receptor, osilodrostat, metyrapone, MC2R inhibitors, pasireotide or combination thereof. In some aspects, the second composition or therapeutic agent targets MC2R. In some aspects, an oligonucleotide or oligonucleotide-ligand conjugate targeting CYP11B1 is administered with at least 2 additional therapeutic agents.

In other aspects, the present disclosure provides a method of treating Cushing's syndrome in a subject, comprising administering to the subject a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand described herein A conjugate or a pharmaceutical composition thereof, wherein the cells in the adrenal gland or adrenal cortex express CYP11B1.

In additional aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein, wherein the The oligonucleotide or oligonucleotide-ligand conjugate reduces the expression, production or activity of CYP11B1.

In some aspects, the present disclosure provides a use of a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein or a pharmaceutical composition thereof to reduce CYP11B1 expression in the adrenal gland .

In other aspects, the present disclosure provides a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate as described herein or a pharmaceutical composition thereof for reducing the symptoms of patients with symptoms associated with CYP11B1 expression. Use of CYP11B1 expression in subjects with diseases, disorders or conditions. In some aspects, the disease, disorder, or condition associated with expression of CYP11B1 is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital Adrenal agenesis and familial Addison's disease group.

In some aspects, the present disclosure provides a pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an MC2R inhibitor, and wherein the second therapeutic agent is an inhibitor of CYPB11 expression agent.

In other aspects, the present disclosure provides a pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an oligonucleotide and is an inhibitor of MC2R expression, and wherein the first therapeutic agent is an oligonucleotide and an inhibitor of MC2R expression, and wherein the first therapeutic agent is The two therapeutic agents are inhibitors of CYPB11 expression.

In additional aspects, the present disclosure provides a pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an inhibitor of MC2R expression, and wherein the second therapeutic agent is an oligopeptide. Nucleotides are inhibitors of CYPB11 expression.

In some aspects, the present disclosure provides a pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an oligonucleotide described herein and is an inhibitor of MC2R expression , and wherein the second therapeutic agent is an oligonucleotide described herein and is an inhibitor of CYPB11 expression.

In another aspect, the present disclosure provides a combination product including: (i) An oligonucleotide that inhibits MC2R comprising an antisense strand that is 15 to 30 nucleotides in length and includes a nucleotide sequence as set forth in any one of SEQ ID NO: 85 to 96; and 15 to a sense strand that is 40 nucleotides in length and includes the nucleotide sequence set forth in any of SEQ ID NO: 37 to 48, and (ii) An oligonucleotide that inhibits CYPB11, which contains an antisense strand that is 15 to 30 nucleotides long and includes a complementary region to a target sequence of CYP11B1, wherein the target sequence is 15 to 30 of SEQ ID NO: 226 contiguous nucleotides; and a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded helix region. In some aspects, (i) and (ii) are formulated as an injectable suspension, gel, oil, pill, tablet, suppository, powder, capsule, mist, ointment, cream, patch or for use Extended and/or slow release galenic means.

In some aspects, combination products described herein are used to treat diseases associated with expression of MC2R or CYP11B1.

In some aspects, the present disclosure provides a method of treating a disease associated with expression of MC2R or CYP11B1, comprising administering to a patient who has received or is receiving an oligonucleotide for reducing expression of CYP11B1 for reducing MC2R. Performance oligonucleotides. In other aspects, the present disclosure provides a method of treating a disease associated with expression of MC2R or CYP11B1, comprising administering to a patient who has received or is receiving an oligonucleotide for reducing expression of MC2R for reducing CYP11B1 Performance oligonucleotides. In additional aspects, the present disclosure provides a method of treating a disease associated with MC2R or CYP11B1 expression, comprising administering a targeted agent to a patient who has received or is receiving an oligonucleotide for reducing CYP11B1 expression. Oligonucleotides or oligonucleotide-ligands for MC2R that reduce MC2R expression. In some aspects, the oligonucleotide used to reduce the expression of CYP11B1 is a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein. In other aspects, the present disclosure provides a method of treating a disease associated with expression of MC2R or CYP11B1, comprising administering to a patient who has received or is receiving an oligonucleotide for reducing expression of MC2R for reducing CYP11B1 Oligonucleotides or oligonucleotide-ligands targeting CYP11B1 have been demonstrated. In some aspects, the oligonucleotide used to reduce MC2R expression is an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein. In some aspects, the disorder associated with expression of MC2R or CYP11B1 is selected from Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal development Insufficiency and familial Addison's disease.

In another aspect, the present disclosure provides a method of treating a disease or condition associated with cortisol synthesis and/or signaling pathways, comprising administering to a subject in need thereof an oligonucleotide for reducing MC2R expression. oligonucleotide, wherein the subject has received or is receiving an oligonucleotide for reducing the expression of CYP11B1. In some aspects, the present disclosure provides a method of treating a disease or condition associated with cortisol synthesis and/or signaling pathways, comprising administering to a subject in need thereof an oligonucleotide for reducing CYP11B1 expression. acid, wherein the subject has received or is receiving an oligonucleotide for reducing the expression of MC2R. In other aspects, the present disclosure provides a method of treating a disease or condition associated with cortisol synthesis and/or signaling pathways, comprising administering to a patient who has received or is receiving an oligonucleotide for reducing CYP11B1 expression. The patient is administered an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein for reducing MC2R expression. In some aspects, the oligonucleotide used to reduce the expression of CYP11B1 is a CYP11B1-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein. In some aspects, the present disclosure provides a method of treating a disease or condition associated with cortisol synthesis and/or signaling pathways, comprising administering to a patient who has received or is receiving an oligonucleotide for reducing MC2R expression. The patient is administered a CYP11B1 -targeting oligonucleotide or oligonucleotide-ligand conjugate described herein for reducing the expression of CYP11B1. In some aspects, the oligonucleotide used to reduce MC2R expression is an MC2R-targeting oligonucleotide or oligonucleotide-ligand conjugate described herein. In some aspects, the disease or condition associated with cortisol synthesis and/or signaling pathways is selected from Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical insufficiency of ACTH. Reactive, congenital adrenal hypoplasia, and familial Addison's disease.

I. definition

As used herein, "about" or "approximately" when applied to one or more values of interest refers to a value that is similar to a stated reference value. In some specific examples, "about" means 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14% in any direction (greater or less than) of the specified reference value. %, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less unless otherwise stated or otherwise It's obvious from the context (unless the number exceeds 100% of possible values).

As used herein, "administer," "administering," "administration" and the like refer to providing a subject with a pharmacologically useful form (e.g., treating a condition). Substances (e.g., oligonucleotides).

As used herein, the term "antisense oligonucleotide" encompasses nucleic acid-based molecules that have a sequence, particularly a seed sequence, that is complementary to all or part of a target mRNA (e.g., MC2R or CYP11B1), thereby being able to form an The mRNA forms a double helix. Therefore, the term "antisense oligonucleotide" as used herein may be referred to as "complementary nucleic acid-based inhibitors."

As used herein, "attenuate," "attenuating," "attenuation" and the like refer to reduction or effective cessation. As a non-limiting example, one or more treatments herein may reduce or effectively prevent the onset or progression of a disorder related to cortisol synthesis and signaling pathways (eg, Cushing's syndrome) in a subject. Such attenuation may be exemplified by, for example, a reduction in one or more aspects of the disease (e.g., symptoms, tissue characteristics, and cellular, inflammatory or immune activity, etc.) associated with cortisol synthesis and signaling pathways, Undetectable progression (exacerbation) of one or more aspects of a disease related to cortisol synthesis and signaling pathways, or an undetectable state of a subject's disease related to cortisol synthesis and signaling pathways that might be expected Like.

As used herein, "combination product," "combination therapy," "polytherapy" and the like refer to therapy that uses more than one therapeutic agent or more than one agent or modality for treating a disease or condition. The therapeutic agents comprising the combination may be administered simultaneously, intermittently, or in any sequence. Combination products may include, for example, two oligonucleotides or oligonucleotides combined with antibodies or small molecule drugs. For such therapies, the dosage of each agent used may be altered to optimize and/or enhance patient outcomes.

As used herein, "complementarity" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to form base pairs with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may be base paired in a Watson-Crick manner or in any other manner that allows the formation of a stable double helix. In some embodiments, as described herein, two nucleic acids can have regions of multiple nucleotides that are complementary to each other to form complementary regions.

As used herein, the term "CYP11B1" refers to cytochrome P450 family subfamily 11 B member 1, which is a protein involved in catalyzing metabolic reactions. In the adrenal cortex, CYP11B1 converts progesterone into cortisol. In some specific examples, inhibition of CYP11B1 refers to inhibition of CYP11B1 protein, inhibition of CYP11B1 gene at the transcription level, or both.

As used herein, "deoxyribonucleotide" refers to a nucleotide that has a hydrogen substituted for the hydroxyl group at the 2' position of its pentose sugar when compared to ribonucleotides. Modified deoxyribonucleotides are deoxyribonucleotides having one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions in sugars, phosphate groups, or bases.

As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA oligonucleotide that is substantially in the form of a double-stranded helix. In some embodiments, complementary base pairing of the double helix region(s) of a dsRNA oligonucleotide is formed between antiparallel nucleotide sequences of covalently separated nucleic acid strands. In some embodiments, complementary base pairing of the double helix region(s) of dsRNA is formed between antiparallel nucleotide sequences of covalently linked nucleic acid strands. In some embodiments, complementary base pairing of the double helix region(s) of the dsRNA is achieved by folding (e.g., via hairpins) of a single nucleic acid strand to provide complementary antiparallel nucleotide sequences that are base paired together. formed. In some embodiments, dsRNA includes two covalently separated nucleic acid strands that form a complete double helix with each other. However, in some embodiments, the dsRNA includes two covalently separated nucleic acid strands that form part of a double helix (eg, have overhangs at one or both ends). In some embodiments, the dsRNA contains partially complementary antiparallel nucleotide sequences and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

As used herein, "double helix" with respect to a nucleic acid (eg, an oligonucleotide) refers to a structure formed by complementary base pairing of two antiparallel nucleotide sequences.

As used herein, "excipient" refers to non-therapeutic agents that may be included in the compositions, for example, to provide or contribute to a desired consistency or stabilizing effect.

As used herein, "hepatocyte" or "hepatocytes" refers to cells of the parenchymal tissue of the liver. These cells make up approximately 70% to 85% of the liver mass and produce serum albumin, FBN, and the prothrombin set of coagulation factors (except factors 3 and 4). Markers for cells of the hepatocyte lineage include, but are not limited to, transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes may include, but are not limited to, cytochrome P450 (Cyp3a11), fumarate acetoacetate hydrolase (Fah), glucose-6-phosphate (G6p), albumin (Alb), and OC2-2F8 . See, e.g., Huch et al. (2013) Nature 494:247-50.

As used herein, "labile linker" refers to a linker that can be cleaved (eg, by acidic pH). "Relatively stable linkers" refers to linkers that cannot be cleaved.

As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., In phosphate buffer (in cells), the two antiparallel regions on either side of the unpaired region hybridize to form a double helix (called the "stem").

As used herein, the term "MC2R" refers to the melanocortin 2 receptor (receptor for adrenocorticotropic hormone (ACTH)), a hormone produced in the pituitary gland. This hormone controls the production of cortisol, which is produced in the adrenal glands. Inhibition of MC2R may refer to inhibition of the MC2R protein, inhibition of the MC2R gene at the transcriptional level, or both.

As used herein, a "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications when compared to a reference internucleotide linkage that includes a phosphodiester bond. . In some embodiments, the modified nucleotides have non-naturally occurring linkages. Generally, a modified internucleotide linkage confers one or more desirable properties to the nucleic acid in which the modified internucleotide linkage is present. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications when compared to the corresponding reference nucleotide, the nucleotide being selected from the group consisting of: adenine ribonucleoside nucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides and thymidine deoxyribonucleotides. In some embodiments, the modified nucleotides are non-naturally occurring nucleotides. In some embodiments, modified nucleotides have one or more chemical modifications in their sugar, nucleobase, and/or phosphate groups. In some embodiments, a modified nucleotide has one or more chemical moieties that are joined to a corresponding reference nucleotide. Generally, a modified nucleotide confers one or more desired properties to the nucleic acid in which the modified nucleotide is present. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

As used herein, "nicked tetracyclic structure" refers to the structure of an RNAi oligonucleotide characterized by separate sense (follower) and antisense (leader) strands, wherein the sense strand has The complementary region of the antisense strand, and at least one strand thereof (usually the sense strand) has four loops configured to stabilize adjacent stem regions formed within the at least one strand.

As used herein, "oligonucleotide" refers to a short nucleic acid (eg, less than about 100 nucleotides long). Oligonucleotides can be single-stranded (ss) or ds. Oligonucleotides may or may not have double helix regions. As a set of non-limiting examples, oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer matrix interfering RNA (dsiRNA), Antisense oligonucleotides, short siRN or ss (single-stranded) siRNA. In some embodiments, the double-stranded (dsRNA) is an RNAi oligonucleotide.

As used herein, an "overhang" refers to the terminal non-base-paired nucleotide(s) resulting from a strand or region that extends beyond the end of a complementary strand to form a double helix with the complementary strand. In some embodiments, the overhang includes one or more unpaired nucleotides extending from the double helix region at the 5' or 3' end of the dsRNA. In some embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of dsRNA.

As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is located at the 5' terminal nucleotide of the oligonucleotide in place of a 5'-phosphate, which is usually susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonate esters such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5' -VP). In some embodiments, the oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar at the 5' terminal nucleotide (termed a "4'-phosphate analog"). Examples of 4'-phosphate analogs are methoxyphosphonates (in which the oxygen atom of the methoxy group is bound to the sugar moiety (eg, at its 4'-carbon)) or analogs thereof. See, for example, U.S. Provisional Patent Application Nos. 62/383,207 (filed September 2, 2016) and 62/393,401 (filed September 12, 2016). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., International Patent Application No. WO 2011/133871; U.S. Patent No. 8,927,513; and Prakash et al . (2015) Nucleic Acids Res . 43: 2993-3011).

As used herein, "reduced expression" of a gene (e.g., MC2R or CYP11B1) refers to a cell, cell population, sample, or subject when compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject) A decrease in the amount or level of the RNA transcript (eg, MC2R or CYP11B1 mRNA) or protein encoded by the gene and/or a decrease in the amount or level of activity of the gene in the subject. For example, when compared to cells without dsRNA treatment, cells treated with an oligonucleotide herein (e.g., an oligonucleotide comprising an antisense strand having a core similar to that comprising MC2R or CYP11B1 mRNA) The behavior of contact with a nucleotide sequence that is complementary to the nucleotide sequence) can lead to a decrease in MC2R or CYP11B1 mRNA, protein and/or activity (for example, degradation of MC2R or CYP11B1 mRNA via the RNAi pathway). Likewise, as used herein, "reducing expression" refers to behavior that results in reduced expression of a gene (eg, MC2R or CYP11B1). As used herein, "reduction in MC2R expression" refers to MC2R mRNA, MC2R in a cell, cell population, sample or subject when compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject) Reduction in the amount or level of protein and/or MC2R activity. As used herein, "reduction in CYP11B1 expression" refers to the amount of CYP11B1 mRNA, CYP11B1 protein in a cell, cell population, sample, or subject when compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject) and/or a decrease in the amount or level of CYP11B1 activity.

As used herein, a "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., dsRNA) that is sufficiently complementary to an antiparallel nucleotide sequence to permit hybridization under appropriate hybridization conditions (e.g., in phosphate buffer , hybridization between two nucleotide sequences in cells, etc.). In some embodiments, oligonucleotides herein comprise a target sequence having a complementary region to an mRNA target sequence (eg, MC2R or CYP11B1).

As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar containing a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides that have one or more atomic modifications or substitutions other than the 2' position, including modifications or substitutions in ribose, phosphate groups or bases.

As used herein, "RNAi oligonucleotide" refers to (a) a dsRNA having a sense strand (follower) and an antisense strand (leader), wherein the antisense strand or a portion of the antisense strand is Argonaute 2 (Ago2) endonuclease for cleavage of target mRNA or (b) ss oligonucleotide with a single antisense strand, wherein the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease Cleavage of target mRNA.

As used herein, "strand" refers to a single contiguous sequence of nucleotides linked together by an internucleotide linkage (eg, a phosphodiester linkage or a phosphorothioate linkage). In some embodiments, the strand has two free ends (e.g., a 5' end and a 3' end).

As used herein, "subject" refers to any mammal, including mice, rabbits, and humans. In a specific example, the subject is a human or NHP. Additionally, "individual" or "patient" and "subject" are used interchangeably.

As used herein, "synthetic" refers to a nucleic acid or other molecule that has been synthesized artificially (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not derived from a natural source (e.g., a cell or organism) from which the molecule is ordinarily produced. ) derived nucleic acids or other molecules.

As used herein, "targeting ligand" refers to a molecule (e.g., carbohydrate, amino sugar, cholesterol, peptide, or lipid) that selectively binds to a cognate molecule of a tissue or cell of interest ( For example, a receptor) and can be coupled to another substance for the purpose of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide to target the oligonucleotide to a specific target tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, when the target ligand is coupled to an oligonucleotide, the oligonucleotide, the target ligand, and the receptor are selectively bound to and include the receptor expressed on the cell surface. Cellular internalization of the complex facilitates delivery of the oligonucleotide to specific cells. In some embodiments, the targeting ligand is coupled to the oligonucleotide via a linker that is cleaved after or during cellular internalization, allowing the oligonucleotide to be released from the targeting ligand in the cell.

As used herein, "tetraloop" refers to a loop that increases the stability of adjacent double helices formed by hybridization of nucleotide flanking sequences. An increase in stability is detectable as an increase in the melting temperature (T _m ) of adjacent stem double helices that is higher than expected for adjacent stem double helices (on average, from a set of randomly selected The _Tm of a loop of considerable length composed of a selected nucleotide sequence). For example, a tetraloop may confer a hairpin containing a double helix at least 2 base pairs (bp) long that has a temperature of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C in 10 mM _NaHPO , a _Tm of at least about 60°C, at least about 65°C, or at least about 75°C. In some embodiments, tetraloops can stabilize bp in adjacent stem double helices through stacking interactions. In addition, interactions between nucleotides in the four-ring ring include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al. (1990) Nature 346:680-82; Heus & Pardi (1991) Science 253:191-94). In some embodiments, the tetracycle contains or consists of 3 to 6 nucleotides, and typically 4 to 5 nucleotides. In certain embodiments, a tetracycle includes or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., they may or may not be associated with the target partially joined). In a specific example, the tetracyclic system consists of 4 nucleotides. Any nucleotide may be used in the tetracycle, and the standard IUPAC-IUB notation for such nucleotides may be used as described in Cornish-Bowden (1985) Nucleic Acids Res . 13:3021-30. For example, the letter "N" can be used to show that any base can be at that position, the letter "R" can be used to show that A (adenine) or G (guanine) can be at that position, and "B" can be used to show that C (cyto) Pyrimidine), G (guanine) or T (thymine) can be at this position. Examples of tetracycles include the UNCG family of tetracycles (e.g., UUCG), the GNRA family of tetracycles (e.g., GAAA), and the CUUG tetracycles (Woese et al . (1990) Proc. Natl. Acad. Sci. USA 87:8467 -8471; Antao et al . (1991) Nucleic Acids Res . 19:5901-05). Examples of DNA tetracycles include the d(GNNA) family of tetracycles (e.g., d(GTTA), d(GNRA)) tetracycles, the d(GNAB) family of tetracycles, the d(CNNG) family of tetracycles, and the d(CNNG) family of tetracycles. The d(TNCG) family of rings (eg, d(TTCG)). See, e.g., Nakano et al . (2002) Biochem. 41:4281-14292; Shinji et al . (2000) Nippon Kagakkai Koen Yokoshu 78:731. In some embodiments, the four rings are contained within a notched four-ring structure.

As used herein, "treat" or "treating" refers to the act of providing care to a subject in need thereof, e.g., by administering to the subject a therapeutic agent (e.g., an oligonucleotide herein acid), in order to improve the health and/or well-being of the subject or to prevent or reduce the likelihood of the condition (e.g., disease, disease) occurring in the subject. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor to a condition (eg, disease, disorder) experienced by the subject. II. Oligonucleotide inhibitors of MC2R and CYP11B1 expression

In particular, the present disclosure provides oligonucleotides that inhibit the expression of MC2R or CYP11B1. In some embodiments, oligonucleotides that inhibit MC2R expression target MC2R mRNA. In some embodiments, oligonucleotides that inhibit the expression of CYP11B1 target CYP11B1 mRNA. Accordingly, the present disclosure provides RNAi therapeutics targeting MC2R or CYP11B1. In some specific examples, RNAi therapeutic agents are used alone or in combination. i. MC2R and CYP11B1 target sequences

In some embodiments, oligonucleotides (eg, RNAi oligonucleotides) herein target a target sequence comprising MC2R mRNA. In some embodiments, the oligonucleotides described herein correspond to target sequences within the MC2R mRNA sequence. In some embodiments, oligonucleotides or portions, fragments or strands thereof (eg, antisense or guide strands of dsRNA) bind or adhere to target sequences including MC2R mRNA, thereby inhibiting MC2R expression.

In some embodiments, oligonucleotides (eg, RNAi oligonucleotides) herein target a target sequence comprising CYP11B1 mRNA. In some embodiments, the oligonucleotides described herein correspond to target sequences within the CYP11B1 mRNA sequence. In some embodiments, oligonucleotides or portions, fragments or strands thereof (eg, antisense or guide strands of dsRNA) bind or adhere to target sequences including CYP11B1 mRNA, thereby inhibiting CYP11B1 expression.

In some specific examples, oligonucleotides target MC2R target sequences to achieve the purpose of inhibiting MC2R expression in vivo. In some embodiments, the amount or degree of inhibition of MC2R expression by an oligonucleotide targeting an MC2R target sequence correlates with the potency of the oligonucleotide. In some embodiments, the amount or degree of inhibition of MC2R expression by an oligonucleotide targeting the MC2R target sequence is consistent with the effect of the oligonucleotide on a subject or patient having a disease, disorder or condition associated with MC2R expression. Relevant to the amount or degree of therapeutic benefit.

In some specific examples, oligonucleotides target CYP11B1 target sequences to achieve the purpose of inhibiting the expression of CYP11B1 in vivo. In some embodiments, the amount or degree of inhibition of CYP11B1 expression by an oligonucleotide targeting a CYP11B1 target sequence correlates with the potency of the oligonucleotide. In some embodiments, an oligonucleotide targeting a CYP11B1 target sequence inhibits expression of CYP11B1 by an amount or degree that is consistent with the effect of a subject or patient treated with the oligonucleotide having a disease, disorder, or condition associated with expression of CYP11B1 Relevant to the amount or degree of therapeutic benefit.

By examining the nucleotide sequences of mRNAs encoding MC2R or CYP11B1, including those from multiple different species (e.g., human, stone crab macaque, and mouse; see, e.g., Example 1), and as a result of in vitro and in vivo experiments (See, e.g., Examples 3 to 7), certain nucleotide sequences of MC2R or CYP11B1 mRNA have been found to be more suitable for oligonucleotide-based inhibition than other nucleotide sequences, and thus may be used as oligonucleotides herein. Target sequence of nucleotides. In some embodiments, the sense strand of an oligonucleotide (eg, dsRNA) described herein includes an MC2R target sequence. In some embodiments, a portion or region of the sense strand of the dsRNA described herein includes the MC2R target sequence. The MC2R gene is conserved between humans and rhesus monkeys. In some specific examples, the MC2R target sequence includes or consists of the sequence of any one of SEQ ID Nos: 227 and 228. In some specific examples, the MC2R target sequence includes or consists of any sequence of SEQ ID No: 228. In some embodiments, the sense strand of an oligonucleotide (eg, dsRNA) described herein includes a CYP11B1 target sequence. In some embodiments, a portion or region of the sense strand of the dsRNA described herein includes the CYP11B1 target sequence. The CYP11B1 gene is conserved between humans and rhesus macaques. In some specific examples, the CYP11B1 target sequence includes or consists of the sequence of any one of SEQ ID Nos: 225 and 226. In some specific examples, the CYP11B1 target sequence includes or consists of the sequence of SEQ ID NO: 226. ii. MC2R and CYP11B1 target sequences

In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have complementary regions to MC2R mRNA (e.g., within the target sequence of MC2R mRNA) to target the mRNA in cells and the purpose of inhibiting its expression. In some embodiments, oligonucleotides herein comprise MC2R target sequences (e.g., antisense or guide strands of dsRNA) that have the ability to interact with the MC2R through complementary (Watson-Crick) base pairing. The complementary region to which the target sequence binds or adheres. The target sequence or complementary region usually has a suitable length and base content so that the oligonucleotide (or strand thereof) can bind or adhere to MC2R mRNA to inhibit its expression.

In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have complementary regions to CYP11B1 mRNA (e.g., within the target sequence of CYP11B1 mRNA) to target the mRNA in cells and the purpose of inhibiting its expression. In some embodiments, oligonucleotides herein comprise a CYP11B1 target sequence (e.g., an antisense or guide strand of a dsRNA) that has the ability to interact with CYP11B1 via complementary (Watson-Crick) base pairing. The complementary region to which the target sequence binds or adheres. The target sequence or complementary region usually has a suitable length and base content so that the oligonucleotide (or strand thereof) can bind or adhere to CYP11B1 mRNA to inhibit its expression.

In some specific examples, the target sequences or complementary regions are at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19 at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29 or at least about 30 nucleotides long. In some embodiments, the target sequence or complementary region is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30 ) nucleotide length. In some embodiments, the target sequence or complementary region is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the target sequence or complementary region is 18 nucleotides long. In some embodiments, the target sequence or complementary region is 19 nucleotides long. In some embodiments, the target sequence or complementary region is 20 nucleotides long. In some embodiments, the target sequence or complementary region is 21 nucleotides long. In some embodiments, the target sequence or complementary region is 22 nucleotides long. In some embodiments, the target sequence or complementary region is 23 nucleotides long. In some embodiments, the target sequence or complementary region is 24 nucleotides long.

In some embodiments, the oligonucleotides herein comprise a target sequence or complementary region that is fully complementary to the MC2R target sequence (eg, the antisense or leader strand of a double-stranded oligonucleotide). In some embodiments, the target sequence or complementary region is partially complementary to the MC2R target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence of MC2R.

In some embodiments, the oligonucleotides herein comprise a target sequence or complementary region that is fully complementary to the CYP11B1 target sequence (eg, the antisense or leader strand of a double-stranded oligonucleotide). In some embodiments, the target sequence or complementary region is partially complementary to the CYP11B1 target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence of CYP11B1.

In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 201 to 212, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to the sequences of SEQ ID NOs: 203 and 209, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 203, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 209, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to a sequence selected from SEQ ID NO: 203 and 209, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 203, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 209, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to a sequence selected from SEQ ID NO: 203 and 209, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 203, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 209, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 228, and the target sequence or complementary region is 21 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 228, and the target sequence or complementary region is 22 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 228, and the target sequence or complementary region is 23 nucleotides long. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 228, and the target sequence or complementary region is 24 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 229 to 420, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 229 to 420, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of any one of SEQ ID NOs: 229 to 420, and the target sequence or complementary region is 20 nucleotides long.

In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203, and 389 The target sequence or complementary region is complementary to the target sequence, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203, and 389 The target sequence or complementary region is complementary to the target sequence, and the target sequence or complementary region is 19 nucleotides long.

In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to a sequence selected from SEQ ID NO: 248, 381, 386, and 203, and the target sequence or complementary region is 18 nucleotides Sour and long. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to a sequence selected from SEQ ID NO: 248, 381, 386, and 203, and the target sequence or complementary region is 19 nucleotides Sour and long.

In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 19 nucleotides long. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 20 nucleotides long. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 21 nucleotides long. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 22 nucleotides long. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence within SEQ ID NO: 226, and the target sequence or complementary region is 23 nucleotides long. In some embodiments, in some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the sequence in SEQ ID NO: 226, and the target sequence or complementary region is 24 nucleotides. long.

In some embodiments, the oligonucleotide includes a target sequence or complementary region that is partially complementary to the sequence of MCR2. In some embodiments, the oligonucleotide includes a target sequence or complementary region that is partially complementary to the sequence of CYP11B1.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is fully complementary to the sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is fully complementary to the sequence of any one of SEQ ID NOs: 201 to 212. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence of SEQ ID NO: 203. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 209. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is fully complementary to the sequence of any one of SEQ ID NOs: 229 to 420. In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203 and 389. Target sequences or complementary regions whose sequences are completely complementary to each other. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 248. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 381. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 386. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 203. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is completely complementary to the target sequence within 15 to 30 contiguous nucleotides of SEQ ID NO: 226.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is partially complementary to the sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is partially complementary to the sequence of any one of SEQ ID NOs: 201 to 212. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence shown in SEQ ID NO: 203. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence shown in SEQ ID NO: 209. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is partially complementary to the sequence of any one of SEQ ID NOs: 229 to 420. In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203 and 389. The target sequence or complementary region of any sequence is partially complementary. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence set forth in SEQ ID NO: 248. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence shown in SEQ ID NO: 381. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence set forth in SEQ ID NO: 386. In some embodiments, MC2R oligonucleotides comprise a target sequence or a complementary region that is partially complementary to the sequence shown in SEQ ID NO: 203. In some embodiments, a CYP11B1 oligonucleotide includes a target sequence or complementary region that is partially complementary to a target sequence within 15 to 30 contiguous nucleotides such as SEQ ID NO: 226.

In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) herein includes a target sequence or complementary region that is complementary to an adjacent nucleotide sequence that includes MC2R or CYP11B1 mRNA, wherein the adjacent nucleic acid sequence The nucleotide sequence is about 12 to about 30 nucleotides long (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides long). In some embodiments, the oligonucleotides herein comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence comprising MC2R or CYP11B1 mRNA, wherein the adjacent nucleotide sequence is 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long. In some embodiments, the oligonucleotides herein comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence comprising MC2R or CYP11B1 mRNA, wherein the adjacent nucleotide sequence is 19 nucleotides long. In some embodiments, the oligonucleotides herein comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence comprising MC2R or CYP11B1 mRNA, wherein the adjacent nucleotide sequence is 20 nucleotides long.

In some embodiments, the target sequence or complementary region of the oligonucleotide that is complementary to nucleotides adjacent to the MC2R or CYP11B1 target sequence spans the entire length of the antisense strand. In some embodiments, the complementary region of an oligonucleotide that is complementary to an adjacent nucleotide of the MC2R or CYP11B1 target sequence spans a portion of the entire length of the antisense strand. In some embodiments, oligonucleotides herein comprise a complementary region that is at least partially (e.g., completely) complementary to a contiguous stretch of nucleotides spanning nucleotides 1 to 20 of the target sequence of MC2R or CYP11B1 (e.g., , on the antisense strand of dsRNA).

In some embodiments, oligonucleotides herein comprise target sequences or complementary regions that have one or more base pairs (bp) mismatches with corresponding MC2R or CYP11B1 target sequences. In some specific examples, the target sequence or complementary region may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding MC2R or CYP11B1 target sequence. Target sequence or complementary region, provided that the ability of the target sequence or complementary region to bind or adhere to MC2R or CYP11B1 mRNA under appropriate hybridization conditions and/or the ability of the oligonucleotide to inhibit the expression of MC2R or CYP11B1 is maintained. Alternatively, the target sequence or complementary region may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding MC2R or CYP11B1 target sequence, provided that the target sequence Or the ability of the complementary region to bind or adhere to MC2R or CYP11B1 mRNA under appropriate hybridization conditions and/or the ability of the oligonucleotide to inhibit the expression of MC2R or CYP11B1 is maintained. In some embodiments, the oligonucleotide includes a target sequence or a complementary region that has one mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or a complementary region that has two mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region that has three mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region that has 4 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region that has 5 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or a complementary region that has more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, where at least 2 (eg, all) mismatches are located contiguously (eg, 2, 3, 4, 5 or more mismatches in a row), or wherein the mismatches are interspersed throughout the target sequence or complementary region. In some embodiments, the oligonucleotide includes a target sequence or a complementary region that has more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, where at least 2 (e.g., all) mismatches are located contiguously (e.g., 2, 3, 4, 5 or more mismatches in a row), or where at least one or more non-mismatched base pairs are located between such mismatches, or its combination.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, wherein the target sequence Or the complementary region may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding MC2R target sequence. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, wherein the target sequence Or the complementary region may have no more than about 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212, wherein the target sequence or complementary region can There are at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding MC2R target sequence. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212, wherein the target sequence or complementary region can There are no more than about 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 203, wherein the target sequence or complementary region may have up to about 1, up to About 2, up to about 3, up to about 4, up to about 5, etc. mismatches to the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 209, wherein the target sequence or complementary region may have no more than about 1, No more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence.

In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 229 to 420, wherein the target sequence or complementary region can There are at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 229 to 420, wherein the target sequence or complementary region can There are no more than about 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence. In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203 and 389. Any adjacent nucleotide sequence complementary target sequence or complementary region, wherein the target sequence or complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most There are approximately 5 mismatches to the corresponding MC2R target sequence. In some embodiments, MC2R oligonucleotides comprise SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203 and 389. Any adjacent nucleotide sequence complementary target sequence or complementary region, wherein the target sequence or complementary region may have no more than about 1, no more than about 2, no more than about 3, no more than about 4 and no more than about 5 mismatches to the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 248, wherein the target sequence or complementary region may have up to about 1, up to About 2, up to about 3, up to about 4, up to about 5, etc. mismatches to the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 381, wherein the target sequence or complementary region may have no more than about 1, No more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 386, wherein the target sequence or complementary region may have no more than about 1, No more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence. In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to the adjacent nucleotide sequence of SEQ ID NO: 203, wherein the target sequence or complementary region may have no more than about 1, No more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding MC2R target sequence.

In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the contiguous sequence of 15 to 30 nucleotides of SEQ ID NO: 226, wherein the target sequence or complementary region may have up to about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding CYP11B1 target sequence. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to the contiguous sequence of 15 to 30 nucleotides of SEQ ID NO: 226, wherein the target sequence or complementary region may have no more than About 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5 mismatches with the corresponding CYP11B1 target sequence. iii. Type of oligonucleotide

A variety of oligonucleotide types and/or structures can be used to target MC2R and/or CYP11B1 in the methods herein, including but not limited to RNAi oligonucleotides, antisense oligonucleotides, miRNA, and the like. Any oligonucleotide type or other described herein is contemplated for use herein as a framework for incorporation of MC2R or CYP11B1 target sequences.

In some embodiments, the oligonucleotides herein inhibit MC2R or CYP11B1 expression by participating in the upstream or downstream RNA interference (RNAi) pathway involving Dicer. For example, RNAi oligonucleotides each having a size of approximately 19 to 25 nucleotides (with at least one 3' overhang of 1 to 5 nucleotides) have been developed (see, e.g., U.S. Patent No. 8,372,968 No.). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, eg, U.S. Patent No. 8,883,996). Further work resulted in extended dsRNA in which at least one end of at least one strand extends beyond the double helix target region, including structures in which one strand includes a thermodynamically stable four-loop structure (see, e.g., U.S. Patent Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010/033225). Such structures may include ss extensions (on one or both sides of the molecule) as well as ds extensions.

In some embodiments, the oligonucleotides herein participate in the RNAi pathway downstream of Dicer involvement (eg, Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, the oligonucleotide has an overhang (e.g., 1, 2, or 3 nucleotides long) at the 3' end of the sense strand. In some embodiments, an oligonucleotide (e.g., siRNA) includes a 21-nucleotide leader strand antisense to the target RNA and a complementary follower strand, where the two strands are bonded to form a 19-bp double helix and in A 2-nucleotide overhang from one or both of the 3' ends. Longer oligonucleotide designs can also be used, including those with a 23-nt leader strand and a 21-nt follower strand, with a blunt end (follower) on the right side of the molecule. The 3' end of the leader strand/the 5' end of the leader strand) and the 3'-leader overhang of 2 nucleotides on the left side of the molecule (the 5' end of the follower strand/the 3' end of the leader strand). In this type of molecule, there is a 21 bp double helix region. See, for example, U.S. Patent Nos. 9,012,138; 9,012,621; and 9,193,753.

In some embodiments, oligonucleotides herein include a sense strand and a sense strand each in the range of about 17 to 26 (eg, 17 to 26, 20 to 25, or 21 to 23) nucleotides in length. Antisense stocks. In some embodiments, the oligonucleotides described herein comprise an antisense strand that is 19 to 30 nucleotides long and a sense strand that is 19 to 50 nucleotides long, wherein the antisense strand and the sense strand are The sense strand is a separate strand that forms an asymmetric double helix region with an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand. In some embodiments, oligonucleotides herein include a sense strand and an antisense strand each ranging from about 19 to 22 nucleotides in length. In some specific examples, the shares of the anti-sense stock and the anti-sense stock are of equal length. In some embodiments, the oligonucleotide includes a sense strand and an antisense strand such that there is a 3'-overhang on the sense strand or the antisense strand or both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having a sense strand and an antisense strand each ranging from about 21 to 23 nucleotides in length, the sense strand, the antisense strand, or the sense strand The 3' overhangs of both the strand and the antisense strand are 1 or 2 nucleotides long. In some embodiments, the oligonucleotide has a 22 nt leader strand and a 20 nt follower strand with a blunt end on the right side of the molecule (3' end of the follower strand/5' end of the leader strand) And there are 2 nucleotides of the 3'-leader overhang on the left side of the molecule (the 5' end of the follower strand/the 3' end of the leader strand). In this type of molecule, there is a 20 bp double helix region.

Other oligonucleotide designs for use with the compositions and methods herein include: 16-nucleotide siRNA (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY. Blackburn (ed.), Royal Society of Chemistry, 2006 ), shRNA (e.g., having a stem of 19 bp or less; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-158), blunt-ended siRNA (e.g., having a stem of 19 bp; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-158) , e.g., Kraynack & Baker (2006) RNA 12:163-176), asymmetric siRNA (aiRNA; see, e.g., Sun et al. (2008) Nat. Biotechnol . 26:1379-1382), asymmetric shorter- Double helix siRNA (see, for example, Chang et al. (2009) Mol. Ther. 17:725-32), forked siRNA (see, for example, Hohjoh (2004) FEBS Lett . 557:193-198), ss siRNA (Elsner (2012) Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNA (see, e.g., Abe et al. (2007) J. Am. Chem. Soc . 129:15108-09), and small internal Segmented interfering RNA (siRNA; see, eg, Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Other non-limiting examples of oligonucleotide structures that can be used to reduce or inhibit expression of MC2R and/or CYP11B1 in some embodiments are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., , Hamilton et al. (2002) EMBO J. 21:4671-79; see also U.S. Patent Application Publication No. 2009/0099115).

In addition, in some specific examples, the oligonucleotide used herein to reduce or inhibit the expression of MC2R and/or CYP11B1 is ss. Such structures may include, but are not limited to, ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, e.g., Matsui et al. (2016) Mol. Ther . 24:946-55). However, in some embodiments, the oligonucleotides herein are antisense oligonucleotides (ASOs). Antisense oligonucleotides are ss oligonucleotides that have a nucleobase sequence that, when written in the 5' to 3' direction, contains the reverse complement of the target fragment of a specific nucleic acid, appropriately modified (e.g., as a gapmer) so as to induce RNaseH-mediated cleavage of its target RNA in the cell, or (e.g., as a mixture) so as to inhibit translation of the target mRNA in the cell. ASOs for use herein may be modified in any suitable manner known in the art, including, for example, as shown in U.S. Patent No. 9,567,587 (including, for example, length, sugar moieties of nucleobases (pyrimidine, purine), and Changes in the heterocyclic portion of the nucleobase). Furthermore, ASOs have been used for decades to reduce the expression of specific target genes (see, e.g., Bennett et al. (2017) Annu. Rev. Pharmacol . 57:81-105).

In some embodiments, antisense oligonucleotides share complementary regions with MC2R or CYP11B1 mRNA. In some embodiments, antisense oligonucleotides target multiple regions of the human MC2R gene identified as NM_000529.2 (SEQ ID NO: 228). In some embodiments, antisense oligonucleotides target multiple regions of the human CYP11B1 gene identified as NM_000497.4 (SEQ ID NO: 226). In some embodiments, antisense oligonucleotides are 15 to 50 nucleotides long. In some embodiments, antisense oligonucleotides are 15 to 25 nucleotides long. In some embodiments, the antisense MC2R oligonucleotide is 22 nucleotides long. In some embodiments, the antisense MC2R oligonucleotide is complementary to any of SEQ ID NOs: 201 to 212 and 229 to 420. In some embodiments, the antisense MC2R oligonucleotide is complementary to any one of SEQ ID NOs: 201 to 212. In some embodiments, the antisense MC2R oligonucleotide is complementary to any of SEQ ID NOs: 229-420. In some embodiments, the antisense oligonucleotide is complementary to any of SEQ ID NO: 203 and 209. In some specific examples, the antisense oligonucleotide is one of SEQ ID NOs: 236, 237, 248, 278, 337, 341, 365, 366, 371, 373, 381, 386, 387, 388, 203 and 389. Either complements the other. In some embodiments, the antisense oligonucleotide is complementary to any of SEQ ID NOs: 248, 381, 386, and 203. In some embodiments, the antisense oligonucleotide is complementary to SEQ ID NO:248. In some embodiments, the antisense oligonucleotide is complementary to SEQ ID NO:381. In some embodiments, the antisense oligonucleotide is complementary to SEQ ID NO:386. In some embodiments, the antisense oligonucleotide is complementary to SEQ ID NO:203. In some embodiments, the antisense CYP11B1 oligonucleotide is complementary to 15 to 30 contiguous nucleotides of SEQ ID NO:226. In some embodiments, antisense oligonucleotides are at least 15 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 19 contiguous nucleotides in length. In some embodiments, antisense oligonucleotides are at least 20 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide differs from the target sequence by 1, 2, or 3 nucleotides. iv. Double-stranded oligonucleotides

The present disclosure provides double-stranded RNA (dsRNA) for targeting MC2R or CYP11B1 and inhibiting MC2R or CYP11B1 expression (e.g., via an RNAi pathway), comprising a sense strand (also referred to herein as a follower strand) and a reverse strand. Loyal shares (also called guiding shares in this article). In some embodiments, the sense and antisense shares are separate shares and are not covalently linked. In some embodiments, the sense and antisense shares are covalently linked. In some embodiments, the sense strand and the antisense strand form a double helix region, wherein the sense strand and the antisense strand, or a portion thereof, complement each other (e.g., by Watson-Crick base pairing) combine.

In some specific examples, the sense strand has a first region (R1) and a second region (R2), wherein R2 includes a first subregion (S1), a four-ring or three-ring (L), and a second subregion ( S2), where L is between S1 and S2, and where S1 and S2 form a second double helix (D2). D2 can be of different lengths. In some embodiments, D2 is about 1 to 6 bp long. In some embodiments, D2 is 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, or 4 to 5 bp long. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp long. In some specific examples, D2 is 6 bp long.

In some specific examples, the sense strand R1 and the antisense strand form a first double helix (D1). In some embodiments, D1 is at least about 15 (eg, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides long. In some embodiments, D1 is in the range of about 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30 or 21 to 30 nucleotides long). In some embodiments, D1 is 12 nucleotides long (eg, at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides long). In some specific examples, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long . In some embodiments, D1 is 20 nucleotides long. In some specific examples, D1 including the legal shares and the anti-sense shares does not span the entire length of the legal shares and/or the anti-sense shares. In some embodiments, D1, which includes the legal stock and the antisense stock, spans the entire length of the legal stock or the antisense stock, or both. In some embodiments, D1, which includes the legal shares and the anti-anti-shares, spans the entire length of both the legal shares and the anti-anti-shares.

In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) herein has a 5' end that is less thermodynamically stable than the other 5' end. In some embodiments, asymmetric oligonucleotides are provided that include a blunt end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the 3'-overhang on the antisense strand is about 1 to 8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length) . In some embodiments, the oligonucleotide has an overhang comprising two (2) nucleotides at the 3' end of the antisense (leader) strand. However, other tabs are also possible. In some embodiments, the overhang is a 3'-overhang, which includes between 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5 or 6 nucleotides in length. However, in some embodiments, the overhang is a 5'-overhang, which includes between 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4 , 5 or 6 nucleotides in length.

In some embodiments, the two (2) terminal nucleotides of the 3' end of the antisense strand are modified. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand are complementary to the target mRNA (eg, MC2R mRNA). In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotides herein are unpaired. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotides herein comprise unpaired GG. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotides herein are not complementary to the target mRNA. In some embodiments, the two (2) terminal nucleotides at the 3' end of each oligonucleotide are GG. In some embodiments, one or both of the two (2) terminal GG nucleotides at the 3' end of each oligonucleotide herein are not complementary to the target mRNA.

In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) mismatch. If there is more than one mismatch between the sense and antisense strands, they can be located consecutively (eg, 2, 3, or more in a row), or scattered throughout the complementation region. In some embodiments, the 3’ end of the equity shares contains one or more mismatches. In some specific examples, two (2) mismatches are incorporated at the 3’ end of the equity shares. In some embodiments, base mismatching or destabilization of the 3' end of the sense strand of the oligonucleotides herein improves or increases the potency of the oligonucleotide.

It should be understood that in some embodiments, when describing the structure of an oligonucleotide or other nucleic acid, reference may be made to the sequences presented in the sequence listing. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., the RNA counterpart of the DNA nucleotide or the RNA counterpart of the RNA nucleotide) as compared to the specific sequence. DNA counterpart) and/or one or more modified nucleotides and/or one or more modified inter-nucleotide linkages and/or one or more other modifications, while retaining the specific Sequences that are substantially identical or similar in complementary nature. a. MC2R double-stranded oligonucleotide

In some aspects, the present disclosure provides double-stranded (ds) RNAi oligonucleotides comprising sense and antisense strands for targeting MC2R mRNA and inhibiting MC2R expression (e.g., via an RNAi pathway). In some embodiments, the sense and antisense shares are covalently linked. In some embodiments, the sense strand and the antisense strand form a double helix region, wherein the sense strand and the antisense strand, or a portion thereof, complement each other (e.g., by Watson-Crick base pairing) combine.

In some embodiments, MC2R oligonucleotides herein comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 201 to 212 and a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 213 to 224 The opposite of stocks.

In some embodiments, MC2R oligonucleotides herein comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 201 to 212 and 229 to 420 and a nucleotide sequence comprising a sequence selected from SEQ ID NOs: 213 to 224 and Antisense strand with nucleotide sequence 421 to 612.

In some embodiments, MC2R oligonucleotides herein comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 229 to 420 and a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 421 to 612 The opposite of stocks.

In some embodiments, MC2R oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand and an antisense strand comprising a nucleotide sequence selected from: (a) SEQ ID NO: 201 and 213 respectively; (b) SEQ ID NO: 202 and 214 respectively; (c) SEQ ID NO: 203 and 215 respectively; (d) SEQ ID NO: 204 and 216 respectively; (e) SEQ ID NO: 205 and 217 respectively; (f) SEQ ID NO: 206 and 218 respectively; (g) SEQ ID NO: 207 and 219 respectively; (h) SEQ ID NO: 208 and 220 respectively; (i) SEQ ID NO: 209 and 221 respectively; (j) SEQ ID NO: 210 and 222 respectively; (k) SEQ ID NO: 211 and 223 respectively; and (l) SEQ ID NO: 212 and 224 respectively.

In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 203, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 215. In some specific examples, the sense strand includes the nucleotide sequence of SEQ ID NO:209, and the antisense strand includes the nucleotide sequence of SEQ ID NO:221.

In some embodiments, MC2R oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand and an antisense strand comprising a nucleotide sequence selected from: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively.

In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 85. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 38, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 86.

In some embodiments, MC2R oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand and an antisense strand comprising a nucleotide sequence selected from: (a) SEQ ID NO: 235 and 427 respectively; (b) SEQ ID NO: 236 and 428 respectively; (c) SEQ ID NO: 237 and 429 respectively; (d) SEQ ID NO: 248 and 440 respectively; (e) SEQ ID NO: 278 and 470 respectively; (f) SEQ ID NO: 337 and 529 respectively; (g) SEQ ID NO: 341 and 533 respectively; (h) SEQ ID NO: 365 and 557 respectively; (i) SEQ ID NO: 366 and 558 respectively; (j) SEQ ID NO: 371 and 563 respectively; (k) SEQ ID NO: 373 and 565 respectively; (l) SEQ ID NO: 381 and 573 respectively; (m) are SEQ ID NO: 386 and 578 respectively; (n) SEQ ID NO: 387 and 579 respectively; (o) SEQ ID NO: 388 and 580 respectively; (p) SEQ ID NO: 389 and 581 respectively; and (q) are SEQ ID NO: 203 and 215 respectively.

In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 235, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 427. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 236, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 428. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 237, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 429. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 248, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 440. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 278, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 470. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 337, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 529. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 341, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 533. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 365, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 557. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 366, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 558. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 371, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 563. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 373, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 565. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 381, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 573. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 386, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 578. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 387, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 579. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 388, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 580. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 389, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 581. In some embodiments, the sense strand includes the nucleotide sequence of SEQ ID NO: 203, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 215.

In some embodiments, MC2R oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand and an antisense strand comprising a nucleotide sequence selected from: (a) SEQ ID NO: 248 and 440 respectively; (b) SEQ ID NO: 381 and 573 respectively; (c) SEQ ID NO: 386 and 578 respectively; and (d) SEQ ID NO: 203 and 215 respectively.

In some embodiments, the MC2R oligonucleotides (e.g., RNAi oligonucleotides) herein comprise a 25 nucleotide sense strand and a 27 nucleotide antisense strand, and when digested by Dicer The enzyme's action causes the antisense strand to be incorporated into mature RISC. In some embodiments, the 25 nucleotide sense strand includes a nucleotide sequence selected from SEQ ID NO: 201 to 212. In some embodiments, the 27 nucleotide antisense strand comprises a nucleotide sequence selected from SEQ ID NO: 213 to 224. In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NO: 201 to 212, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NO: 201 to 212, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

In some embodiments, the 25 nucleotide sense strand includes a nucleotide sequence selected from SEQ ID NOs: 201 to 212 and 229 to 420. In some embodiments, the 27 nucleotide antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 213 to 224 and 421 to 612. In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NOs: 201 to 212 and 229 to 420, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides) . In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NOs: 201 to 212 and 229 to 420, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

In some embodiments, the MC2R oligonucleotides (e.g., RNAi oligonucleotides) herein comprise a 25 nucleotide sense strand and a 27 nucleotide antisense strand, and when digested by Dicer The enzyme's action causes the antisense strand to be incorporated into mature RISC. In some embodiments, the 25 nucleotide sense strand includes a nucleotide sequence selected from SEQ ID NO: 229 to 420. In some embodiments, the 27 nucleotide antisense strand comprises a nucleotide sequence selected from SEQ ID NO: 421 to 612. In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NO: 229 to 420, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide includes a nucleotide sequence selected from SEQ ID NO: 229 to 420, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, and includes a length of 1 to the 5'-overhang between 6 nucleotides. In some embodiments, the MC2R oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 201 to 212 and 229 to 420, wherein the oligonucleotide comprises 1 to 6 nuclei in length. 5'-overhang between nucleotides. In some embodiments, MC2R oligonucleotides comprise antisense strands comprising nucleotide sequences selected from SEQ ID NOs: 213 to 224 and 421 to 612, wherein the oligonucleotides comprise 1 to 6 nuclei in length. 5'-overhang between nucleotides. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 201 to 212 and 229 to 420 and a sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 213 to 224 and 421 to 621. An antisense strand of a nucleotide sequence, wherein the oligonucleotide includes a 5'-overhang of between 1 and 6 nucleotides in length.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212, and comprise a length of 1 to 6 nuclei. 5'-overhang between nucleotides. In some embodiments, the MC2R oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 201 to 212, wherein the oligonucleotide comprises a length of between 1 and 6 nucleotides. 5'-Protruding end. In some embodiments, the MC2R oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 213 to 224, wherein the oligonucleotide comprises a length of between 1 and 6 nucleotides 5'-Protruding end. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 201 to 212 and an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 213 to 224 strand, wherein the oligonucleotide includes a 5'-overhang of between 1 and 6 nucleotides in length.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 229 to 420, and comprise a length of 1 to 6 nuclei 5'-overhang between nucleotides. In some embodiments, the MC2R oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 229 to 420, wherein the oligonucleotide comprises a length of between 1 and 6 nucleotides. 5'-Protruding end. In some embodiments, the MC2R oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 421 to 612, wherein the oligonucleotide comprises a length of between 1 and 6 nucleotides 5'-Protruding end. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 229 to 420 and an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 421 to 612 strand, wherein the oligonucleotide includes a 5'-overhang of between 1 and 6 nucleotides in length.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212, wherein the oligonucleotides herein are The two (2) terminal nucleotides at the 3' end of the antisense strand contain unpaired GG. In some embodiments, MC2R oligonucleotides comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 213 to 224, wherein the antisense strand comprises an unpaired GG at the 3' end of the strand. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 201 to 212 and an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 213 to 224 strand, where the antisense strand contains the unpaired GG at the 3' end of the strand.

In some embodiments, the sense and antisense strands of MC2R oligonucleotides herein comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85, respectively ; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 respectively and 89; (f) SEQ ID NO: 42 and 90, respectively; (g) SEQ ID NO: 43 and 91, respectively; (h) SEQ ID NO: 44 and 92, respectively; (i) SEQ ID NO: 43 and 91, respectively : 45 and 93; (j) are SEQ ID NO: 46 and 94 respectively; (k) are SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, in which the rights shares There are one or more (eg, 1, 2, 3, 4 or 5) mismatches with the antisense stock.

In some embodiments, MC2R oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand and an antisense strand comprising a nucleotide sequence selected from: (a) SEQ ID NO: 37 and 85, respectively; and (b) SEQ ID NO: 38 and 86, respectively, wherein one or more (e.g., 1, 2, 3, 4 or 5) mismatches.

In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 229 to 420, wherein the oligonucleotides herein are The two (2) terminal nucleotides at the 3' end of the antisense strand contain unpaired GG. In some embodiments, MC2R oligonucleotides comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 421 to 612, wherein the antisense strand comprises an unpaired GG at the 3' end of the strand. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 229 to 420 and an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 421 to 612 strand, where the antisense strand contains the unpaired GG at the 3' end of the strand.

In some specific examples, the sense and antisense strands of the MC2R oligonucleotides herein comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811, respectively ; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 respectively and 854; (f) SEQ ID NO: 721 and 913, respectively; (g) SEQ ID NO: 725 and 917, respectively; (h) SEQ ID NO: 749 and 941, respectively; (i) SEQ ID NO: 725 and 917, respectively :750 and 942; (j) are SEQ ID NO: 755 and 947 respectively; (k) are SEQ ID NO: 757 and 949 respectively; (l) are SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 757 and 949 respectively; (m) are SEQ ID NO: 757 and 949 respectively; ID NOs: 770 and 962; (n) SEQ ID NOs: 771 and 963, respectively; (o) SEQ ID NOs: 772 and 964, respectively; (p) SEQ ID NOs: 773 and 965, respectively; and (q) SEQ ID NOs: 38 and 86, respectively, wherein there are one or more (eg, 1, 2, 3, 4 or 5) mismatches between the sense strand and the antisense strand.

In some specific examples, the sense and antisense strands of the MC2R oligonucleotides herein comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824, respectively ; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) SEQ ID NO: 38 and 86 respectively, wherein the sense stock and the antisense There are one or more (for example, 1, 2, 3, 4 or 5) mismatches between stocks. b. CYP11B1 double-stranded oligonucleotide

In some aspects, the present disclosure provides double-stranded (ds) RNAi oligonucleotides comprising sense and antisense strands for targeting CYP11B1 mRNA and inhibiting CYP11B1 expression (eg, via an RNAi pathway). In some embodiments, the sense and antisense shares are covalently linked. In some embodiments, the sense strand and the antisense strand form a double helix region, wherein the sense strand and the antisense strand, or a portion thereof, complement each other (e.g., by Watson-Crick base pairing). combine.

In some embodiments, the CYP11B1 dsRNA herein includes a sense strand including 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and a nucleotide including at least 15 nucleotides complementary to the sense strand The antisense stock of the sequence.

In some embodiments, the CYP11B1 oligonucleotides herein comprise a 25 nucleotide sense strand and a 27 nucleotide antisense strand, which when acted upon by the Dicer enzyme results in the incorporation of the antisense strand Mature RISC. In some embodiments, the CYP11B1 oligonucleotide comprises at least 15 phosphate-linked nucleotide sequences of SEQ ID NO: 226, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides). In some embodiments, the sense strand of the CYP11B1 oligonucleotide includes at least 15 contiguous nucleotide sequences of SEQ ID NO: 226, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region that is complementary to 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, and includes a sequence of 1 to 6 nucleotides in length. Between the 5'-protruding ends. In some embodiments, the CYP11B1 oligonucleotide comprises a sense strand comprising 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, wherein the oligonucleotide comprises 1 to 6 nucleotides in length 5'-protruding end between. In some embodiments, the CYP11B1 oligonucleotide comprises an antisense strand comprising 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, wherein the oligonucleotide comprises 1 to 6 nucleotides in length 5'-protruding end between. In some embodiments, the CYP11B1 oligonucleotide includes a sense strand including 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226 and 15 to 30 contiguous nucleotide sequences including the sense strand. The antisense strand, wherein the oligonucleotide includes a 5'-overhang between 1 and 6 nucleotides in length.

In some embodiments, a CYP11B1 oligonucleotide includes a target region or complementary region complementary to 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, wherein the antisense strand of the oligonucleotide herein is The two (2) terminal nucleotides at the 3' end contain unpaired GG. In some embodiments, the CYP11B1 oligonucleotide comprises an antisense strand comprising 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, wherein the antisense strand comprises an unpaired 3' end of the strand. GG. In some embodiments, the CYP11B1 oligonucleotide includes an antisense strand that includes 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226 and an antisense strand that includes a nucleotide sequence that is complementary to the sense strand. , where the antisense strand contains the unpaired GG at the 3' end of the strand.

In some embodiments, the CYP11B1 oligonucleotide includes a sense strand including 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226 and an antisense strand including a nucleotide sequence complementary to the sense strand. , where there are one or more (for example, 1, 2, 3, 4 or 5) mismatches between the sense shares and the anti-sense shares. c. Antisense stocks

In some embodiments, oligonucleotides (eg, RNAi oligonucleotides) disclosed herein for targeting MC2R include as set forth in any one of SEQ ID NOs: 213 to 224 and 421 to 612 sequence or antisense stocks composed of them. In some embodiments, MC2R oligonucleotides comprise at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or an antisense strand consisting thereof. Oligonucleotides (eg, RNAi oligonucleotides) disclosed herein for targeting MC2R include antisenses that include or consist of the sequence set forth in any of SEQ ID NOs: 213 to 224. share. In some embodiments, the MC2R oligonucleotide comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or an antisense strand composed thereof. In some embodiments, MC2R oligonucleotides comprise antisense strands that include or consist of the sequence shown in SEQ ID NO: 215. In some embodiments, MC2R oligonucleotides comprise antisense strands that include or consist of the sequence shown in SEQ ID NO: 221.

In some embodiments, oligonucleotides disclosed herein for targeting MC2R (e.g., and RNAi oligonucleotides) comprise a sequence set forth in any one of SEQ ID NOs: 421 to 612 or The anti-contra stocks composed of them. In some embodiments, the MC2R oligonucleotide comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or an antisense strand composed thereof. In some embodiments, MC2R oligonucleotides comprise antisense strands including the sequence of SEQ ID NO: 440 or consisting thereof. In some embodiments, MC2R oligonucleotides comprise antisense strands including or consisting of the sequence of SEQ ID NO: 573. In some embodiments, MC2R oligonucleotides comprise antisense strands including or consisting of the sequence of SEQ ID NO: 578. In some embodiments, MC2R oligonucleotides comprise antisense strands including or consisting of the sequence of SEQ ID NO: 579. In some embodiments, MC2R oligonucleotides comprise antisense strands including the sequence of SEQ ID NO: 215 or consisting thereof.

In some embodiments, the oligonucleotides (eg, RNAi oligonucleotides) disclosed herein for targeting CYP11B1 include 15 to 30 contiguous sequences as shown in SEQ ID NO: 226 or the sequence thereof. The antithesis of the composition. In some embodiments, the CYP11B1 oligonucleotide comprises at least about 12 (e.g., at least 12, at least 13, at least 14) of sequences complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides or antisense consisting thereof share.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) herein include up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17 or up to 12 nucleotides long). In some embodiments, the dsRNA includes up to about 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17 or up to 12 nucleotides long) antisense strand. In some embodiments, the oligonucleotide can be at least about 12 nucleotides long (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27 , at least 30, at least 35 or at least 38 nucleotides long) antisense strands. In some embodiments, the oligonucleotide can have about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides long. In some specific examples, the oligonucleotide can have 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, Antisense strands 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides long. In some embodiments, the oligonucleotide contains an antisense strand that is 15 to 30 nucleotides long. In some specific examples, the antisense strands of any of the oligonucleotides disclosed herein are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides long. In some embodiments, the oligonucleotide contains an antisense strand that is 22 nucleotides long.

In some embodiments, the antisense strand of an oligonucleotide may be referred to as the "lead strand." For example, if the antisense strand can bind to the RNA-induced silencing complex (RISC) and bind to an Argonaute protein (such as Ago), or to one or more similar factors and guide silencing of the target gene , it may be called a leading stock. In some specific examples, stocks that are complementary to leading stocks may be called "following stocks." d. Loyal shares

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein for targeting MC2R include as set forth in any one of SEQ ID NOs: 201 to 212 and 229 to 420 Sequence or antisense sequence consisting of it. In some embodiments, MC2R oligonucleotides comprise at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or a sense strand composed thereof. In some embodiments, oligonucleotides disclosed herein for targeting MC2R (e.g., and RNAi oligonucleotides) comprise a sequence set forth in any one of SEQ ID NOs: 201 to 212, or a sequence thereof. Composed of beneficial shares. In some embodiments, the MC2R oligonucleotide comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or a sense strand composed thereof. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of the sequence of SEQ ID NO: 203. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of the sequence of SEQ ID NO: 209. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of a sequence selected from SEQ ID NO: 37 to 47. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of a sequence selected from SEQ ID NO: 37 and 38. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of a sequence selected from SEQ ID NOs: 37 to 47 and 613 to 804.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein for targeting MC2R comprise or consist of a sequence set forth in any of SEQ ID NOs: 229 to 420. Composed of beneficial shares. In some embodiments, MC2R oligonucleotides comprise at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19) contiguous nucleotides or a sense strand composed thereof. In some embodiments, MC2R oligonucleotides comprise the nucleotide sequence of SEQ ID NO: 248 or a sense strand consisting thereof. In some embodiments, MC2R oligonucleotides comprise the nucleotide sequence of SEQ ID NO: 381 or a sense strand consisting thereof. In some embodiments, MC2R oligonucleotides comprise the nucleotide sequence of SEQ ID NO: 386 or a sense strand consisting thereof. In some embodiments, MC2R oligonucleotides comprise the nucleotide sequence of SEQ ID NO: 387 or a sense strand consisting thereof. In some embodiments, MC2R oligonucleotides comprise the nucleotide sequence of SEQ ID NO: 203 or a sense strand consisting thereof. In some embodiments, MC2R oligonucleotides comprise a sense strand comprising or consisting of a sequence selected from SEQ ID NO: 613 to 804. In some specific examples, the oligonucleotides include those selected from the group consisting of SEQ ID NO: 619, 621, 632, 662, 721, 725, 749, 750, 755, 757, 765, 770, 771, 772, 38, and 773 A sequence or a group of shares consisting of it. In some embodiments, the oligonucleotide includes a sense strand that includes or consists of a sequence selected from SEQ ID NO: 632, 765, 770, and 38.

In some specific examples, the oligonucleotides (eg, RNAi oligonucleotides) disclosed herein for targeting CYP11B1 comprise 15 to 30 contiguous sequences of SEQ ID NO: 226 or consist of Loyal shares. In some embodiments, the CYP11B1 oligonucleotide comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18) of the sequence including SEQ ID NO: 226. , at least 19, at least 20, at least 21, at least 22 or at least 23) contiguous nucleotides or a sense strand composed thereof.

In some embodiments, the oligonucleotides comprise up to about 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, sense strand (or follower strand) up to 17 or up to 12 nucleotides long). In some embodiments, the oligonucleotide can be about 12 nucleotides long (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, At least 36 or at least 38 nucleotides long) sense strand. In some embodiments, the oligonucleotides can have between about 12 and about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36 , 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40 ) has a sense strand within the range of nucleotide length. In some specific examples, the oligonucleotide can have 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides long sense strand.

In some embodiments, oligonucleotides disclosed herein for targeting MC2R mRNA and inhibiting MC2R expression comprise the sense sequence set forth in any of SEQ ID NOs: 37 and 38. In some embodiments, the MC2R oligonucleotides herein have at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 , at least 20, at least 21, at least 22 or at least 23) the sense strand of contiguous nucleotides of the sequence shown in any one of SEQ ID NO: 37 and 38.

In some specific examples, the oligonucleotides disclosed herein for targeting MC2R mRNA and inhibiting MC2R expression include SEQ ID NOs: 619, 621, 632, 662, 721, 725, 749, 750, 755, 757 , 765, 770, 771, 772, 38 and 773 any one of the positive stock sequences shown. In some embodiments, the MC2R oligonucleotides herein have at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 , at least 20, at least 21, at least 22 or at least 23) such as SEQ ID NO: 619, 621, 632, 662, 721, 725, 749, 750, 755, 757, 765, 770, 771, 772, The sense strand of the adjacent nucleotides of the sequence shown in any one of 38 and 773.

In some specific examples, the oligonucleotides disclosed herein for targeting MC2R mRNA and inhibiting MC2R expression include the sense sequence shown in any one of SEQ ID NOs: 632, 765, 770, and 38 . In some embodiments, the MC2R oligonucleotides herein have at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 , at least 20, at least 21, at least 22 or at least 23) the sense strand of contiguous nucleotides of the sequence shown in any one of SEQ ID NO: 632, 765, 770 and 38.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand that includes a stem-loop structure at its 3' end. In some embodiments, stem-loop systems are formed by intrastrand base pairing. In some embodiments, the sense strand includes a stem-loop structure at its 5' end. In some embodiments, the stem of the stem loop includes a double helix that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In some embodiments, the stem of the stem loop includes a double helix that is 2 nucleotides long. In some embodiments, the stem of the stem loop includes a 3 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 4 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 5 nucleotide long double helix. In some embodiments, the stem of the stem-loop includes a 6 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 7 nucleotide long double helix. In some embodiments, the stem of the stem-loop includes a double helix that is 8 nucleotides long. In some embodiments, the stem of the stem loop includes a 9 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 10 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a double helix that is 11 nucleotides long. In some embodiments, the stem of the stem loop includes a 12 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 13 nucleotide long double helix. In some embodiments, the stem of the stem loop includes a 14 nucleotide long double helix.

In some embodiments, the stem-loop provides protection of the oligonucleotide from degradation (e.g., enzymatic degradation), facilitates or improves targeting and/or delivery to target cells, tissues, or organs (e.g., liver), or both . For example, in some embodiments, the stem-loop loop is composed of nucleotides that include one or more modifications that promote, improve, or increase targeting, targeting of target mRNA (e.g., MC2R or CYP11B1 mRNA). Inhibition of gene expression (eg, MC2R or CYP11B1 expression), and/or delivery, uptake, and/or penetration into target cells, tissues, or organs (eg, liver), or combinations thereof. In some embodiments, the stem loop itself or the modification(s) to the stem loop does not affect or substantially affect the inherent gene expression inhibiting activity of the oligonucleotide, but rather promotes, improves, or increases stability (e.g., provides protection protection from degradation) and/or delivery, uptake and/or penetration of the oligonucleotide into target cells, tissues or organs (e.g., liver). In certain embodiments, the oligonucleotides herein include a sense strand that includes (e.g., at its 3' end) a stem loop as shown below: S1-L-S2, where S1 and S2 Complementary, and wherein L forms a linked nucleoside between S1 and S2 up to about 10 nucleotides long (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long) Single-stranded ring of acid. In some embodiments, loop (L) is 3 nucleotides long. In some embodiments, loop (L) is 4 nucleotides long. In some embodiments, loop (L) is 5 nucleotides long. In some embodiments, loop (L) is 6 nucleotides long. In some embodiments, loop (L) is 7 nucleotides long. In some embodiments, loop (L) is 8 nucleotides long. In some embodiments, loop (L) is 9 nucleotides long. In some embodiments, loop (L) is 10 nucleotides long.

In some embodiments, the tetracycle includes the sequence 5'-GAAA-3'. In some embodiments, the stem loop includes the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 198).

In some embodiments, the sense strand includes a stem-loop structure at its 3' end. In some embodiments, the sense strand includes a stem-loop structure at its 5' end. In some embodiments, the stem is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 bp long double helix. In some embodiments, the stem-loop provides protection of the molecule from degradation (eg, enzymatic degradation) and promotes targeting properties for delivery to target cells. For example, in some embodiments, the loop provides added nucleotides that can be modified without substantially affecting the gene expression inhibiting activity of the oligonucleotide. In certain embodiments, as for the oligonucleotides herein, the sense strand includes (e.g., at its 3' end) a stem loop as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 that is up to about 10 nucleotides long (eg, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long). In some embodiments, the MC2R oligonucleotide comprises a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420, and the oligonucleotide The acid contains a sense strand that contains (e.g., at its 3' end) a stem loop as follows: S1-L-S2, where S1 is complementary to S2, where L is between S1 and S2 to form 4 A single-stranded loop of nucleotide length. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212, and the oligonucleotide includes a sense strand, the sense strand contains (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, where S1 is complementary to S2, and where L forms 4 nucleotides long between S1 and S2 The single strand ring. In some embodiments, the MC2R oligonucleotide includes a target sequence or complementary region that is complementary to an adjacent nucleotide sequence of any one of SEQ ID NOs: 229 to 420, and the oligonucleotide includes a sense strand, the sense strand contains (e.g., at its 3' end) a stem loop as shown below: S1-L-S2, where S1 is complementary to S2, where L is formed between S1 and S2 and is 4 nucleosides in length Acid long single-stranded ring. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region complementary to 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226, and the oligonucleotide includes a sense strand, the The sense strand contains (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, where S1 is complementary to S2, and where L forms a single strand 4 nucleotides long between S1 and S2 ring. Figure 1A depicts non-limiting examples of such oligonucleotides.

In some embodiments, ring (L) of the stem loop having the structure S1-L-S2 as described herein is a tricyclic ring. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region and a triloop that are complementary to the adjacent nucleotide sequence of any of SEQ ID NOs: 201 to 212 and 229 to 420. In some embodiments, ring (L) of the stem loop having the structure S1-L-S2 as described herein is a tricyclic ring. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region and a triloop that are complementary to the contiguous nucleotide sequence of any of SEQ ID NOs: 201 to 212. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region and a triloop that are complementary to the contiguous nucleotide sequence of any of SEQ ID NOs: 229 to 420. In some embodiments, the CYP11B1 oligonucleotide includes a target sequence or complementary region and a triloop that are complementary to 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226. In some embodiments, tricycles include ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (eg, delivery ligands), and combinations thereof.

In some embodiments, ring (L) of the stem loop having the structure S1-L-S2 as described herein is a tetracyclic ring. In some embodiments, MC2R oligonucleotides herein comprise a target sequence or complementary region complementary to the adjacent nucleotide sequence of any one of SEQ ID NOs: 201 to 212 and 229 to 420 and a tetraloop . In some specific examples, the ring (L) of the stem ring having the above-mentioned structure S1-L-S2 is a tetracyclic ring. In some embodiments, MC2R oligonucleotides herein comprise a target sequence or complementary region and a tetraloop that are complementary to the contiguous nucleotide sequence of any of SEQ ID NOs: 201 to 212. In some specific examples, the ring (L) of the stem ring having the structure S1-L-S2 is a tetracyclic ring. In some embodiments, MC2R oligonucleotides comprise a target sequence or complementary region and a tetraloop that are complementary to the contiguous nucleotide sequence of any of SEQ ID NOs: 229 to 420. In some embodiments, a CYP11B1 oligonucleotide herein includes a target sequence or complementary region complementary to 15 to 30 contiguous nucleotide sequences of SEQ ID NO: 226 and a tetraloop. In some embodiments, tricycles include ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (eg, delivery ligands), and combinations thereof.

In some embodiments, the stem-loop ring (F) is a tetracyclic ring (eg, within a notched tetracyclic ring structure). Tetracycles may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, tetracycles have 4 to 5 nucleotides. e.The length of the double helix

In some specific examples, the number of double helices formed between the sense strand and the antisense strand is at least 12 (for example, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21) nucleotides long. In some specific examples, the double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides long). In some specific examples, the double helix formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 12 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 13 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 14 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 15 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 16 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 17 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 18 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 19 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 20 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 21 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 22 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 23 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 24 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 25 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 26 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 27 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 28 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 29 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand is 30 nucleotides long. In some embodiments, the double helix formed between the sense strand and the antisense strand does not span the entire length of the sense strand and/or the antisense strand. In some embodiments, the double helix between the sense and antisense strands spans the entire length of the sense or antisense strand. In some embodiments, the double helix between the sense and antisense strands spans the entire length of both the sense and antisense strands.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, The double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (for example, 12 to 30, 12 to 27, 12 to 22, 15 to 25 , 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides long).

In some embodiments, the double helix between the sense and antisense strands spans the entire length of the sense or antisense strand. In some embodiments, the double helix between the sense and antisense strands spans the entire length of both the sense and antisense strands.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, The double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (for example, 12 to 30, 12 to 27, 12 to 22, 15 to 25 , 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides long).

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, The double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (for example, 12 to 30, 12 to 27, 12 to 22, 15 to 25 , 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides long).

In some embodiments, the double helix between the sense and antisense strands spans the entire length of the sense or antisense strand. In some embodiments, the double helix between the sense and antisense strands spans the entire length of both the sense and antisense strands.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively, The double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (for example, 12 to 30, 12 to 27, 12 to 22, 15 to 25 , 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides long).

In some specific examples, a CYP11B1 oligonucleotide includes: 1) a sense strand, which includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) an antisense strand, which includes the same nucleotide as the sense strand. A complementary nucleotide sequence in which the double helix formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides long). v. Oligonucleotide modification a. Sugar modification

In some embodiments, oligonucleotides (eg, RNAi oligonucleotides) described herein comprise modified sugars. In some embodiments, modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, at the 2', 3', 4', and/or 5 of the sugar. 'One or more modifications occur at the carbon position. In some embodiments, modified sugars may also include non-natural alternative carbon structures, such as those found in locked nucleic acids ("LNA"; see, e.g., Koshkin et al. (1998) Tetrahedon 54: 3607-30. Unlocked nucleic acids (“UNA”; see, e.g., Snead et al. (2013) Mol. Ther-Nucl. Acids 2:e103) and bridging nucleic acids (“BNA”; see, e.g., Imanishi & Obika (2002) ) Chem Commun. (Camb) 21:1653-59).

In some embodiments, nucleotide modifications in sugars include 2'-modifications. In some specific examples, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F) , 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2 -(Methylamino)-2-Pendantoxyethyl] (2'-O-NMA) or 2'-deoxy-2'-fluoro-β-d-arabinucleic acid (2'-FANA). In some specific examples, the modification is 2'-F, 2'-OMe or 2'-MOE. In some embodiments, the modification in the sugar includes modification of the sugar ring, which may include modification of one or more carbons of the sugar ring. For example, modification of a sugar in a nucleotide may include linking the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethanol linkage. base or methylene bridge. In some embodiments, modified nucleotides have acyclic sugars that lack the bond between the 2'-carbon and the 3'-carbon. In some embodiments, the modified nucleotide has a thiol group (e.g., at the 4' position of the sugar).

In some embodiments, the oligonucleotides described herein comprise at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30 , at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 or more) modified nucleotides. In some embodiments, the sense strand of the oligonucleotide includes at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or more) modified nucleotides. In some embodiments, the antisense strand of the oligonucleotide includes at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20 or more) modified Nucleotides.

In some embodiments, all nucleotides in the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the oligonucleotide (ie, both the sense and antisense strands) are modified. In some embodiments, modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d- arabic nucleic acid). In some embodiments, the modified nucleotide includes a 2'-modification (e.g., 2'-F or 2'-OMe).

In some embodiments, the present disclosure provides oligonucleotides with different modification patterns. In some embodiments, oligonucleotides herein include a sense strand having a modification pattern as shown in the Examples and Sequence Listing and an antisense strand having a modification pattern as shown in the Examples and Sequence Listing.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein comprise antisense strands having 2'-F modified nucleotides. In some embodiments, oligonucleotides herein include antisense strands including nucleotides modified with 2'-F and 2'-OMe. In some embodiments, oligonucleotides disclosed herein comprise a sense strand having a 2'-F modified nucleotide. In some embodiments, the oligonucleotides disclosed herein comprise the sense strand of nucleotides modified with 2'-F and 2'-OMe.

In some embodiments, the oligonucleotides described herein comprise a sense strand, wherein the sense strand is about 10 to 15%, 10%, 11%, 12%, 13%, 14% of the nucleotides. % or 15% contains 2'-fluoro modifications. In some embodiments, approximately 11% of the nucleotides in the sense strand contain 2'-fluoro modifications. In some embodiments, the oligonucleotides described herein comprise antisense strands, wherein the nucleotides of the antisense strands comprise about 25 to 35%, 25%, 26%, 27%, 28%, 29 %, 30%, 31%, 32%, 33%, 34% or 35% contain 2'-fluoro modification. In some embodiments, approximately 32% of the nucleotides of the antisense strand contain 2'-fluoro modifications. In some embodiments, the oligonucleotide has a ratio of about 15 to 25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% Nucleotides contain 2'-fluoro modifications. In some embodiments, approximately 19% of the nucleotides in the dsRNAi oligonucleotide contain 2'-fluoro modifications.

In some specific examples, one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group. In some specific examples, one or more of positions 3, 8, 9, 10, 12, 13 and 17 of the sense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with a 2'-F group. In some specific examples, one or more of positions 2, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand is modified with a 2'-F group. In some specific examples, one or more of positions 2, 3, 4, 5, 7, 8, 10, 14, 16, and 19 of the antisense strand is modified with a 2'-F group. In some embodiments, the sugar moiety of each nucleotide at positions 1 to 7 and 12 to 20 in the sense strand is modified with 2'-OMe. In some specific examples, the sugar portion of each nucleotide at positions 1 to 7, 12 to 27, and 31 to 36 in the sense strand is modified with 2’-OMe. In some specific examples, the sugar moiety of each nucleotide at positions 1 to 2, 4 to 7, 11, 14 to 16, and 18 to 20 in the sense strand is modified with 2'-OMe. In some specific examples, the sugar moiety of each nucleotide at positions 1 to 2, 4 to 7, 11, 14 to 16, and 18 to 27, and 31 to 36 in the sense strand is modified with 2'-OMe. In some embodiments, the sugar portion of each nucleotide at positions 3, 4, 6, 9, 11, 13, 15, 17, 18, and 20 to 22 in the antisense strand is modified with 2'-OMe. In some embodiments, the sugar portion of each nucleotide at positions 4, 6, 8, 9, 11, 12, 13, and 15 to 22 in the antisense strand is modified with 2'-OMe. In some embodiments, the sugar portion of each nucleotide at positions 6, 8, 9, 11 to 13, and 15 to 22 in the antisense strand is modified with 2'-OMe.

In some specific examples, one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modified with a 2'-F group. In some embodiments, the sugar moiety of each nucleotide in positions 1 to 7 and 12 to 20 in the sense strand is modified with 2'-OMe. In some embodiments, the sugar moiety of each nucleotide at positions 1, 6, 8, 9, 11 to 13, and 15 to 22 in the antisense strand is modified with 2'-OMe.

The present disclosure provides oligonucleotides with different modification patterns. In some embodiments, the modified oligonucleotide includes a sense sequence having a modification pattern as shown in Figure 1A and an antisense sequence having a modification pattern as shown in Figure 1A . In some embodiments, for these oligonucleotides, one or more of positions 8, 9, 10, or 11 of the sense strand is modified with a 2'-F group. In other embodiments, for these oligonucleotides, the sugar moiety of each nucleotide at positions 1 to 7 and 12 to 20 in the sense strand is modified with 2'-OMe.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, One or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively; One or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, One or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively,

One or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group. In some specific examples, CYP11B1 oligonucleotides include: 1) The sense strand, which contains 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) Antisense strand, which contains a nucleotide sequence complementary to the sense strand, One or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

In some embodiments, oligonucleotides provided herein comprise antisense strands having a 2 '-F modified sugar moiety, and the sugar moiety of each remaining nucleotide of the antisense strand is selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2' -Ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O -[2-(methylamino)-2-side-oxyethyl](2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabinucleic acid (2'-FANA ) modification of the group composed of. In some embodiments, oligonucleotides provided herein comprise antisense strands having 2'-F modified sugars at each of nucleotide positions 2, 3, 5, 7, 10, and 14. moiety, and the sugar moiety of each remaining nucleotide of the antisense strand is selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2 '-Aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-( The group consisting of methylamino)-2-oxyethyl] (2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabinic acid (2'-FANA) The modification modification.

In some embodiments, oligonucleotides provided herein comprise antisense strands having a 2'-F modified sugar moiety at each nucleotide at positions 2 to 5, 7, 10, and 14 , and the sugar moiety of each remaining nucleotide of the antisense strand is selected from 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'- Aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methyl Modification of the group consisting of amino)-2-side oxyethyl] (2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabinic acid (2'-FANA) Grooming.

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Antisense strands with a 2'-F modified sugar moiety at position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21 or position 22.

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Antisense strands with a 2'-OMe modified sugar moiety at position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21 or position 22.

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21 or position 22 has a compound selected from the group consisting of 2'-O-propargyl, 2'-O- Propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl ( 2'-MOE), 2'-O-[2-(methylamino)-2-side-oxyethyl](2'-O-NMA), and 2'-deoxy-2'-fluoro-β -A group consisting of d-Arabic nucleic acid (2'-FANA).

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Position 12, Position 13, Position 14, Position 15, Position 16, Position 17, Position 18, Position 19, Position 20, Position 21, Position 22, Position 23, Position 24, Position 25, Position 26, Position 27, Position 28 , position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 sense strands with a 2'-F modified sugar moiety.

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Position 12, Position 13, Position 14, Position 15, Position 16, Position 17, Position 18, Position 19, Position 20, Position 21, Position 22, Position 23, Position 24, Position 25, Position 26, Position 27, Position 28 , position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 sense strands with a 2'-OMe modified sugar moiety.

In some specific examples, the oligonucleotides provided herein include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Position 12, Position 13, Position 14, Position 15, Position 16, Position 17, Position 18, Position 19, Position 20, Position 21, Position 22, Position 23, Position 24, Position 25, Position 26, Position 27, Position 28 , position 29, position 30, position 31, position 32, position 33, position 34, position 35 or position 36 has a group selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side-oxyethyl](2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabinucleic acid (2' -FANA). b. 5' terminal phosphate

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) described herein include a sense strand and an antisense strand, wherein the antisense strand includes a 5' terminal phosphate. In some embodiments, the 5' terminal phosphate group of the oligonucleotide enhances the interaction with Ago2. However, oligonucleotides containing 5'-phosphate groups may be easily degraded by phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5' phosphates that are resistant to such degradation. In some embodiments, the phosphate analog may be methoxyphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 1' end of the oligonucleotide strand is attached with a chemical moiety that mimics the electrostatic and steric properties of the natural 5'-phosphate group ("phosphate mimetic").

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide contains a 5' terminal phosphate, optionally a 5' terminal phosphate analogue.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains a 5' terminal phosphate, optionally a 5' terminal phosphate analogue.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains a 5' terminal phosphate, optionally a 5' terminal phosphate analogue.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively,

Wherein the oligonucleotide contains a 5' terminal phosphate, optionally a 5' terminal phosphate analogue. In some specific examples, CYP11B1 oligonucleotides include: 1) The sense strand, which contains 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) Antisense strand, which contains a nucleotide sequence complementary to the sense strand, Wherein the oligonucleotide contains a 5' terminal phosphate, optionally a 5' terminal phosphate analogue.

In some embodiments, the oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog"). See, for example, International Patent Application Publication No. WO 2018/045317. In some embodiments, oligonucleotides herein include a 4'-phosphate analog at the 5' terminal nucleotide. In some embodiments, the phosphate analog is a methoxyphosphonate, wherein the oxygen atom of the methoxy group is bonded to a sugar moiety (eg, at its 4'-carbon) or an analog thereof. In other specific examples, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is in contact with a sugar. Partial 4'-carbon or its analogue is bound. In certain embodiments, the 4'-phosphate analog is methoxyphosphonate. In some specific examples, the methoxyphosphonate is represented by the formula -O-CH ₂ -PO(OH) ₂ or -O-CH ₂ -PO(OR) ₂ , wherein R is independently selected from H, CH _3. Alkyl group, CH ₂ CH ₂ CN, CH ₂ OCOC(CH ₃ ) ₃ , CH ₂ OCH ₂ CH ₂ Si(CH ₃ ) ₃ or protecting group. In some embodiments, the alkyl group is CH ₂ CH ₃ . More typically _, R is independently selected from H, _CH3 or _CH2CH3 . In some embodiments, the 4'-phosphate analog is 4'-methoxyphosphonate. In some embodiments, the modified nucleotide with a 4'-phosphonate analog is uridine. In some embodiments, the modified nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine.

In some embodiments, oligonucleotides provided herein comprise an antisense strand comprising a 4'-phosphate analogue at the 5' terminal nucleotide, wherein the 5' terminal nucleotide comprises the following structure: 4'-O-Monomethylphosphonate-2'-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs]. c. Modified nucleoside internal linkage

In some embodiments, oligonucleotides can include modified internucleoside linkages. In some embodiments, the phosphate modification or substitution can result in an oligonucleotide comprising at least about 1 (eg, at least 1, at least 2, at least 3, or at least 5) modified internucleotide linkages. acid. In some embodiments, any of the oligonucleotides disclosed herein includes about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified inter-nucleotide linkages. In some embodiments, any of the oligonucleotides disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified inter-nucleotide linkages.

The modified inter-nucleotide linkage can be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, or a thiocarbonylalkyl linkage. thionoalkylphosphotriester linkage, phosphite linkage, phosphonate linkage or boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any of the oligonucleotides disclosed herein is a phosphorothioate linkage.

In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 3 and 3 of the antisense strand. 4. There is a phosphorothioate linkage between one or more of positions 20 and 21 of the antisense strand and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 20 and 2 of the antisense strand. There is a phosphorothioate linkage between each of positions 21 and 22 of the antisense strand.

In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 20 and 2 of the antisense strand. There is a phosphorothioate linkage between 21 and each of positions 21 and 22 of the antisense strand.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide comprises modified inter-nucleotide linkages.

In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 20 and 2 of the antisense strand. There is a phosphorothioate linkage between 21 and each of positions 21 and 22 of the antisense strand. In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises modified inter-nucleotide linkages.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises modified inter-nucleotide linkages.

In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 20 and 2 of the antisense strand. There is a phosphorothioate linkage between 21 and each of positions 21 and 22 of the antisense strand. In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises modified inter-nucleotide linkages.

In some specific examples, the CYP11B1 oligonucleotide includes: 1) a sense strand, which includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) an antisense strand, which includes the sense strand. A complementary nucleotide sequence, wherein the oligonucleotide contains modified inter-nucleotide linkages. d.Base modification

In some embodiments, oligonucleotides (eg, RNAi oligonucleotides) herein have one or more modified nucleobases. In some embodiments, a modified nucleobase (also referred to herein as a base analog) is attached at the 1' position of the sugar moiety of the nucleotide. In some embodiments, the modified nucleobase is a nitrogen-containing base. In some embodiments, the modified nucleobase does not contain nitrogen atoms. See, for example, US Patent Application Publication No. 2008/0274462. In some embodiments, modified nucleotides include universal bases. However, in certain embodiments, the modified nucleotide does not contain nucleobases (abase).

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide contains one or more modified nucleobases.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains one or more modified nucleobases.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains one or more modified nucleobases.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains one or more modified nucleobases.

In some specific examples, CYP11B1 oligonucleotides include: 1) The sense strand, which contains 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) Antisense strand, which contains a nucleotide sequence complementary to the sense strand, Wherein the oligonucleotide contains one or more modified nucleobases.

In some embodiments, the universal base is the heterocyclic moiety located at the 1' position of the nucleotide sugar moiety in the modified nucleotide or at the equivalent position in the substitution of the nucleotide sugar moiety when present in a double helix In this case, the universal base can be positioned opposite more than one type of base without substantially changing the structure of the double helix. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is completely complementary to the target nucleic acid, a single-stranded nucleic acid containing universal bases forms a double-stranded helix with the target nucleic acid, which has a ratio to that of the complementary nucleic acid. The resulting double helix has a lower _Tm . However, in some embodiments, when compared to a reference single-stranded nucleic acid in which the universal base has been base-substituted to create a single mismatch, the single-stranded nucleic acid containing the universal base forms a double helix with the target nucleic acid, which Have a higher _Tm than the double helix formed with the nucleic acid containing the mismatched base.

Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3- Nitropyrrole (see, U.S. Patent Application Publication No. 2007/0254362; Van Aerschot et al . (1995) Nucleic Acids Res . 23:4363-4370; Loakes et al . (1995) Nucleic Acids Res . 23:2361- 2366; and Loakes & Brown (1994) Nucleic Acids Res . 22:4039-4043). e. Reversible modification

Although certain modifications can be made to protect the oligonucleotide from the in vivo environment before reaching the target cell, such modifications can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made so that the molecule retains desired properties outside the cell, and then these reversible modifications are removed upon entering the cytosolic environment of the cell. Reversible modifications can be removed, for example, by the action of intracellular enzymes or by chemical conditions within the cell (eg, by reduction of intracellular glutathione).

In some embodiments, the reversibly modified nucleotide includes a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charges generated by inter-nucleotide diphosphate linkages and to improve cellular uptake and nuclease resistance. See US Patent Application Publication No. 2011/0294869, International Patent Application Publication Nos. WO 2014/088920 and WO 2015/188197, and Meade et al . (2014) Nat. Biotechnol . 32:1256-1263. This reversible modification of the internucleotide diphosphate linkage is designed for intracellular cleavage by the reducing environment of the cytosol (eg, glutathione). Early examples include neutralizing phosphotriester modifications that are reported to be cleavable within cells (see Dellinger et al . (2003) J. Am. Chem. Soc . 125:940-50).

In some embodiments, such reversible modifications allow for protection during in vivo administration (e.g., via the blood and/or lysosomal/endosomal compartments of cells) where the oligonucleotide will be exposed to nucleases and other severe environmental conditions (e.g., pH). When released into the cytosol of cells where glutathione levels are higher than in the extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. The use of reversible, glutathione-sensitive moieties may introduce more sterically larger chemical groups into the oligonucleotide of interest than available options using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and therefore should not interfere with the biological activity of the oligonucleotide within the cytosol of the cell. As a result, these larger chemical groups have been engineered to confer various advantages to nucleotides or oligonucleotides, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be modified to alter the kinetics of its release.

In some embodiments, the glutathione-sensitive moiety is attached to a sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, particularly when the modified nucleotide is the 5' nucleotide of an oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, particularly when the modified nucleotide is the 3' terminal nucleotide of an oligonucleotide. In some embodiments, the glutathione-sensitive moiety includes a sulfonyl group. See, for example, U.S. Provisional Patent Application No. 62/378,635, filed on August 23, 2016, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof . vi. Target ligand

In some embodiments, it is desirable to target the oligonucleotides of the present disclosure to one or more cells or one or more organs. Such strategies can help avoid undesirable effects on other organs or avoid excessive loss of oligonucleotide to cells, tissues or organs that would not benefit from the oligonucleotide. Targeting of oligonucleotides to one or more cells or one or more organs can be accomplished by a variety of methods. Conjugation of oligonucleotides to tissue- or cell-specific antibodies, small molecules, or targeting ligands can facilitate delivery of the oligonucleotide to and into one or more target cells or tissues. Accumulation of oligonucleotides (Chernolovskaya et al . (2019) Front Pharmacol. 10:444). For example, conjugation of oligonucleotides to saturated fatty acids (eg, C22 ) can facilitate delivery to cells or tissues such as adipose tissue, which uptake such ligands more readily than traditional oligonucleotide ligands. Accordingly, in some embodiments, the oligonucleotides disclosed herein are modified to facilitate tissue, cell, or organ targeting and/or delivery (eg, to facilitate delivery of the oligonucleotide to the liver). In certain embodiments, the oligonucleotides disclosed herein are modified to facilitate delivery of the oligonucleotide to hepatocytes of the liver. In certain embodiments, the oligonucleotides disclosed herein are modified to facilitate delivery of the oligonucleotide to the adrenal gland.

In some embodiments, an oligonucleotide includes at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) bound to one or more targeting ligands. .

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide comprises a targeting ligand bound to at least one nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises a targeting ligand bound to at least one nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises a targeting ligand bound to at least one nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively,

Wherein the oligonucleotide comprises a targeting ligand bound to at least one nucleotide. In some specific examples, CYP11B1 oligonucleotides include: 1) The sense strand, which contains 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) Antisense strand, which contains a nucleotide sequence complementary to the sense strand, Wherein the oligonucleotide comprises a targeting ligand bound to at least one nucleotide.

In some embodiments, the targeting ligands comprise carbohydrates, amino sugars, cholesterol, peptides, polypeptides, proteins or portions of proteins (eg, antibodies or antibody fragments), or lipids. In some embodiments, the target ligand is an aptamer. In some embodiments, the target ligand is an adrenocorticotropic hormone (ACTH) ligand. In some embodiments, the ACTH ligand targets the adrenal gland. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

In some embodiments, one or more (eg, 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, each of 2 to 4 nucleotides of the oligonucleotide is conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is attached to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the targeting ligand is attached to the 5' end of the sense or antisense strand). or a 2 to 4 nucleotide overhang or extended junction at the 3' end), such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, the oligonucleotide can include a stem loop at the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop can be individually coupled to a target ligand. In some embodiments, the oligonucleotides (e.g., dsRNA) provided by the present disclosure include a stem loop at the 3' end of the sense strand, wherein the stem loop loop includes three loops or four loops, and wherein respectively Three or four nucleotides containing three or four rings are individually linked to the target ligand. a. GalNAc target ligand

GalNAc is a high-affinity ligand for ASGPR that is primarily expressed on the sinusoidal surface of hepatocytes and plays a role in binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins) main character. Conjugation (indirect or direct) of a GalNAc moiety to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to ASGPR expressed on cells. In some embodiments, oligonucleotides of the present disclosure are conjugated to at least one or more GalNAc moieties, wherein the GalNAc moiety targets the oligonucleotide to human liver cells (e.g., human liver cells) ASGPR expressed on hepatocyte)). In some embodiments, the GalNAc moiety targets the oligonucleotide to the liver.

In some embodiments, oligonucleotides of the disclosure are conjugated directly or indirectly to monovalent GalNAc. In some embodiments, the oligonucleotide is directly or indirectly conjugated to more than one monovalent GalNA (i.e., to 2, 3, or 4 monovalent GalNAc moieties, and typically to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties. In some embodiments, the bivalent, trivalent, or tetravalent GalNAc moiety is conjugated to the oligonucleotide via a branched linker. In some embodiments, the monovalent GalNAc moiety is conjugated to the first nucleotide, and the divalent, trivalent, or tetravalent GalNAc moiety is conjugated to the second nucleotide via a branched linker.

In some embodiments, one or more (eg, 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of each of the four loops are conjugated to a separate GalNAc. In some embodiments, each of 1 to 3 nucleotides of the tricycle is conjugated to a separate GalNAc. In some embodiments, the target ligand is attached to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is attached to the 5' or 3' end of the sense or antisense strand). The 2 to 4 nucleotide overhangs or extensions at the ' end make the GalNAc portion resemble the bristles of a toothbrush and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is conjugated to the nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in four loops of the sense strand, with each GalNAc moiety conjugated to 1 nucleotide.

In some embodiments, the tetracycle is any combination of adenine and guanine nucleotides.

In some embodiments, tetracycle (L) has a monovalent GalNAc moiety attached to any one or more guanine nucleotides of the tetracycle via any linker described herein, as depicted below (X = heteroatom ):

In some embodiments, tetracycle (L) has a monovalent GalNAc moiety attached to any one or more adenine nucleotides of the tetracycle via any linker described herein, as depicted below (X = heteroatom ):

In some embodiments, the oligonucleotides herein comprise a monovalent oligonucleotide with a guanine nucleotide attached (referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc). GalNAc portion, as depicted below:

In some embodiments, the oligonucleotides herein comprise a monovalent oligonucleotide with a guanine nucleotide attached (referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc). GalNAc section, as shown below:

An example of such a junction is shown below, which is a stem attachment point containing the nucleotide sequence GAAA (L = linker, X = heteroatom) from 5' to 3'. As shown in Figure 1A , such loops may be present, for example, at positions 27 to 30 of the prosthetic strand. In the chemical formula, Used to describe the point of attachment of oligonucleotide strands.

Appropriate methods or chemistry (eg, click chemistry) can be used to link the target ligand to the nucleotide. In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, an acetal linker is used to conjugate a targeting ligand to a nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016/100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable. An example of a loop containing the nucleotide GAAA from 5' to 3' is shown below, where the GalNAc moiety is attached to the nucleotides of the loop using an acetal linker. Such loops may be present, for example, at positions 27 to 30 of any of the sense strands shown in Figure 1A . In the chemical formula, It is the attachment point for oligonucleotide strands. or

As mentioned above, various suitable methods or chemical synthesis techniques (eg, click chemistry) can be used to connect the target ligand and the nucleotide. In some embodiments, a click linker is used to join the targeting ligand to the nucleotide. In some embodiments, an acetal linker is used to conjugate a targeting ligand to a nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016/100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable.

In some embodiments, a double helix extension (eg, up to 3, 4, 5, or 6 bp long) is provided between the targeting ligand (eg, the GalNAc moiety) and the dsRNA. In some embodiments, the oligonucleotides herein do not have GalNAc conjugated thereto.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to the nucleotide.

In some specific examples, the CYP11B1 oligonucleotide includes: 1) a sense strand, which includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) an antisense strand, which includes the sense strand. A complementary nucleotide sequence, wherein the oligonucleotide includes at least one GalNAc moiety conjugated to the nucleotide. b. Lipid targeting ligand

In some embodiments, the present disclosure provides an oligonucleotide-ligand conjugate comprising an oligonucleotide for inhibiting a target expressed in cells of the adrenal gland or adrenocortex. The expressed nucleotide sequence of the mRNA (eg, MC2R and CYP11B1) and one or more targeting ligands that bind the oligonucleotide. In some embodiments, cells of the adrenal gland or adrenal cortex are epithelial cells. In some embodiments, oligonucleotide-ligand conjugates described herein comprise a nucleotide sequence and one or more target ligands, wherein the nucleotide sequence comprises a combination with one or more of the formula: The target ligand shown in Ia binds one or more nucleosides (nucleic acids): Or its medically acceptable salt, wherein: B is a nucleobase; R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R , -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or R ¹ and R ² on the same carbon together with their intervening atoms form 0 to 3 independently selected from nitrogen, oxygen and a 3 to 7-membered saturated or partially unsaturated ring of sulfur heteroatoms; each R ^A is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, 4 to 7 membered saturated or partially unsaturated heterocycles having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur to a 6-membered heteroaromatic ring; each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaryls having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur Ring; or two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaryl having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur ring; each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain 0 to 10 methylene units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O- , -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR- substitution; Each -Cy- is independently a bivalent ring that is optionally substituted, The divalent ring system is selected from the group consisting of a phenylene group, an 8- to 10-membered bicyclic aryl group, a 4- to 7-membered saturated or partially unsaturated carbocyclic group, and a 4 to 11-membered saturated or partially unsaturated spirocyclic carbocyclic group. radical, an 8- to 10-membered bicyclic saturated or partially unsaturated carbocyclyl group, a 4 to 7-membered saturated or partially unsaturated heterocyclyl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. , having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4 to 11 membered saturated or partially unsaturated spirocyclic heterocyclyl group, having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur An 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic aryl group having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 5 8- to 10-membered bicyclic heteroarylene groups independently selected from nitrogen, oxygen or sulfur heteroatoms; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched chain C _1-50 hydrocarbon chains, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C( O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-, -V ¹ CR ² W ¹ -or Substitution; m is 1 to 50; X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-; Y is hydrogen , Suitable hydroxyl protecting group, or ; R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from X ² is ^{O ,} S or _NR ; The linking group; Y ² is hydrogen, a suitable protecting group, a phosphoramidite analogue, an internucleotide linking group attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a solid support attached and Z is -O-, -S-, -NR- or -CR ₂ -.

In some embodiments, the oligonucleotide-ligand conjugate comprises one or more nucleic acids conjugated to a target ligand represented by Formula II-a : or a medically acceptable salt thereof.

In some embodiments, the oligonucleotide-ligand conjugate includes one or more nucleoside nucleic acids conjugated to a target ligand represented by Formula II-b or II-c : Or a medically acceptable salt thereof, wherein: L ¹ is a covalent bond, a monovalent or divalent saturated or unsaturated, straight or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene The base units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O) -, -S(O) ₂ -, -P(O)OR-, -P(S)OR-or Replacement; R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and R ⁵ is adamantyl or a saturated or unsaturated, straight or branched C _1-50 hydrocarbon chain, wherein 0 to 10 of the hydrocarbon chain The methylene units are independently -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S)OR substitution.

In some specific examples, R ⁵ is selected from

In some specific examples, ^R5 is selected from:

In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series . In some specific examples, the R ⁵ series .

In some embodiments, the nucleotide sequence includes one or more nucleic acids coupled to a target ligand represented by Formula II-b or II-c : or its medically acceptable salt; wherein B is a nucleobase or hydrogen; m is 1 to 50; X ¹ is -O- or -S-; Y is hydrogen, or ; ^{R 3} is hydrogen or a suitable protecting group; ^{X 2} is O or S; A linker at the 2' or 3'end; Y ² is hydrogen, a phosphoramidite analog, an inter-nucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or an attached solid The linking group of the support; R ⁵ is an adamantyl group or a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -O- , -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P( O)OR- or -P(S)OR substitution; and R is hydrogen, a suitable protecting group, or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heterocycles having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur Heteroaromatic rings.

In some specific examples, R ⁵ is selected from

In some specific examples, the R ⁵ series .

In some embodiments, the nucleotide sequence of the oligonucleotide contains 1 to 10 targeting ligands. In some embodiments, the nucleotide sequence contains 1, 2, or 3 targeting ligands.

In some embodiments, the oligonucleotide of the oligonucleotide-ligand conjugate is a double-stranded molecule. In some embodiments, the oligonucleotide is an RNAi molecule. In some embodiments, the double-stranded oligonucleotide contains a stem loop. In some embodiments, the ligand is attached to any nucleotide in the stem loop. In some embodiments, the ligand is attached to the first nucleotide from 5' to 3' in the stem loop. In some embodiments, the ligand is attached to the second nucleotide from 5' to 3' in the stem loop. In some embodiments, the ligand is attached to the third nucleotide from 5' to 3' in the stem loop. In some embodiments, the ligand is attached to the fourth nucleotide from 5' to 3' in the stem loop. In some embodiments, the ligand is attached to one, two, three, or four nucleotides in the stem loop. In some embodiments, the ligand is attached to three nucleotides in the stem loop.

In some embodiments, the oligonucleotide-ligand conjugate includes a sense strand of 36 nucleotides from 5' to 3' at positions 1 to 36. In some embodiments, the oligonucleotide-ligand conjugate includes a lipid bound to position 1 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a lipid conjugated to position 27 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a lipid conjugated to position 28 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a lipid conjugated to position 29 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a lipid bound to position 30 of the 36-nucleotide sense strand.

In some embodiments, the oligonucleotide-ligand conjugate includes a C8-C30 hydrocarbon chain joined to position 1 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a C22 hydrocarbon chain joined to position 1 of the 36-nucleotide sense strand.

In some embodiments, the oligonucleotide-ligand conjugate includes a lipid conjugated to the 5' nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand conjugate includes a C8-C30 hydrocarbon chain bonded to the 5' end nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand includes a C22 hydrocarbon chain bonded to the 5' end nucleotide of the sense strand.

In some embodiments, the oligonucleotide-ligand conjugate includes an antisense strand of 15 to 30 nucleotides and a sense strand of 15 to 40 nucleotides, wherein the sense strand and the antisense strand The strands form a double helix region, wherein the antisense strand contains a complementary region to a target sequence expressed in the adrenal gland or adrenal cortex, and wherein the sense strand contains a stem loop at its 3' end, the stem loop includes 4 Tetracyclic nucleosides, one or more of the four nucleosides are represented by formula II-Ib: , wherein B is selected from adenine and guanine nucleobases, and wherein R ⁵ is a hydrocarbon chain. In some embodiments, m is 1, X is ^O , Y is ^an internucleotide linker attached to the 5' end of the nucleoside, and Y is represented by As shown, Y ¹ is a linker attached to the 2' or 3' end of the nucleotide, X ² is O, X ³ is O, and R ³ is H. In some specific examples, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some specific examples, the hydrocarbon chain is a C22 hydrocarbon chain. In some specific examples, the C22 hydrocarbon chain is composed of shown. In some specific examples, the four nucleosides of the tetracyclic ring are numbered from 1 to 4 from 5' to 3', and position 1 is represented by Formula II-Ib. In some embodiments, position 2 is represented by Formula II-Ib. In some embodiments, position 3 is represented by Formula II-Ib. In some embodiments, position 4 is represented by Formula II-Ib. In some embodiments, the sense strand is 36 nucleotides having 36 nucleotides from 5' to 3' at positions 1 to 36, wherein the stem loop is included at positions 21 to 36 The nucleotides, and one or more nucleosides at positions 27 to 30 are represented by formula II-Ib. In some embodiments, the antisense strand is 22 nucleotides long.

In some aspects, the present disclosure provides oligonucleotide-ligands for targeting a target mRNA (e.g., a target mRNA that mediates immunosuppression) and inhibiting or reducing target gene expression (e.g., via an RNAi pathway) A conjugate, wherein the oligonucleotide-ligand conjugate is a double-stranded (ds) nucleic acid molecule comprising a sense strand (also referred to herein as a follower strand) and an antisense strand (also referred to herein as a leader strand). In some embodiments, the sense and antisense strands are separate chains and are not covalently linked. In some embodiments, the sense and antisense shares are covalently linked. In some embodiments, the sense strand and the antisense strand form a double helix region, wherein the sense strand and the antisense strand, or a portion thereof, complement each other (e.g., by Watson-Crick base pairing) To combine or bond.

In some embodiments, the oligonucleotide-ligand conjugate includes an antisense strand of 15 to 30 nucleotides and a sense strand of 15 to 40 nucleotides, wherein the sense strand and the antisense strand The strands form a double-stranded helix region, wherein the antisense strands comprise a complementary region to a target sequence expressed in the adrenal gland or adrenal cortex, and wherein the 5' end nucleotide of the sense strands comprises a nucleotide represented by Formula II-Ib Nucleosides: , wherein B is selected from adenine and guanine nucleobases, and wherein R ⁵ is a hydrocarbon chain. In some embodiments, m is 1, X is ^O , Y is ^an internucleotide linker attached to the 5' end of the nucleoside, and Y is represented by As shown, Y ¹ is a linker attached to the 2' or 3' end of the nucleotide, X ² is O, X ³ is O, and R ³ is H. In some specific examples, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some specific examples, the hydrocarbon chain is a C22 hydrocarbon chain. In some specific examples, the C22 hydrocarbon chain is composed of

shown.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) are SEQ ID NO: 48 and 96 respectively, Wherein the oligonucleotide contains at least one C22 moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; and (b) SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains at least one C22 moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively, Wherein the oligonucleotide contains at least one C22 moiety conjugated to the nucleotide.

In some embodiments, the sense and antisense strands of the MC2R oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) are SEQ ID NO: 38 and 86 respectively,

Wherein the oligonucleotide contains at least one C22 moiety conjugated to the nucleotide. In some specific examples, a CYP11B1 oligonucleotide includes: 1) a sense strand, which includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 2) an antisense strand, which includes the sense strand. A complementary nucleotide sequence, wherein the oligonucleotide includes at least one C22 moiety conjugated to the nucleotide. vii. Exemplary oligonucleotides a. Exemplary MC2R oligonucleotides

In some embodiments, an oligonucleotide targeting MC2R includes a sense strand and an antisense strand as shown in Table 4 , wherein the oligonucleotide includes a double-stranded stem having about 2 to 6 base pairs and A stem-loop structure of a loop of 3 to 4 nucleotides, and wherein the sense strand and the antisense strand comprise a modification pattern as shown in Figure 1A . In some embodiments, an oligonucleotide targeting MC2R includes a sense strand and an antisense strand as shown in Table 4 , wherein the oligonucleotide includes a double-stranded stem having about 2 to 6 base pairs and A stem-loop structure of a loop of 3 to 4 nucleotides, wherein the sense strand and the antisense strand comprise the modification pattern as shown in Figure 1A , and wherein the antisense strand is located at 4' of the 5' end nucleotide 'The carbon is modified with methoxyphosphonate. In some embodiments, the oligonucleotide comprises a stem loop including the nucleotide sequence of SEQ ID NO:198. In some embodiments, the oligonucleotide includes a 6 base pair double-stranded stem and a 4 nucleotide stem loop containing one, two, three, or four nucleotides conjugated to GalNAc. In some specific examples, the nucleotide conjugated to GalNAc is a monovalent GalNAc conjugated to an adenine nucleotide, called [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as follows Shown: .

In some embodiments, the stem loop includes a double-stranded stem of 6 base pairs and a loop including the nucleotide sequence GAAA, where each adenine nucleotide is ademA-GalNAc.

In some embodiments, the oligonucleotide targeting MC2R includes the sense strand and the antisense strand as shown in Table 8 , wherein the sense strand and the antisense strand include the modification pattern as shown in Figure 1A . In some embodiments, the oligonucleotide targeting MC2R includes a sense strand and an antisense strand as shown in Table 8 , wherein the sense strand and the antisense strand include a modification pattern as shown in Figure 1A , and The antisense strand is modified with phosphonate methoxylate at the 4' carbon of the 5' end nucleotide. In some embodiments, the oligonucleotide comprises a stem loop including the nucleotide sequence of SEQ ID NO:198. In some embodiments, the oligonucleotide includes a 6 base pair double-stranded stem and a 4 nucleotide stem loop containing one, two, three, or four nucleotides conjugated to GalNAc. In some specific examples, the nucleotide conjugated to GalNAc is a monovalent GalNAc conjugated to an adenine nucleotide, called [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as follows Shown:

In some embodiments, the stem loop includes a double-stranded stem of 6 base pairs and a loop including the nucleotide sequence GAAA, where each adenine nucleotide is ademA-GalNAc.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing MC2R expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, GalNAc-conjugated nucleotides at positions 28, 29, and 30; and at position 1 a phosphorothioate linkage between , 2'-OMe of 11 to 13 and 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22, and inclusion at position 1 The 5'-terminal nucleotide of the 4'-phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate- 4O-mU]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and positions 27 to 30 A loop forming the stem loop, optionally wherein positions 27 to 30 comprise four loops, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise a member selected from The nucleotide sequences of the group formed: (a) are SEQ ID NO: 37 and 85 respectively; (b) are SEQ ID NO: 38 and 86 respectively; (c) are SEQ ID NO: 39 and 87 respectively; ( d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively ; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 respectively and 95; and (1) are SEQ ID NOs: 48 and 96, respectively.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing MC2R expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, GalNAc-conjugated nucleotides at positions 28, 29, and 30; and at position 1 a phosphorothioate linkage between , 2'-OMe of 11 to 13 and 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and The 5' terminal nucleotide comprising a 4'-phosphate analogue at position 1, optionally wherein the 5' terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyl Uridine [MePhosphonate-4O-mU]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, and positions 21 to 36 of the sense strand form a stem loop, wherein positions 27 to 30 form a loop of the stem loop, optionally wherein positions 27 to 30 comprise four loops, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise Nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 621 and 813 respectively; (g) SEQ ID NO: 662 and 854 respectively NO: 725 and 917; (h) SEQ ID NO: 749 and 941, respectively; (i) SEQ ID NO: 750 and 942, respectively; (j) SEQ ID NO: 755 and 947, respectively; (k) SEQ ID NO: 755 and 947, respectively SEQ ID NO: 757 and 949; (l) SEQ ID NO: 765 and 957, respectively; (m) SEQ ID NO: 770 and 962, respectively; (n) SEQ ID NO: 771 and 963, respectively; (o) SEQ ID NOs: 772 and 964, respectively; (p) SEQ ID NOs: 773 and 965, respectively; and (q) SEQ ID NOs: 38 and 86, respectively.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing MC2R expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, nucleotides conjugated to C22 lipids at position 28; and nucleotides at positions 1 and 2 phosphorothioate linkages between; antisense strands, which include 2'-F modified nucleotides at positions 2 to 5, 7, 10 and 14, and nucleotides at positions 1, 6, 8 to 9, 11 to 13 and 2'-OMe of 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22, and the inclusion of 4'- at position 1 The 5'-terminal nucleotide of the phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU ]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and positions 27 to 30 form the stem a ring of rings, optionally including four rings at positions 27 to 30, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand and the antisense strand comprise a group selected from the group consisting of: The nucleotide sequences of the group: (a) are SEQ ID NO: 37 and 85 respectively; (b) are SEQ ID NO: 38 and 86 respectively; (c) are SEQ ID NO: 39 and 87 respectively; (d) are respectively are SEQ ID NO: 40 and 88; (e) are SEQ ID NO: 41 and 89 respectively; (f) are SEQ ID NO: 42 and 90 respectively; (g) are SEQ ID NO: 43 and 91 respectively; (h ) are SEQ ID NO: 44 and 92 respectively; (i) are SEQ ID NO: 45 and 93 respectively; (j) are SEQ ID NO: 46 and 94 respectively; (k) are SEQ ID NO: 47 and 95 respectively; and (1) are SEQ ID NO: 48 and 96 respectively.

In some specific examples, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing MC2R expression include: A sense strand comprising 2'-F modified nucleotides at positions 8 to 11, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, 28 nucleotide bound to the C22 lipid; and a phosphorothioate linkage between positions 1 and 2; Antisense strands comprising 2'-F modified nucleotides at positions 2 to 5, 7, 10 and 14, 2'- at positions 1, 6, 8 to 9, 11 to 13 and 15 to 22 OMe, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and 4'-phosphate analogues at position 1 The 5' end nucleotide, optionally wherein the 5' end nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; wherein the Positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and positions 27 to 30 form a loop of the stem loop, Optionally wherein positions 27 to 30 comprise four rings, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand and the antisense strand comprise nucleosides selected from the group consisting of Acid sequence: (a) SEQ ID NO: 619 and 811 respectively; (b) SEQ ID NO: 620 and 812 respectively; (c) SEQ ID NO: 621 and 813 respectively; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 and 917 respectively; (h) SEQ ID NO: 749 and 941 respectively; (i) SEQ ID NO: 750 and 942 respectively; (j) SEQ ID NO: 755 and 947 respectively; (k) SEQ ID NO: 757 and 949 respectively; (l) SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) SEQ ID NO: 771 and 963 respectively; (o) SEQ ID NO: 772 and 964 respectively; (p) SEQ ID NO: 773 and 965 respectively; and (q) are SEQ ID NO: 38 and 86 respectively.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing MC2R expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, nucleotides conjugated to C22 lipids at position 1; and nucleotides at positions 1 and 2 phosphorothioate linkages between; antisense strands, which include 2'-F modified nucleotides at positions 2 to 5, 7, 10 and 14, and nucleotides at positions 1, 6, 8 to 9, 11 to 13 and 2'-OMe of 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and at position 1 The 5' terminal nucleotide comprising a 4'-phosphate analogue, optionally wherein the 5' terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [ MePhosphonate-4O-mU]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and position 27 to 30 to form a loop of the stem loop, optionally wherein positions 27 to 30 comprise four loops, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise a loop selected from the following The nucleotide sequences of the groups composed of each: (a) are SEQ ID NO: 619 and 811 respectively; (b) are SEQ ID NO: 620 and 812 respectively; (c) are SEQ ID NO: 621 and 813 respectively ; (d) SEQ ID NO: 632 and 824 respectively; (e) SEQ ID NO: 662 and 854 respectively; (f) SEQ ID NO: 721 and 913 respectively; (g) SEQ ID NO: 725 respectively and 917; (h) SEQ ID NO: 749 and 941, respectively; (i) SEQ ID NO: 750 and 942, respectively; (j) SEQ ID NO: 755 and 947, respectively; (k) SEQ ID NO: 755 and 947, respectively :757 and 949; (l) are SEQ ID NO: 765 and 957 respectively; (m) are SEQ ID NO: 770 and 962 respectively; (n) are SEQ ID NO: 771 and 963 respectively; (o) are SEQ ID NO: 771 and 963 respectively; (o) are SEQ ID NO: 770 and 962 respectively; ID NOs: 772 and 964; (p) are SEQ ID NOs: 773 and 965, respectively; and (q) are SEQ ID NOs: 38 and 86, respectively.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-GalNAc = GalNAc with adenine nucleotide attached.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with:

where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-GalNAc = GalNAc with adenine nucleotide attached.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-C22 = C22 carbon chain attached to the adenine nucleotide.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-C22 = C22 carbon chain attached to the adenine nucleotide.

In some specific examples, the present disclosure provides an oligonucleotide (e.g., RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a group selected from the group consisting of: The sense and antisense nucleotide sequences of and 190; (d) SEQ ID NO: 177 and 191, respectively; (e) SEQ ID NO: 178 and 192, respectively; (f) SEQ ID NO: 179 and 193, respectively; (g) SEQ ID NO: 179 and 193, respectively : 180 and 194; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 182 and 196 respectively; ID NOs: 184 and 198; and (1) are SEQ ID NOs: 185 and 199, respectively.

In some specific examples, the present disclosure provides an oligonucleotide (e.g., RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a group selected from the group consisting of: The nucleotide sequences of the sense and antisense strands: (a) are SEQ ID NO: 186 and 188 respectively; and (b) are SEQ ID NO: 187 and 189 respectively.

In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 186 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 186. The antisense strand of the nucleotide sequence shown in NO: 188. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 187 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 187. The antisense strand of the nucleotide sequence shown in NO: 189.

In some specific examples, the present disclosure provides an oligonucleotide (e.g., RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a group selected from the group consisting of: The nucleotide sequences of the sense and antisense strands: (a) are SEQ ID NO: 174 and 188 respectively; and (b) are SEQ ID NO: 175 and 189 respectively.

In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 174 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 174. The antisense strand of the nucleotide sequence shown in NO: 188. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 175 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 175. The antisense strand of the nucleotide sequence shown in NO: 189.

In some specific examples, the present disclosure provides an oligonucleotide (e.g., RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a group selected from the group consisting of: The sense and antisense nucleotide sequences of and 1003; and (d) are SEQ ID NO: 1000 and 1004 respectively.

In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 997 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 997. The antisense strand of the nucleotide sequence shown in NO:1001. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 998 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 998. The antisense strand of the nucleotide sequence shown in NO: 1002. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 999 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 999. The antisense strand of the nucleotide sequence shown in NO: 1003. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 1000 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 1000. The antisense strand of the nucleotide sequence shown in NO: 1004.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademX-C22 = C22 carbon chain attached to the nucleotide.

In some specific examples, the present disclosure provides an oligonucleotide (e.g., RNAi oligonucleotide) for reducing MC2R expression, wherein the oligonucleotide includes a group selected from the group consisting of: The sense and antisense nucleotide sequences of and 1003; and (d) are SEQ ID NOs: 1008 and 189 respectively.

In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 1005 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 1005. The antisense strand of the nucleotide sequence shown in NO:1001. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include a sense strand including the nucleotide sequence shown in SEQ ID NO: 1006 and a sense strand including the nucleotide sequence shown in SEQ ID NO: 1006. The antisense strand of the nucleotide sequence shown in NO: 1002. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 1007 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 1007. The antisense strand of the nucleotide sequence shown in NO: 1003. In some specific examples, the MC2R-targeting oligonucleotides provided by the present disclosure for reducing MC2R expression include the sense strand including the nucleotide sequence shown in SEQ ID NO: 1008 and the oligonucleotide including the nucleotide sequence shown in SEQ ID NO: 1008. The antisense strand of the nucleotide sequence shown in NO: 189.

In some embodiments, MC2R-targeting oligonucleotides comprise structures as shown in Figures 31A - 31B . In some embodiments, MC2R-targeting oligonucleotides comprise structures as shown in Figures 32A - 32B . Oligonucleotides targeting MC2R include the structures shown in Figures 33A - 33B . Oligonucleotides targeting MC2R include the structures shown in Figures 34A - 34B . b. Exemplary modified CYP11B1 oligonucleotides

In some embodiments, an oligonucleotide targeting CYP11B1 includes a sense strand that is 15 to 50 nucleotides long and includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 15 to 40 An antisense strand that is nucleotide long and includes a nucleotide sequence complementary to 15 to 30 nucleotides of the sense strand, wherein the oligonucleotide includes a double-stranded stem having about 2 to 6 base pairs and a stem-loop structure of a loop of 3 to 4 nucleotides, and wherein the sense strand and the antisense strand comprise a modification pattern as shown in Figure 1A . In some embodiments, an oligonucleotide targeting CYP11B1 includes a sense strand that is 15 to 50 nucleotides long and includes 15 to 30 contiguous nucleotides of SEQ ID NO: 226, and 15 to 40 An antisense strand that is nucleotide long and includes a nucleotide sequence complementary to 15 to 30 nucleotides of the sense strand, wherein the oligonucleotide includes a double-stranded stem having about 2 to 6 base pairs and a stem-loop structure of a loop of 3 to 4 nucleotides, and wherein the sense strand and the antisense strand comprise a modification pattern as shown in Figure 1A , wherein the antisense strand is at 4 of the 5' end nucleotides 'The carbon is modified with methoxyphosphonate. In some embodiments, a CYP11B1 oligonucleotide comprises a stem loop including the nucleotide sequence of SEQ ID NO:198. In some embodiments, a CYP11B1 oligonucleotide includes a 6 base pair double-stranded stem and a 4 nucleotide stem loop (including one, two, three, or four nucleotides conjugated to GalNAc). In some specific examples, the nucleotide conjugated to GalNAc is a monovalent GalNAc conjugated to an adenine nucleotide, called [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as follows Shown: .

In some embodiments, the stem loop includes a double-stranded stem of 6 base pairs and a loop including the nucleotide sequence GAAA, where each adenine nucleotide is ademA-GalNAc.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing CYP11B1 expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, GalNac-conjugated nucleotides at positions 28, 29, and 30; and at position 1 a phosphorothioate linkage between , 2'-OMe of 11 to 13 and 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22, and inclusion at position 1 The 5'-terminal nucleotide of the 4'-phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate- 4O-mU]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and positions 27 to 30 A loop of the stem loop is formed, optionally wherein positions 27 to 30 comprise four loops, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand comprises 19 contiguous SEQ ID NO: 226 The nucleotides and the antisense strand comprise nucleotides complementary to the 19 contiguous nucleotides of SEQ ID NO:226.

In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein for reducing CYP11B1 expression include: a sense strand comprising a 2'-F modification at positions 8 to 11 nucleotides, 2'-OMe modified nucleotides at positions 1 to 7, 12 to 27, and 31 to 36, nucleotides conjugated to C22 lipids at position 28; and nucleotides at positions 1 and 2 phosphorothioate linkages between; antisense strands, which include 2'-F modified nucleotides at positions 2 to 5, 7, 10 and 14, and nucleotides at positions 1, 6, 8 to 9, 11 to 13 and 2'-OMe of 15 to 22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22, and the inclusion of 4'- at position 1 The 5'-terminal nucleotide of the phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU ]; wherein positions 1 to 20 of the antisense strand and positions 1 to 20 of the sense strand form a double helix region, wherein positions 21 to 36 of the sense strand form a stem loop, and positions 27 to 30 form the stem a loop of loops, optionally wherein positions 27 to 30 comprise four loops, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand comprises the 19 contiguous nucleotides of SEQ ID NO: 226 And the antisense strand contains nucleotides complementary to 19 contiguous nucleotides of SEQ ID NO:226.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing CYP11B1 expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-GalNac = GalNac with adenine nucleotide attached.

In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing CYP11B1 expression, wherein the oligonucleotide includes a sense strand and an antisense strand according to : Crossed with: where mX = 2'- O -methyl modified nucleotide, fX = 2'-fluoro modified nucleotide, - S - = phosphorothioate linkage, - = phosphodiester linkage, [ MePhosphonate-4O-mX] = 5'-4-O-monomethylphosphonate-2'-O-methyl modified nucleotide and ademA-C22 = C22 carbon chain attached to adenine nucleotide . III. Formulation

A variety of formulations have been developed to facilitate the application of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotide in the formulation. In some embodiments, oligonucleotides are formulated in buffer solutions, such as phosphate buffered saline, liposomes, microcells, and capsids.

Formulations of oligonucleotides and cationic lipids can be used to facilitate transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (eg, polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, the formulations include lipid nanoparticles. In some specific examples, the excipients include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres or nanoparticles, or may be separately formulated for administration to cells, tissues, or cells of subjects in need thereof. Organ or body administration (see, for example, Remington: THE SCIENCE AND PRACTICE OF PHARMACY, ^22nd edition, Pharmaceutical Press, 2013).

In some embodiments, the formulations herein include excipients. In some embodiments, excipients confer increased stability, increased absorption, increased solubility, and/or therapeutic enhancement to the active ingredients of the composition. In some specific examples, the excipient is a buffer (eg, sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or vehicle (eg, buffer solution, petroleum jelly, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its storage life and then made into solution prior to use (eg, administration to a subject). Accordingly, the excipient in a composition comprising any of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidine ketones) or disintegration temperature modifiers (e.g., dextran, Ficoll™ or gelatin).

In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (eg, inhalation), transdermal (eg, topical), transmucosal, and rectal administration.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it will be preferable to include an isotonic agent (eg, sugar), polyol (eg, mannitol, sorbitol, sodium chloride) in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotide in the desired amount with one or a combination of the ingredients enumerated above in the solvent of choice, followed by filtered sterilization.

In some embodiments, the compositions may contain at least about 0.1% or more therapeutic agent, although the percentage of active ingredient(s) may range from about 1% to about 80% or more by weight or volume of the total composition. between. Those skilled in the art will consider factors (such as solubility, bioavailability, biological half-life, route of administration, storage life, and other pharmacological considerations) when preparing such pharmaceutical compositions, and accordingly, various dosages and treatment regimens Possibly whatever you want. a. MC2R and CYP11B1 oligonucleotide combination formulations

In some embodiments, pharmaceutical compositions include first and second therapeutic agents described herein. In some embodiments, the first therapeutic agent is an MC2R oligonucleotide described herein. In some embodiments, the second therapeutic agent is a CYP11B1 oligonucleotide described herein.

In some embodiments, the pharmaceutical composition is a combination product comprising the MC2R oligonucleotide described herein and the CYP11B1 oligonucleotide described herein.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NOs: 213 to 224 and 421 to 612 and an antisense strand comprising any one of SEQ ID NOs: 201 to 212 and 229 to 420 Loyal shares; and ii) CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NOs: 188 to 199 and 1001 to 1004 and any one selected from the group consisting of SEQ ID NOs: 174 to 187 and 997 to 1000 its righteous shares; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NOs: 1001 to 1003 and 189 and a sense strand selected from any one of SEQ ID NOs: 1005 to 1008; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NOs: 213 to 224 and a sense strand comprising any one of SEQ ID NOs: 201 to 212; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NO: 188 to 199 and a sense strand selected from any one of SEQ ID NO: 174 to 187; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NOs: 421 to 612 and a sense strand comprising any one of SEQ ID NOs: 229 to 420; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some specific examples, the combination product includes: i) MC2R oligonucleotide comprising an antisense strand comprising any one of SEQ ID NO: 1001 to 1004 and a sense strand selected from any one of SEQ ID NO: 997 to 1000; and ii) A CYP11B1 oligonucleotide comprising an antisense strand comprising a nucleotide sequence complementary to 15 to 30 contiguous nucleotides of SEQ ID NO: 226 and comprising 15 to 30 contiguous nucleotides of SEQ ID NO: 226 The sense strand of the nucleotide.

In some embodiments, the combination product is formulated as an injectable suspension, gel, oil, pill, tablet, suppository, powder, capsule, mist, ointment, cream, patch or for prolongation and/or Galenic form of slow release means. In some embodiments, the combination product is formulated as an injectable suspension. IV. Usage i. Reduce MC2R and / or CYP11B1 expression in cells

The present disclosure provides methods for contacting or delivering to a cell or cell population an effective amount of any of the oligonucleotides herein for the purpose of reducing MC2R expression. The present disclosure further provides methods of contacting or delivering to a cell or cell population an effective amount of any of the oligonucleotides herein for the purpose of reducing CYP11B1 expression. The present disclosure further provides methods of contacting or delivering to a cell or cell population an effective amount of any of the oligonucleotides herein for the purpose of reducing MC2R and CYP11B1 expression. Such methods may include the steps described herein, and these may, but need not, be performed in the order described. However, other sequences are also conceivable. Furthermore, single or multiple steps may be performed in parallel and/or overlapping in time and/or performed individually or in multiple iterations of steps. Additionally, such methods may include additional, unspecified steps.

The methods herein can be used with any suitable cell type. In some embodiments, the cell is any cell that expresses mRNA (e.g., hepatocytes, macrophages, cells derived from monocytes, prostate cancer cells, brain cells, endocrine tissue, bone marrow, lymph nodes, lungs, gallbladder, liver , duodenum, small intestine, pancreas, kidney, gastrointestinal tract, bladder, fat and soft tissue, and skin). In some embodiments, the cells are primary cells obtained from the subject. In some embodiments, the primary cells have undergone a limited number of passages such that the cells substantially maintain their native phenotypic properties. In some embodiments, the cell to which the oligonucleotide is delivered is ex vivo or in vitro (ie, delivery can be to the cell in culture or to the organism in which the cell is located).

In some embodiments, the oligonucleotides herein are delivered using appropriate nucleic acid delivery methods, including but not limited to injection of a solution containing the oligonucleotide, bombardment of particles covered with the oligonucleotide, and exposure of cells or cell populations. Electroporation of cell membranes in solutions containing oligonucleotides or in the presence of oligonucleotides. Other suitable methods can be used to deliver oligonucleotides to cells, such as lipid-mediated vector transport, chemically mediated transport, and cationic liposome transfection (such as calcium phosphate), among others.

In some embodiments, reduction in MC2R and/or CYP11B1 expression can be achieved by assessing one or more properties or characteristics of cells or cell populations associated with MC2R and/or CYP11B1 expression using appropriate assays or techniques (e.g., using MC2R and or CYP11B1 expression biomarkers), or by assays or techniques that assess molecules that are direct indicators of MC2R and/or CYP11B1 expression (e.g., MC2R and/or CYP11B1 mRNA or MC2R and/or CYP11B1 protein). In some embodiments, MC2R and/or CYP11B1 expression in cells or cell populations contacted with an oligonucleotide is compared to an appropriate control (e.g., an appropriate control that is not exposed to the oligonucleotide or exposed to a control oligonucleotide). cells or cell populations), and evaluate the extent to which the oligonucleotides herein reduce the expression of MC2R and/or CYP11B1. In some embodiments, the appropriate control level for expression of mRNA into protein after delivery of the RNAi molecule can be a predetermined level or value, such that the control level does not need to be measured every time. The predetermined level or value can take many forms. In some embodiments, the predetermined level or value may be a single cutoff value, such as a median or mean.

In some embodiments, administration of the oligonucleotides herein results in reduced expression of MC2R in a cell or population of cells. In some embodiments, MC2R exhibits a reduction of about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% compared to an appropriate mRNA control level. % or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower. An appropriate control level may be the level of mRNA expression and/or protein translation in cells or cell populations that have not been exposed to the oligonucleotides herein. In some embodiments, the effectiveness of delivering oligonucleotides to cells according to the methods herein is assessed after a limited period of time. For example, at least about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1, 2, 3, 4, 5, 6, 7, or even up to 14 days after introduction of the oligonucleotide into the cell. Analysis of mRNA levels in cells. For example, in some embodiments, at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days or about 84 days or longer, determine MC2R expression in cells or cell populations. In some embodiments, at least about 1 month, about 2 months, about 3 months, about 4 months after contacting the oligonucleotide with the cell or cell population or delivering the oligonucleotide to the cell or cell population. , about 5 months or about 6 months or more, to determine MC2R expression in the cell or cell population.

In some embodiments, administration of the oligonucleotides herein results in reduced expression of CYP11B1 in a cell or population of cells. In some embodiments, when compared to an appropriate mRNA control level, CYP11B1 exhibits a reduction of about 1% or less, about 5% or less, about 10% or less, about 15% or less, About 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less. An appropriate control level may be the level of mRNA expression and/or protein translation in cells or cell populations that have not been exposed to the oligonucleotides herein. In some embodiments, the effect of delivering oligonucleotides to cells according to the methods herein is evaluated after a limited period of time. For example, at least about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1, 2, 3, 4, 5, 6, 7, or even up to 14 days after introduction of the oligonucleotide into the cell. Analysis of mRNA levels in cells. For example, in some embodiments, at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days or about 84 days or longer, to determine CYP11B1 expression in cells or cell populations. In some embodiments, at least about 1 month, about 2 months, about 3 months, about 4 months after contacting the oligonucleotide with the cell or cell population or delivering the oligonucleotide to the cell or cell population. , about 5 months, or about 6 months or more, to determine the expression of CYP11B1 in the cell or cell population.

In some embodiments, the oligonucleotide is delivered in the form of a transgene that is engineered to express the oligonucleotide or strands containing the oligonucleotide (eg, its sense and antisense strands) in the cell. In some embodiments, oligonucleotides are delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. Transgenes can be delivered using viral vectors (eg, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (eg, plasmids or synthetic mRNA). In some embodiments, the transgene can be injected directly into the subject. ii.Medical purposes

The present disclosure also provides oligonucleotides for use in or suitable for use in the treatment of a subject that would benefit from reduced expression of MC2R (eg, a human suffering from a disease, disorder, or condition associated with expression of MC2R). In some aspects, the present disclosure provides oligonucleotides useful or suitable for use in treating a subject suffering from a disease, disorder or condition associated with expression of MC2R. The present disclosure also provides oligonucleotides useful or suitable for use in the manufacture of medicaments or pharmaceutical compositions for the treatment of diseases, disorders, or conditions associated with expression of MC2R. In some embodiments, oligonucleotides used or suitable for use target MC2R mRNA and reduce MC2R expression (eg, via an RNAi pathway). In some embodiments, oligonucleotides used or suitable for use target MC2R mRNA and reduce the amount or level of MC2R mRNA, MC2R protein, and/or MC2R activity.

The present disclosure also provides oligonucleotides for use in or suitable for use in the treatment of a subject that would benefit from reduced expression of CYP11B1 (eg, a human suffering from a disease, disorder or condition associated with expression of CYP11B1). In some aspects, the present disclosure provides oligonucleotides useful or suitable for use in treating a subject suffering from a disease, disorder or condition associated with expression of CYP11B1. The present disclosure also provides oligonucleotides useful or suitable for use in the manufacture of medicaments or pharmaceutical compositions for the treatment of diseases, disorders, or conditions associated with expression of CYP11B1. In some embodiments, oligonucleotides used or suitable for use target CYP11B1 mRNA and reduce CYP11B1 expression (eg, via an RNAi pathway). In some embodiments, oligonucleotides used or suitable for use target CYP11B1 mRNA and reduce the amount or level of CYP11B1 mRNA, CYP11B1 protein and/or CYP11B1 activity.

Additionally, the following methods may include selecting subjects who have or are susceptible to a disease, disorder or condition associated with expression of MC2R and/or CYP11B1. In some cases, methods may include selecting individuals with markers for, or susceptibility to, a disease associated with expression of MC2R and/or CYP11B1 (such as elevated blood corticosterone levels). In some embodiments, methods include selecting individuals with loss of expression of CYP11B1 (eg, individuals with congenital adrenal insufficiency). In some embodiments, methods include selecting individuals based on adrenal plasma steroid levels before and after adrenocorticotropic hormone (ACTH) stimulation.

In some embodiments, oligonucleotides used or suitable for use inhibit or reduce tumor growth in a subject. In some embodiments, the tumor is a pituitary tumor.

Likewise, as described in detail below, the methods may also include steps such as measuring or obtaining baseline values for markers of MC2R and/or CYP11B1 expression and then comparing such obtained values to one or more other baseline values or value obtained after administration of the oligonucleotide) to assess the effectiveness of the treatment. iii. Treatment methods

The present disclosure also provides methods of treating a subject having, being suspected of having, or being at risk of developing a disease, disorder, or condition using the oligonucleotides herein. In some aspects, the present disclosure provides methods of using the oligonucleotides herein to treat or attenuate the onset or progression of a disease, disorder or condition associated with expression of MC2R and/or CYP11B1. In other aspects, the present disclosure provides methods of using oligonucleotides herein to achieve one or more therapeutic benefits in a subject suffering from a disease, disorder or condition associated with expression of MC2R and/or CYP11B1. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of any one or more oligonucleotides herein. In some embodiments, treatment includes reducing MC2R and/or CYP11B1 expression. In some instances, the subject receives treatment. In some embodiments, the subject is treated prophylactically. In some embodiments, the subject has received or is receiving a treatment for reducing MC2R (e.g., an oligonucleotide targeting MC2R) and is administered a treatment for reducing expression of CYP11B1 (e.g., an oligonucleotide targeting CYP11B1 glycosides). In some embodiments, the subject has received or is receiving a treatment for reducing CYP11B1 (e.g., an oligonucleotide targeting CYP11B1), and is administered a treatment for reducing the expression of MC2R (e.g., an oligonucleotide targeting MC2R glycosides).

In some embodiments of the methods herein, one or more oligonucleotides, or a pharmaceutical composition comprising one or more oligonucleotides, herein are administered to a subject suffering from a disease, disorder, or condition associated with expression of MC2R. object, causing the subject's MC2R performance to decrease, thereby treating the subject. In some embodiments, the subject has a reduced amount or level of MC2R mRNA. In some embodiments, the subject has a reduced amount or level of MC2R protein. In some embodiments, the subject has a reduced amount or level of MC2R activity. In some embodiments, the subject has a reduced amount or level of plasma corticosterone. In some embodiments, an oligonucleotide targeting MC2R reduces plasma corticosterone levels by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, oligonucleotides targeting MC2R reduce plasma corticosterone levels by approximately 70%. In some embodiments, a single dose of an oligonucleotide targeting MC2R reduces plasma corticosterone levels by approximately 70%. In some embodiments, oligonucleotides targeting MC2R reduce plasma corticosterone levels to baseline. In some embodiments, oligonucleotides targeting MC2R reduce plasma corticosterone levels to healthy or normal levels. In some embodiments, the subject has reduced or altered MC2R expression, the amount or level of MC2R mRNA, the amount or level of MC2R protein, the amount or level of MC2R activity, and the amount or level of plasma corticosterone.

In some embodiments of the methods herein, one or more oligonucleotides, or a pharmaceutical composition comprising one or more oligonucleotides, herein are administered to a subject suffering from a disease, disorder, or condition associated with expression of CYP11B1 A substance that reduces the subject's CYP11B1 expression, thereby treating the subject. In some embodiments, the subject has a reduced amount or level of CYP11B1 mRNA. In some embodiments, the subject has a reduced amount or level of CYP11B1 protein. In some embodiments, the subject has a reduced amount or level of CYP11B1 activity. In some embodiments, the subject has a reduced amount or level of plasma corticosterone. In some embodiments, an oligonucleotide targeting CYP11B1 reduces plasma corticosterone levels by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, oligonucleotides targeting CYP11B1 reduce plasma corticosterone levels by approximately 40%. In some embodiments, a single dose of an oligonucleotide targeting CYP11B1 reduces plasma corticosterone levels by approximately 50%. In some embodiments, a single dose of an oligonucleotide targeting CYP11B1 reduces plasma corticosterone levels by approximately 40 to 50%. In some embodiments, oligonucleotides targeting CYP11B1 reduce plasma corticosterone levels to baseline. In some embodiments, oligonucleotides targeting CYP11B1 reduce plasma corticosterone levels to healthy or normal levels. In some embodiments, the subject has a decrease or alteration in any combination of: CYP11B1 expression, the amount or level of CYP11B1 mRNA, the amount or level of CYP11B1 protein, the amount or level of CYP11B1 activity, and the amount or level of plasma corticosterone.

In some embodiments of the methods herein, a subject suffering from a disease, disorder, or condition associated with expression of MC2R and CYP11B1 is administered one or more oligonucleotides herein, or a composition comprising one or more oligonucleotides. The pharmaceutical composition reduces the expression of MC2R and CYP11B1 in the subject, thereby treating the subject. In some embodiments, the subject has reduced amounts or levels of MC2R and CYP11B1 mRNA. In some embodiments, the subject has reduced amounts or levels of MC2R and CYP11B1 proteins. In some embodiments, the subject has a reduced amount or level of MC2R and CYP11B1 activity. In some embodiments, the subject has a reduced amount or level of plasma corticosterone. In some embodiments, a combination of oligonucleotides targeting MC2R and CYP11B1 reduces plasma corticosterone levels by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, combinations of oligonucleotides targeting MC2R and CYP11B1 reduce plasma corticosterone levels by approximately 80%. In some embodiments, combinations of oligonucleotides targeting MC2R and CYP11B1 reduce plasma corticosterone levels to baseline. In some embodiments, combinations of oligonucleotides targeting MC2R and CYP11B1 reduce plasma corticosterone levels to healthy or normal levels. In some embodiments, the subject is reducing or altering any combination of: MC2R and CYP11B1 expression, the amount or level of MC2R and CYP11B1 mRNA, the amount or level of MC2R and CYP11B1 protein, the amount or level of MC2R and CYP11B1 activity, and The amount or level of plasma corticosterone.

In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject suffering from a disease, disorder, or condition associated with MC2R such that the administration of The subject's MC2R performance is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55% when compared to the previous MC2R performance of one or more oligonucleotides or pharmaceutical compositions , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , reference or control subject), the subject's MC2R performance is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65 %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject suffering from a disease, disorder, or condition associated with CYP11B1 such that the administration of The subject's CYP11B1 performance is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55% when compared to the previous CYP11B1 performance of one or more oligonucleotides or pharmaceutical compositions , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , reference or control subject), the subject's CYP11B1 performance is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide or oligonucleotides herein or comprising the oligonucleotide or A pharmaceutical composition of a plurality of oligonucleotides such that the amount or level of MC2R mRNA in the subject is reduced by at least about 30% when compared to the amount or level of MC2R mRNA prior to administration of the oligonucleotide or pharmaceutical composition. , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , a reference or control subject), the amount or level of MC2R mRNA in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55% , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide or oligonucleotides herein or comprising the oligonucleotide or A pharmaceutical composition of a plurality of oligonucleotides such that the amount or level of CYP11B1 mRNA in the subject is reduced by at least about 30% when compared to the amount or level of CYP11B1 mRNA prior to administration of the oligonucleotide or pharmaceutical composition. , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , a reference or control subject), the amount or level of CYP11B1 mRNA in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% %, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide or oligonucleotides herein or comprising the oligonucleotide or A pharmaceutical composition of a plurality of oligonucleotides such that the amount or level of MC2R protein in the subject is reduced by at least about 30% when compared to the amount or level of MC2R protein before administration of the oligonucleotide or pharmaceutical composition. , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , a reference or control subject), the amount or level of MC2R protein in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55 %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide or oligonucleotides herein or comprising the oligonucleotide or A pharmaceutical composition of a plurality of oligonucleotides such that the amount or level of CYP11B1 protein in the subject is reduced by at least about 30% when compared to the amount or level of CYP11B1 protein before administration of the oligonucleotide or pharmaceutical composition. , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, when compared with a subject who did not receive the oligonucleotide or oligonucleotides or pharmaceutical compositions or who received a control oligonucleotide or oligonucleotides, pharmaceutical compositions or treatments (e.g. , a reference or control subject), the amount or level of CYP11B1 protein in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55% , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide or oligonucleotides herein are administered to a subject suffering from a disease, disorder or condition associated with MC2R and CYP11B1 or comprising the oligonucleotide. or a pharmaceutical composition of a plurality of oligonucleotides such that the subject has an amount or level of MC2R and CYP11B1 activity/expression when compared to the amount or level of MC2R and CYP11B1 activity prior to administration of the oligonucleotide or pharmaceutical composition or levels reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85 %, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, when compared to the amount of MC2R and CYP11B1 activity in a subject that did not receive an oligonucleotide or pharmaceutical composition or that received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject) or The amount or level of MC2R and CYP11B1 activity in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject suffering from a disease, disorder, or condition associated with expression of MC2R such that when compared with The amount or level of plasma corticosterone in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more than 99% . In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject suffering from a disease, disorder, or condition associated with expression of CYP11B1 such that when compared with The amount or level of plasma corticosterone in the subject is reduced by at least about 30%, about 35%, about 40%, about 45% compared to the amount or level of plasma corticosterone before administration of the oligonucleotide or pharmaceutical composition. %, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, when compared with the amount of plasma corticosterone in a subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject) or The amount or level of plasma corticosterone in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of MC2R and/or CYP11B1, the amount or level of MC2R and/or CYP11B1 mRNA, or the amount or activity of MC2R and/or CYP11B1 protein in a subject or a sample from a subject are known in the art to which the invention belongs. By. Additionally, the examples presented herein illustrate methods for determining MC2R and/or CYP11B1 expression.

In some embodiments, reducing cells (e.g., adrenal cells), cell populations or groups of cells (e.g., organoids), organs (e.g., adrenal glands), blood or portions thereof (e.g., plasma), tissues (e.g., adrenal tissue) ), MC2R expression, MC2R mRNA amount or level, MC2R protein, MC2R activity in a sample (e.g., adrenal biopsy sample) or any other appropriate biological material obtained or isolated from the subject. In some embodiments, the reduction is in more than one type of cell (e.g., adrenal gland cells and one or more other cell types), in more than one cell population, in more than one organ (e.g., in the adrenal gland and one or more other organs), in more than one A blood portion (e.g., plasma and one or more other blood portions), more than one type of tissue (e.g., adrenal tissue and one or more other types of tissue), or more than one type of sample (e.g., an adrenal biopsy sample and one or more other types of tissue) or various other types of biometric samples), the amount or level of MC2R mRNA, MC2R protein, MC2R activity, plasma corticosterone, or any combination thereof.

In some embodiments, reducing cells (e.g., adrenal cells), cell populations or groups of cells (e.g., organoids), organs (e.g., adrenal glands), blood or portions thereof (e.g., plasma), tissues (e.g., adrenal tissue) ), CYP11B1 expression, amount or level of CYP11B1 mRNA, CYP11B1 protein, CYP11B1 activity in a sample (e.g., adrenal biopsy sample) or any other appropriate biological material obtained or isolated from the subject. In some embodiments, the reduction is in more than one type of cell (e.g., adrenal gland cells and one or more other cell types), in more than one cell population, in more than one organ (e.g., in the adrenal gland and one or more other organs), in more than one A blood portion (e.g., plasma and one or more other blood portions), more than one type of tissue (e.g., adrenal tissue and one or more other types of tissue), or more than one type of sample (e.g., an adrenal biopsy sample and one or more other types of tissue) or various other types of biometric samples), the amount or level of CYP11B1 mRNA, CYP11B1 protein, CYP11B1 activity, plasma corticosterone, or any combination thereof.

In some embodiments, reducing cells (e.g., adrenal cells), cell populations or groups of cells (e.g., organoids), organs (e.g., adrenal glands), blood or portions thereof (e.g., plasma), tissues (e.g., adrenal tissue) ), expression of MC2R and CYP11B1, amount or level of MC2R and CYP11B1 mRNA, MC2R and CYP11B1 protein, MC2R and CYP11B1 activity in a sample (e.g., adrenal biopsy sample) or any other appropriate biological material obtained or isolated from a subject . In some embodiments, the reduction is in more than one type of cell (e.g., adrenal gland cells and one or more other cell types), in more than one cell population, in more than one organ (e.g., in the adrenal gland and one or more other organs), in more than one A blood portion (e.g., plasma and one or more other blood portions), more than one type of tissue (e.g., adrenal tissue and one or more other types of tissue), or more than one type of sample (e.g., an adrenal biopsy sample and one or more other types of tissue) or various other types of biometric samples), the amount or level of MC2R and CYP11B1 mRNA, MC2R and CYP11B1 protein, MC2R and CYP11B1 activity, plasma corticosterone, or any combination thereof.

Examples of diseases, disorders, or conditions associated with manifestations of MC2R include, but are not limited to, Cushing's disease, Cushing's syndrome, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal insufficiency Hypoplasia, congenital adrenal agenesis, familial Addison's disease, and other metabolic-related conditions and diseases associated with MC2R. Examples of diseases, disorders, or conditions associated with expression of CYP11B1 include, but are not limited to, Cushing's disease, Cushing's syndrome, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal insufficiency Hypoplasia, familial Addison's disease, and other metabolism-related conditions and diseases associated with CYP11B1. In some embodiments, diseases, disorders or conditions associated with expression of MC2R and CYP11B1 include, but are not limited to, Cushing's disease, Cushing's syndrome, familial glucocorticoid deficiency, hereditary adrenocortical insensitivity to ACTH. Reactive, congenital adrenal insufficiency, familial Addison's disease, and other metabolic disorders and diseases associated with MC2R. In some embodiments, the present disclosure provides methods of treating congenital adrenal insufficiency in a subject comprising administering an MC2R-targeting oligonucleotide described herein.

In some embodiments, oligonucleotides targeting MC2R and CYP11B1 described herein can be used to treat diseases or conditions that cause adrenal enlargement. In some embodiments, oligonucleotides targeting MC2R alone or in combination with oligonucleotides targeting CYP11B1 reduce adrenal gland size. In some embodiments, oligonucleotides targeting MC2R and CYP11B1 described herein can be used to treat diseases or conditions that result in adrenal cortex thickening. In some embodiments, oligonucleotides targeting MC2R alone or in combination with oligonucleotides targeting CYP11B1 reduce adrenal cortex thickness. In some embodiments, oligonucleotides targeting MC2R and CYP11B1 described herein can be used to treat diseases or conditions that result in ZF vacuolization and/or ZG extrusion. In some embodiments, oligonucleotides targeting MC2R alone or in combination with oligonucleotides targeting CYP11B1 reduce ZF vacuolation and/or ZG extrusion.

In some embodiments, the present disclosure provides a method of inhibiting or reducing tumor growth in a subject, comprising administering an oligonucleotide described herein. In some embodiments, the tumor is a pituitary tumor.

Due to the high specificity of the oligonucleotides herein, they specifically target the mRNA of target genes in diseased cells and tissues. In preventing disease, the target gene may be a gene that is necessary to initiate or maintain the disease, or a gene that has been identified as being associated with a higher risk of contracting the disease. In treating a disease, the oligonucleotide can be brought into contact with cells or tissues that exhibit the disease. For example, oligonucleotides that are substantially identical to all or a portion of a wild-type (i.e., native) or mutant gene associated with a disorder or condition associated with expression of MC2R can be administered to a cell or tissue type of interest, such as hepatocytes. or other hepatocytes) contact or introduce the cell or tissue type of interest. Oligonucleotides that are substantially identical to all or a portion of a wild-type (i.e., native) or mutant gene associated with a disorder or condition associated with expression of CYP11B1 can be administered to a cell or tissue type of interest, such as hepatocytes or other Hepatocytes) are contacted or introduced into the cell or tissue type of interest.

In some embodiments, the MC2R gene can be an MC2R gene from any mammal, such as a human target. Any MC2R gene can be silenced according to the methods described herein. In some embodiments, the CYP11B1 gene can be a CYP11B1 gene from any mammal, such as a human target. Any CYP11B1 gene can be silenced according to the methods described herein.

The methods described herein generally involve administering to a subject an effective amount of one or more oligonucleotides, ie, an amount capable of producing the desired therapeutic result. A therapeutically acceptable amount may be an amount that treats the disease or condition. The appropriate dosage for any subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition intended to be administered, the active ingredient(s) in the composition, the time and route of administration, and general health. , and other medications taken at the same time.

In some embodiments, enterally (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or via the rectum), parenterally (e.g., subcutaneous injection, intravenous injection, or Infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecally), topically (e.g., epidermis, inhalation, via eye drops or via mucosa), or by direct injection administered to the target organ (eg, the subject's liver). Typically, oligonucleotides herein are administered intravenously or subcutaneously.

As one set of non-limiting examples, oligonucleotides herein are typically administered quarterly (once every three months), bimonthly (once every two months), monthly, or weekly. For example, the oligonucleotide can be administered weekly or at intervals of two or three weeks. Alternatively, the oligonucleotide can be administered daily. In some embodiments, a subject is administered one or more loading doses of an oligonucleotide, followed by one or more maintenance doses of an oligonucleotide.

In some embodiments, the oligonucleotides herein are administered individually or in combination. In some embodiments, the oligonucleotides herein are administered simultaneously, sequentially (in any order), or intermittently in combination. For example, two oligonucleotides (i.e., a MC2R oligonucleotide described herein and a CYP11B1 oligonucleotide described herein) can be co-administered simultaneously. Alternatively, one oligonucleotide can be administered and a second oligonucleotide administered any amount of time later (e.g., one hour, one day, one week, or one month). In some embodiments, MC2R oligonucleotides described herein are administered simultaneously with CYP11B1 oligonucleotides described herein. In some embodiments, a MC2R oligonucleotide described herein is administered prior to a CYP11B1 oligonucleotide described herein. In some embodiments, a CYP11B1 oligonucleotide described herein is administered prior to a MC2R oligonucleotide described herein.

In some embodiments, an oligonucleotide herein is administered alone or in combination with at least a second therapeutic agent or procedure (e.g., a second, third, or fourth therapeutic agent or procedure). ). In some embodiments, at least the second therapeutic agent or procedure is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production, or the effects of glucocorticoids on peripheral tissues; surgical procedures; radiation procedures; gene therapy; and any combination thereof. In some embodiments, the inhibitor of cortisol production is selected from the group consisting of ketoconazole, mitotane, metyrapone, and etomidate. In some embodiments, the therapeutic agent is selected from the group consisting of: mifepristone (a glucocorticoid receptor antagonist and competitive inhibitor of cortisol at the receptor), oxirin Lorestat or metyrapone (CYP11B1 inhibitor), pasireotide (lowers the production of ACTH in the pituitary gland), and any combination thereof.

In some embodiments, at least the second therapeutic agent or procedure is selected from, but is not limited to, inhibitors of pituitary ACTH secretion, adrenal cortisol production, or the effects of glucocorticoids on peripheral tissues; surgical procedures; radiation procedures; genetics Treatment; and any combination thereof. In some embodiments, the second therapeutic agent is selected from the group consisting of: mifepristone (a glucocorticoid receptor antagonist and competitive inhibitor of cortisol at the receptor), Osilodrostat or metyrapone (CYP11B1 inhibitors), pasireotide (lowers the production of ACTH in the pituitary gland), and any combination thereof.

In some embodiments, at least a second therapeutic agent described herein is administered with a MC2R oligonucleotide described herein. In some embodiments, at least a second therapeutic agent described herein is administered with a CYP11B1 oligonucleotide described herein. In some embodiments, at least a second therapeutic agent described herein is administered with a MC2R oligonucleotide and a CYP11B1 oligonucleotide described herein.

In some embodiments, MC2R oligonucleotides described herein are administered with at least a second therapeutic agent or procedure selected from the group consisting of: pituitary ACTH secretion , inhibitors of adrenal cortisol production or the effects of glucocorticoids on peripheral tissues; surgical procedures; radiation procedures; gene therapy; and any combination thereof. In some embodiments, MC2R oligonucleotides described herein are combined with at least a second therapeutic agent or procedure selected from the group consisting of: mifepristone (a sugar corticosteroid receptor antagonists and competitive inhibitors of cortisol at this receptor), osilodrostat or metyrapone (CYP11B1 inhibitors), pasireotide (reduces the production of ACTH in the pituitary gland) Vote together with any combination thereof.

In some embodiments, a CYP11B1 oligonucleotide described herein is administered with at least a second therapeutic agent or procedure selected from the group consisting of: Pituitary ACTH secretion , inhibitors of adrenal cortisol production or the effects of glucocorticoids on peripheral tissues; surgical procedures; radiation procedures; gene therapy; and any combination thereof. In some embodiments, a CYP11B1 oligonucleotide described herein is administered with at least a second therapeutic agent or procedure selected from the group consisting of: mifepristone (a glucocorticoid receptor antagonist and competitive inhibitor of cortisol at this receptor), osilodrostat or metyrapone (CYP11B1 inhibitors), pasireotide (reduces ACTH in the pituitary gland) ) and any combination thereof.

In some embodiments, MC2R oligonucleotides and CYP11B1 oligonucleotides described herein are administered with at least a second therapeutic agent or procedure selected from the group consisting of: Group: Inhibitors of pituitary ACTH secretion, adrenal cortisol production, or the effects of glucocorticoids on peripheral tissues; surgical procedures; radiation procedures; gene therapy; and any combination thereof. In some embodiments, MC2R oligonucleotides and CYP11B1 oligonucleotides described herein are administered with at least a second therapeutic agent or procedure selected from the group consisting of: Group: mifepristone (a glucocorticoid receptor antagonist and competitive inhibitor of cortisol at this receptor), osilodrostat or metyrapone (CYP11B1 inhibitors), pasireotide (reduces the production of ACTH in the pituitary gland) and any combination thereof.

In some embodiments, an oligonucleotide herein is administered concurrently, sequentially (in any order), or intermittently with at least a second therapeutic agent described herein (e.g., a second, third, or fourth therapeutic agents). For example, two oligonucleotides (ie, a MC2R oligonucleotide described herein and a CYP11B1 oligonucleotide described herein) are co-administered simultaneously with a third therapeutic agent described herein. In some embodiments, an oligonucleotide is co-administered with at least a second therapeutic agent. Alternatively, one oligonucleotide can be administered and at least a second therapeutic agent described herein administered any amount of time later (eg, one hour, one day, one week, or one month). In some embodiments, a MC2R oligonucleotide and/or a CYP11B1 oligonucleotide described herein is administered simultaneously with at least a second therapeutic agent described herein. In some embodiments, a MC2R oligonucleotide and/or a CYP11B1 oligonucleotide described herein is administered before at least a second therapeutic agent described herein. In some embodiments, at least a second therapeutic agent (eg, a second, third or fourth therapeutic agent) is administered prior to administration of a MC2R oligonucleotide and/or CYP11B1 oligonucleotide described herein.

In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include livestock, such as dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and animals, such as mice, rats, guinea pigs, and hamsters. V.Set _

In some embodiments, the present disclosure provides a kit comprising an oligonucleotide herein and instructions for use. In some embodiments, a kit includes an oligonucleotide herein and instructions containing instructions for use of the kit and/or any components thereof. In some embodiments, a kit includes an oligonucleotide herein, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art to which this invention pertains, in suitable containers. In some embodiments, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the oligonucleotide is placed (and, in some cases, appropriately aliquoted). In some embodiments where additional ingredients are provided, the kit contains additional containers in which the ingredients are placed. The kit may also include tools for containing the oligonucleotide and any other reagents in a confined space for commercial sale. Such containers may include injection or blow molded plastic containers in which the desired vials are retained. The container and/or set may include a label containing instructions for use and/or warnings.

In some embodiments, a kit includes an MC2R oligonucleotide herein and a medically acceptable carrier or a pharmaceutical composition comprising the oligonucleotide and a combination for treating or delaying a subject in need thereof. Instructions for the development of a disease, disorder or condition associated with MC2R expression. In some embodiments, a kit includes a CYP11B1 oligonucleotide herein and a medically acceptable carrier or a pharmaceutical composition comprising the oligonucleotide and a combination of CYP11B1 and CYP11B1 in a subject in need thereof. Instructions for the development of a disease, disease, or condition related to the manifestation. In some embodiments, the kit includes the MC2R oligonucleotide and CYP11B1 oligonucleotide herein and a medically acceptable carrier or a pharmaceutical composition comprising the oligonucleotide and used to treat or delay the disease. Instructions for the development of diseases, disorders, or conditions associated with expression of MC2R and CYP11B1 that are subject to the requirement.

In some embodiments, a kit includes an MC2R oligonucleotide herein and a medically acceptable carrier or a pharmaceutical composition comprising the oligonucleotide and is used to treat patients who have received or are receiving a CYP11B1 inhibitor. Instructions for administering the oligonucleotide to a subject in need of treatment. In some embodiments, a kit includes a CYP11B1 oligonucleotide herein and a medically acceptable carrier or a pharmaceutical composition comprising the oligonucleotide and is used to administer the drug to patients who have received or are undergoing treatment. Instructions for administering the oligonucleotide to a subject in need thereof who is receiving treatment with an MC2R inhibitor. Other specific examples

This disclosure relates to the following specific examples. In this section, the specific example of the term is abbreviated as "E", followed by the ordinal number. For example, E1 is equivalent to Example 1.

E1. An oligonucleotide for reducing the expression of melanocortin 2 receptor (MC2R), the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and an antisense strand of 15 to 40 nucleotides in length. A sense strand, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has a complementary region to the target sequence of MC2R as set forth in any of SEQ ID NOs: 201 to 212 .

E2. The oligonucleotide of E1, wherein the complementary region is completely complementary to the target sequence of MC2R.

E3. The oligonucleotide of any one of E1 or E2, wherein the antisense strand is 19 to 27 nucleotides long.

E4. The oligonucleotide of any one of E1 to E3, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long.

E5. The oligonucleotide of E1, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long.

E6. The oligonucleotide of any one of E1 to E5, wherein the double-stranded helix region is at least 19 nucleotides long.

E7. The oligonucleotide of any one of E1 to E6, wherein the double-stranded helix region is at least 20 nucleotides long, optionally wherein the double-stranded helix region is 21 nucleotides long.

E8. The oligonucleotide of any one of E1 to E7, wherein the complementary region is at least 19 contiguous nucleotides long.

E9. The oligonucleotide of any one of E1 to E8, wherein the complementary region is at least 21 contiguous nucleotides long.

E10. The oligonucleotide of any one of E1 to E9, wherein the antisense strand comprises the sequence shown in any one of SEQ ID NO: 37 to 48.

E11. The oligonucleotide of any one of E1 to E10, wherein the sense strand comprises the sequence shown in any one of SEQ ID NOs: 85 to 96.

E12. The oligonucleotide of any one of E1 to E11, wherein the sense strand comprises a stem loop at its 3' end as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L is in S1 A 3 to 5 nucleotide long loop is formed between S2 and S2.

E13. An oligonucleotide for reducing the performance of MC2R, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and has the same characteristics as SEQ ID NO : The complementary region of the target sequence of MC2R shown in any one of 201 to 212, wherein the sense strand includes a stem loop shown below at its 3' end: S1-L-S2, wherein S1 is complementary to S2 , and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a double helix region of at least 19 nucleotides long.

E14. The oligonucleotide of E13, wherein the complementary region is at least 19 nucleotides long and is completely complementary to the target sequence.

E15. The oligonucleotide of any one of E12 to E14, wherein L is a tetracyclic ring.

E16. The oligonucleotide of any one of E12 to E15, wherein L is 4 nucleotides long.

E17. The oligonucleotide of any one of E12 to E16, wherein L includes the sequence represented by GAAA.

E18. The oligonucleotide of any one of E1 to E17, wherein (i) the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long, or (ii) the antisense strand is 22 nucleotides long and the sense strand is 36 nucleotides long.

E19. The oligonucleotide of E18, wherein the antisense strand and the sense strand form a double helix region that is 25 nucleotides long or 20 nucleotides long.

E20. The oligonucleotide of any one of E1 to E19, wherein the antisense strand comprises a 3' overhang sequence of one or more nucleotides, optionally wherein the 3' overhang sequence is 2 nucleotides acid length, optionally wherein the 3' overhang sequence is GG.

E21. The oligonucleotide of any of the preceding embodiments, wherein the oligonucleotide comprises at least one modified nucleotide.

E22. The oligonucleotide of E21, wherein the modified nucleotide comprises a 2'-modification.

E23. The oligonucleotide of E22, wherein the 2'-modification is selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and Modification of 2'-deoxy-2'-fluoro-β-d-arabinic acid.

E24. The oligonucleotide of any one of E20 to E23, wherein approximately 10 to 15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotides of the sense strand comprise 2 '-Fluorine modification.

E25. The oligonucleotide of any one of E20 to E24, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31% of the nucleotide of the antisense strand %, 32%, 33%, 34% or 35% contain a 2'-fluoro modification.

E26. The oligonucleotide of any one of E20 to E25, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% contained 2'-fluoro modifications.

E27. The oligonucleotide of any one of E20 to E26, wherein the sense strand comprises 36 nucleotides from 5' to 3 at positions 1 to 36, wherein positions 8 to 11 comprise a 2'-fluoro modification .

E28. The oligonucleotide of any one of E20 to E27, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, and positions 2, 3, 4, and 5 thereof , 7, 10 and 14 contain 2'-fluoro modifications.

E29. The oligonucleotide of any one of E23 to E28, wherein the remaining nucleotides comprise a 2'-O-methyl modification.

E30. The oligonucleotide of E27, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications.

E31. The oligonucleotide of E28 or E30, wherein positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand comprise a 2'-O-methyl modification.

E32. The oligonucleotide of any one of E20 to E31, wherein all nucleotides of the oligonucleotide are modified.

E33. The oligonucleotide of any of the preceding embodiments, wherein the oligonucleotide comprises at least one modified inter-nucleotide linkage.

E34. The oligonucleotide of E33, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

E35. The oligonucleotide of E34, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

E36. The oligonucleotide of E34 or E35, wherein the antisense strand comprises 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand comprises positions 1 and 2, 2 and phosphorothioate linkages between 3, 20 and 21, and 21 and 22.

E37. The oligonucleotide of any of the preceding embodiments, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog.

E38. The oligonucleotide of E37, wherein the phosphate analog is methoxyphosphonate, vinylphosphonate or malonylphosphonate.

E39. The oligonucleotide of any of the preceding embodiments, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more target ligands.

E40. The oligonucleotide of E39, wherein the nucleotide is bound to more than one target ligand, wherein the target ligands are the same or different.

E41. The oligonucleotide of E39 or E40, wherein the one or more target ligands are selected from carbohydrates, amino sugars, cholesterol, polypeptides or lipids.

E42. The oligonucleotide of E35 or E36, wherein the one or more target ligands are saturated or unsaturated fatty acid moieties.

E43. The oligonucleotide of E39 or E40, wherein the target ligand is a saturated fatty acid moiety ranging in size from C10 to C24.

E44. The oligonucleotide of E43, wherein the target ligand is a C16 saturated fatty acid moiety.

E45. The oligonucleotide of E43, wherein the target ligand is a C18 saturated fatty acid moiety.

E46. The oligonucleotide of E43, wherein the target ligand is a C22 saturated fatty acid moiety.

E47. The oligonucleotide of E39 or E40, wherein the target ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

E48. The oligonucleotide of E47, wherein the GalNAc part is a monovalent GalNAc part, a divalent GalNAc part, a trivalent GalNAc part or a tetravalent GalNAc part.

E49. The oligonucleotide of any one of E12 to E48, wherein up to 4 nucleotides of L of the stem loop are each conjugated to a monovalent GalNAc moiety.

E50. The oligonucleotide of any one of E1 to E49, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively.

E51. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 85.

E52. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 38, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 86.

E53. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NO: 176 to 187.

E54. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NO: 174 to 175.

E55. The oligonucleotide of any one of E1 to E49 and E54, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NO: 188 to 199.

E56. The oligonucleotide of any one of E1 to E49, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 186 and 188 respectively; (b) SEQ ID NO: 187 and 189 respectively; (c) SEQ ID NO: 176 and 190 respectively; (d) SEQ ID NO: 177 and 191 respectively; (e) SEQ ID NO: 178 and 192 respectively; (f) SEQ ID NO: 179 and 193 respectively; (g) SEQ ID NO: 180 and 194 respectively; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 184 and 198 respectively; and (l) SEQ ID NO: 185 and 199 respectively.

E57. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 186, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 188.

E58. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 187, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 189.

E59. The oligonucleotide of any one of E1 to E49, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 174 and 188 respectively; and (b) SEQ ID NO: 175 and 189 respectively.

E60. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 174, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 188.

E61. The oligonucleotide of any one of E1 to E49, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 175, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 189.

E62. An oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence that is complementary to a target sequence in MC2R mRNA region and (ii) one or more target ligands,

The oligonucleotide-ligand conjugate is represented by formula Ea :

Ea ;

or a medically acceptable salt thereof, wherein:

B is the nucleobase;

R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R, -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or

R ¹ and R ² on the same carbon together with their intervening atoms form a 3 to 7 membered saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

Each ^RA is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic rings and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur;

Each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from nitrogen, oxygen and 4 to 7 membered saturated or partially unsaturated heterocyclic rings having heteroatoms of sulfur and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; or

Two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaromatic ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur;

Each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has a range of 0 to The 10 methylene units are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, - S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-replacement;

Each -Cy- is independently an optionally substituted divalent ring selected from the group consisting of phenylene, 8 to 10-membered bicyclic aryl, and 4 to 7-membered saturated or partially unsaturated carbocyclic ring. group, a 4 to 11-membered saturated or partially unsaturated spirocyclic carbocyclic group, an 8 to 10-membered bicyclic saturated or partially unsaturated carbocyclic group, having 1 to 3 independently selected from nitrogen, oxygen and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic heterocyclic groups with 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4 to 11 membered saturated or partially unsaturated spirocyclic heterocyclic groups Cyclic group, an 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic cyclic group having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and A 5- to 6-membered heteroaryl group with a heteroatom of sulfur or an 8- to 10-membered bicyclic heteroaryl group with 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur;

n ranges from 1 to 10;

L is a covalent bond or a divalent saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O )OR-, -P(S)OR-, -V ¹ CR ² W ¹ -OR replacement; replacement

m ranges from 1 to 50;

X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-;

Y is hydrogen, suitable hydroxyl protecting group, or ;

R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from nitrogen , 4 to 7 membered saturated or partially unsaturated heterocyclic rings with oxygen and sulfur heteroatoms and 5 to 6 membered heteroaromatic rings with 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur;

X ² is O, S or NR;

X ³ is -O-, -S-, -BH ₂ - or covalent bond;

Y ¹ is a linker attached to the 2' or 3' end of a nucleoside, nucleotide or oligonucleotide;

Y ² is hydrogen, a suitable protecting group, a phosphoramidite analog, an internucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a linker attached to a solid support ;as well as

Z is -O-, -S-, -NR- or -CR ₂ -.

E63. The oligonucleotide-ligand conjugate of E62 is represented by formula IEb or IEc :

EB

IEc

or a medically acceptable salt thereof, wherein:

L ¹ is a covalent bond or a divalent saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, -O- , -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P( O)OR-, -P(S)OR-or replacement; replacement

R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and

R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C(O)NR-, - NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S) OR replacement.

E64. The oligonucleotide-ligand conjugate of E63, wherein:

R ⁵ series selected from

.

E65. The oligonucleotide-ligand conjugate of E63, wherein R ⁵ is selected from

.

E66. Oligonucleotide-ligand conjugate of E63, wherein R ⁵ is .

E67. An oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence that is complementary to a target sequence in MC2R mRNA region and (ii) one or more target ligands,

Wherein the oligonucleotide-ligand conjugate is represented by formula IEIb or IEIc :

EIib

iEc

or a medically acceptable salt thereof; wherein

B is the nucleobase;

m ranges from 1 to 50;

X ¹ is -O- or -S-;

Y is hydrogen, or ;

R ³ is hydrogen or a suitable protecting group;

X ² is O or S;

X ³ is -O-, -S- or covalent bond;

Y ¹ is a linker attached to the 2'- or 3' end of a nucleoside, nucleotide or oligonucleotide;

Y ² is hydrogen, a phosphoramidite analog, an internucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a linker attached to a solid support;

R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C(O)NR-, - NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S) OR substitution; and

R is hydrogen, a suitable protecting group, or an optionally substituted group selected from the group consisting of C _1-6 aliphatic, phenyl, and a heterogenous group having 1 to 2 independently selected from nitrogen, oxygen, and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic rings of atoms and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

E68. The oligonucleotide-ligand conjugate of E67, wherein R ⁵ is selected from

.

E69. The oligonucleotide-ligand conjugate of any one of E67 to E68, wherein R ⁵ is

.

E70. The oligonucleotide-ligand conjugate of any one of E62 to E69, wherein the

The nucleotide sequence contains 1 to 10 targeting ligands.

E71. The oligonucleotide-ligand conjugate of any one of E62 to E70, wherein the

Nucleotide sequences contain 1, 2 or 3 targeting ligands.

E72. The oligonucleotide-ligand conjugate of any one of E62 to E71, wherein in the

These cells in the adrenal cortex or adrenal gland are epithelial cells.

E73. The oligonucleotide-ligand conjugate of any one of E62 to E72, wherein

The oligonucleotide is single-stranded, optionally wherein the single-stranded oligonucleotide is from 15 to

30 nucleotides long.

E74. The oligonucleotide-ligand conjugate of any one of E62 to E72, wherein the

Oligonucleotides are double-stranded.

E75. The oligonucleotide-ligand conjugate of E74, wherein the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the The sense strand and the antisense strand form a double helix region, wherein the antisense strand includes a complementary region to the target sequence, and wherein the complementary region is at least 15 contiguous nucleotides long.

E76. The oligonucleotide-ligand conjugate of E75, wherein the antisense strand is 19 to 27 nucleotides long.

E77. The oligonucleotide-ligand conjugate of E75 or E76, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long.

E78. The oligonucleotide-ligand conjugate of any one of E75 to 77 or E64, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleosides Sour and long.

E79 The oligonucleotide-ligand conjugate of any one of E.75 to E78, wherein the double helix region is at least 19 nucleotides long.

E80. The oligonucleotide-ligand conjugate of any one of E75 to E79, wherein the double-stranded helix region is at least 20 nucleotides long, optionally wherein the double-stranded helix region is 21 nucleotides long.

E81. The oligonucleotide-ligand conjugate of any one of E75 to E80, wherein the complementary region is at least 19 contiguous nucleotides long.

E82. The oligonucleotide-ligand conjugate of any one of E75 to E81, wherein the complementary region is at least 21 contiguous nucleotides long.

E83. The oligonucleotide-ligand conjugate of any one of E75 to E82, wherein the sense strand includes a stem loop as shown below at its 3' end: S1-L-S2, wherein S1 is complementary to S2 , and where L forms a 3 to 5 nucleotide long loop between S1 and S2.

E84. The oligonucleotide-ligand conjugate of E83, wherein L is a tetracyclic ring.

E85. The oligonucleotide-ligand conjugate of any one of E83 to E84, wherein L includes the sequence represented by GAAA.

E86. The oligonucleotide-ligand conjugate of any one of E83 to E85, wherein the one or more target ligands are bonded to the stem loop.

E87. The oligonucleotide-ligand conjugate of E86, wherein the one or more target ligands are conjugated to one or more nucleotides of L.

E88. The oligonucleotide-ligand conjugate of E87, wherein L includes 4 nucleotides at positions numbered 1 to 4 from 5' to 3', and one or more target ligands thereof The base is engaged with position 2.

E89. The oligonucleotide-ligand conjugate of any one of E75 to E88, wherein the antisense strand includes a 3' overhang sequence of one or more nucleotides, optionally wherein the 3' overhang The sequence is 2 nucleotides long, optionally where the 3' overhang sequence is GG.

E90. The oligonucleotide-ligand conjugate of E62 to E89, wherein the oligonucleotide comprises at least one modified nucleotide.

E91. The oligonucleotide-ligand conjugate of E90, wherein the modified nucleotide contains a 2'-modification.

E92. The oligonucleotide-ligand conjugate of E90, wherein the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O- Modification of methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinic acid.

E93. The oligonucleotide-ligand conjugate of any one of E90 to E92, wherein about 10 to 15%, 10%, 11%, 12%, 13%, 14% of the nucleotide of the sense strand % or 15% contains 2'-fluoro modifications.

E94. The oligonucleotide-ligand conjugate of any one of E90 to E93, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29% of the nucleotide of the antisense strand %, 30%, 31%, 32%, 33%, 34% or 35% contain a 2'-fluoro modification.

E95. The oligonucleotide-ligand conjugate of any one of E90 to E94, wherein the oligonucleotide is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% contained 2'-fluoro modifications.

E96. The oligonucleotide-ligand conjugate of any one of E90 to E95, wherein the sense strand includes 36 nucleotides from positions 1 to 36 from 5' to 3', wherein positions 8 to 11 Contains 2'-fluoro modification.

E97. The oligonucleotide-ligand conjugate of any one of E90 to E96, wherein the antisense strand comprises 22 nucleotides from 5' to 3' at positions 1 to 22, and wherein position 2 , 3, 4, 5, 7, 10 and 14 contain 2'-fluoro modifications.

E98. The oligonucleotide-ligand conjugate of any one of E90 to E97, wherein the remaining nucleotides comprise a 2'-O-methyl modification.

E99. The oligonucleotide-ligand conjugate of E96, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications.

E100. The oligonucleotide-ligand conjugate of E97 or E99, wherein positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand comprise 2'-O-methyl modifications.

E101. The oligonucleotide-ligand of any one of E90 to E100, wherein all nucleotides of the oligonucleotide are modified.

E102. The oligonucleotide-ligand conjugate of any one of E62 to E101, wherein the oligonucleotide contains at least one modified internucleotide linkage.

E103. The oligonucleotide-ligand conjugate of E102, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

E104. The oligonucleotide-ligand conjugate of E103, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

E105. The oligonucleotide-ligand conjugate of E103 or E104, wherein the antisense strand comprises 22 nucleotides from 3' to 5' at positions 1 to 22, and wherein the antisense strand comprises Phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22.

E106. The oligonucleotide-ligand conjugate of any one of E75 to E105, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analogue.

E107. The oligonucleotide-ligand conjugate of E106, wherein the phosphate analog is methoxyphosphonate, vinylphosphonate or malonylphosphonate.

E108. The oligonucleotide-ligand conjugate of any one of E67 to E107, wherein R ⁵ is

.

E109. The oligonucleotide-ligand conjugate of any one of E67 to E107, wherein R ⁵ is

.

E110. The oligonucleotide-ligand conjugate of any one of E57 to E107, wherein R ⁵ is

.

E111. The oligonucleotide-ligand conjugate of any one of E75 to E110, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively.

E112. The oligonucleotide-ligand conjugate of any one of E75 to E110, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 174, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 188 Nucleotide sequence.

E113. The oligonucleotide-ligand conjugate of any one of E75 to E110, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 175, and the antisense strand comprises the core of SEQ ID NO: 189 nucleotide sequence.

E114. A pharmaceutical composition comprising the oligonucleotide or oligonucleotide-ligand conjugate of any of the aforementioned specific examples and a medically acceptable carrier, delivery agent or excipient.

E115. A kit comprising the oligonucleotide or oligonucleotide-ligand conjugate of any one of E1 to E113, a medically acceptable carrier as needed, and instructions, which instructions include Instructions for administration to subjects with diseases, disorders, or conditions associated with MC2R expression.

E116. A kit of E115 containing instructions for administration to a subject who has received or is receiving a CYP11B1 inhibitor.

E117. A method for treating a subject suffering from a disease, disorder or condition associated with MC2R expression, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or oligonucleotide of any one of E1 to E113 Acid-ligand conjugate or pharmaceutical composition of E114.

E118. The method of E117, wherein the disease, disorder or condition associated with MC2R expression is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, Group consisting of congenital adrenal agenesis and familial Addison's disease.

E119. The method of E117, wherein the disease, disorder or condition associated with MC2R expression is Cushing's syndrome.

E120. The method of any one of E117 to E119, wherein the oligonucleotide or pharmaceutical composition is administered together with a second composition or therapeutic agent.

E121. The method of E120, wherein the second composition or therapeutic agent is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production or glucocorticoid effects on surrounding tissue, surgical procedures , radiation procedures, gene therapy, mifepristone, glucocorticoid receptor antagonists and competitive inhibitors of cortisol at this receptor, osilodrostat, metyrapone, CYP11B1 inhibitors, pa Reitide or combinations thereof.

E122. The method of E120, wherein the second composition or therapeutic agent targets CYP11B1.

E123. The method of any one of E117 to E122, wherein the oligonucleotide or pharmaceutical composition is administered together with at least 2 additional therapeutic agents.

E124. A method of treating Cushing's syndrome in a subject, comprising administering to the subject an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E1 to E113, or a pharmaceutical composition of E114, wherein The cells in the adrenal gland or adrenal cortex express MC2R.

E125. A pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E1 to E113, wherein the oligonucleotide or oligonucleotide-ligand Base conjugates reduce the expression, production or activity of MC2R.

E126. The use of an oligonucleotide or oligonucleotide-ligand conjugate of any one of E1 to E113 or a pharmaceutical composition of E114 in reducing MC2R expression in the adrenal gland.

E127. The oligonucleotide or oligonucleotide-ligand conjugate of any one of E1 to E113, for use in reducing MC2R expression in the adrenal gland of a subject.

E128. An oligonucleotide or oligonucleotide-ligand conjugate of any one of E1 to E113 or a pharmaceutical composition of E114 for reducing MC2R expression in a subject suffering from a disease, disorder or condition associated with MC2R expression purpose.

E129. The use of E128, wherein the disease, disorder or condition associated with MC2R expression is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, Group consisting of congenital adrenal agenesis and familial Addison's disease.

E130. The oligonucleotide or oligonucleotide-ligand conjugate of any one of E1 to E113 for use in reducing MC2R expression in a subject suffering from a disease, disorder or condition associated with MC2R expression.

E131. The oligonucleotide or oligonucleotide-ligand conjugate of E130, wherein the disease, disorder or condition associated with MC2R expression is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoids Deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal agenesis, and familial Addison's disease.

E132. An oligonucleotide for reducing the expression of cytochrome P450 family 11 subfamily B member 1 (CYP11B1), the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and 15 to 40 nuclei A sense strand with a long nucleotide, wherein the sense strand and the antisense strand form a double-stranded helix region, wherein the antisense strand has a complementary region to the target sequence of CYP11B1, wherein the target sequence is SEQ ID NO: 226 15 to 30 contiguous nucleotides, and wherein the complementary region is at least 15 contiguous nucleotides long.

E133. The oligonucleotide of E132, wherein the complementary region is completely complementary to the target sequence of CYP11B1.

E134. The oligonucleotide of any one of E132 or E133, wherein the antisense strand is 19 to 27 nucleotides long.

E135. The oligonucleotide of any one of E132 to E134, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long.

E136. The oligonucleotide of any one of E132 to E135, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long.

E137. The oligonucleotide of any one of E132 to E136, wherein the double helix region is at least 19 nucleotides long.

E138. The oligonucleotide of any one of E132 to E137, wherein the double-stranded helix region is at least 20 nucleotides long, optionally wherein the double-stranded helix region is 21 nucleotides long.

E139. The oligonucleotide of any one of E132 to E138, wherein the complementary region is at least 19 contiguous nucleotides long.

E140. The oligonucleotide of any one of E132 to E139, wherein the complementary region is at least 21 contiguous nucleotides long.

E141. The oligonucleotide of any one of E132 to E140, wherein the sense strand comprises at its 3' end a stem loop as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L is in S1 A 3 to 5 nucleotide long loop is formed between S2 and S2.

E142. An oligonucleotide for reducing the expression of CYP11B1, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and has a target sequence similar to CYP11B1 The complementary region, wherein the target sequence is 15 to 30 contiguous nucleotides of SEQ ID NO: 226, wherein the sense strand includes a stem loop as shown below at its 3' end: S1-L-S2, where S1 is complementary to S2, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a double-stranded helix structure that is at least 19 nucleotides long. .

E143. The oligonucleotide of E142, wherein the complementary region is at least 19 contiguous nucleotides in length and is completely complementary to the target sequence.

E144. The oligonucleotide of any one of E142 to E143, wherein L is a tetracyclic ring.

E145. The oligonucleotide of any one of E142 to E144, wherein L is 4 nucleotides long.

E146. The oligonucleotide of any one of E142 to E145, wherein L includes the sequence represented by GAAA.

E147. The oligonucleotide of any one of E132 to E146, wherein (i) the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long, or (ii) the antisense strand is 22 nucleotides long and the sense strand is 36 nucleotides long.

E148. The oligonucleotide of E147, wherein the antisense strand and the sense strand form a double helix region that is 25 nucleotides long or 20 nucleotides long.

E149. The oligonucleotide of any one of E132 and E148, wherein the antisense strand comprises a 3' overhang sequence of one or more nucleotides in length, optionally wherein the 3' overhang sequence is 2 nucleosides. acid length, optionally wherein the 3' overhang sequence is GG.

E150. The oligonucleotide of any one of E132 to E149, wherein the oligonucleotide comprises at least one modified nucleotide.

E151. The oligonucleotide of E150, wherein the modified nucleotide comprises a 2'-modification.

E152. The oligonucleotide of E151, wherein the 2'-modification is selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and Modification of 2'-deoxy-2'-fluoro-β-d-arabinic acid.

E153. The oligonucleotide of any one of E150 to E152, wherein approximately 10 to 15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotides of the sense strand comprise 2 '-Fluorine modification.

E154. The oligonucleotide of any one of E150 to E153, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31% of the nucleotide of the antisense strand %, 32%, 33%, 34% or 35% contain a 2'-fluoro modification.

E155. The oligonucleotide of any one of E150 to E154, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% contained 2'-fluoro modifications.

E156. The oligonucleotide of any one of E150 to E155, wherein the sense strand comprises 36 nucleotides from 5' to 3' at positions 1 to 36, wherein positions 8 to 11 comprise 2'-fluoro Grooming.

E157. The oligonucleotide of any one of E150 to E156, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, and positions 2, 3, 4, and 5 thereof , 7, 10 and 14 contain 2'-fluoro modifications.

E158. The oligonucleotide of any one of E153 to E157, wherein the remaining nucleotides comprise a 2'-O-methyl modification.

E159. The oligonucleotide of E156, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications.

E160. The oligonucleotide of E157 or E159, wherein positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand comprise a 2'-O-methyl modification.

E161. The oligonucleotide of any one of E150 to E160, wherein all nucleotides of the oligonucleotide are modified.

E162. The oligonucleotide of any one of E132 to E161, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

E163. The oligonucleotide of E162, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

E164. The oligonucleotide of E163, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

E165. The oligonucleotide of E163 or E164, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand includes positions 1 and 2, 2 and phosphorothioate linkages between 3, 20 and 21, and 21 and 22.

E166. The oligonucleotide of any one of E132 to E165, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog.

E167. The oligonucleotide of E166, wherein the phosphate analog is methoxyphosphonate, vinylphosphonate or malonylphosphonate.

E168. The oligonucleotide of E132 to E167, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more target ligands.

E169. The oligonucleotide of E166, wherein the nucleotide is bound to more than one target ligand, wherein the target ligands are the same or different.

E170. The oligonucleotide of E168 or E169, wherein the one or more target ligands are selected from carbohydrates, amino sugars, cholesterol, polypeptides or lipids.

E171. The oligonucleotide of E168 or E169, wherein the one or more target ligands are saturated or unsaturated fatty acid moieties.

E172. The oligonucleotide of E168 or E169, wherein the target ligand is a saturated fatty acid moiety ranging in size from C10 to C24.

E173. The oligonucleotide of E172, wherein the target ligand is a C16 saturated fatty acid moiety.

E174. The oligonucleotide of E172, wherein the target ligand is a C18 saturated fatty acid moiety.

E175. The oligonucleotide of E172, wherein the target ligand is a C22 saturated fatty acid moiety.

E176. The oligonucleotide of E168 or E169, wherein the target ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

E177. The oligonucleotide of E176, wherein the GalNAc portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion or a tetravalent GalNAc portion.

E178. The oligonucleotide of any one of E141 to E177, wherein up to 4 nucleotides of L of the stem loop are each conjugated to a monovalent GalNAc moiety.

E179. An oligonucleotide-ligand conjugate for reducing CYP11B1 mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence that is complementary to a target sequence in CYP11B1 mRNA region and (ii) one or more target ligands,

The oligonucleotide-ligand conjugate is represented by formula Ea :

Ea ;

or a medically acceptable salt thereof, wherein:

B is the nucleobase;

R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R, -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or

R ¹ and R ² on the same carbon together with their intervening atoms form a 3 to 7 membered saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

Each ^RA is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic rings and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur;

Each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from nitrogen, oxygen and 4 to 7 membered saturated or partially unsaturated heterocyclic rings having heteroatoms of sulfur and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; or

Two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaromatic ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur;

Each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has a range of 0 to The 10 methylene units are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, - S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-replacement;

Each -Cy- is independently an optionally substituted divalent ring selected from the group consisting of phenylene, 8 to 10-membered bicyclic aryl, and 4 to 7-membered saturated or partially unsaturated carbocyclic ring. group, a 4 to 11-membered saturated or partially unsaturated spirocyclic carbocyclic group, an 8 to 10-membered bicyclic saturated or partially unsaturated carbocyclic group, having 1 to 3 independently selected from nitrogen, oxygen and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic heterocyclic groups with 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4 to 11 membered saturated or partially unsaturated spirocyclic heterocyclic groups Cyclic group, an 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic cyclic group having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and A 5- to 6-membered heteroaryl group with a heteroatom of sulfur or an 8- to 10-membered bicyclic heteroaryl group with 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur;

n ranges from 1 to 10;

L is a covalent bond or a divalent saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O )OR-, -P(S)OR-, -V ¹ CR ² W ¹ -OR replacement; replacement

m ranges from 1 to 50;

X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-;

Y is hydrogen, suitable hydroxyl protecting group, or ;

R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from nitrogen , 4 to 7 membered saturated or partially unsaturated heterocyclic rings with oxygen and sulfur heteroatoms and 5 to 6 membered heteroaromatic rings with 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur;

X ² is O, S or NR;

X ³ is -O-, -S-, -BH ₂ - or covalent bond;

Y ¹ is a linker attached to the 2' or 3' end of a nucleoside, nucleotide or oligonucleotide;

Y ² is hydrogen, a suitable protecting group, a phosphoramidite analog, an internucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a linker attached to a solid support ;as well as

Z is -O-, -S-, -NR- or -CR ₂ -.

E180. The oligonucleotide-ligand conjugate of E179 is represented by formula IEb or IEc :

EB

IEc

or a medically acceptable salt thereof, wherein:

L ¹ is a covalent bond, monovalent or divalent saturated or unsaturated, straight or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently modified by -Cy-, - O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, - P(O)OR-, -P(S)OR-or replacement; replacement

R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and

R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C(O)NR-, - NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S) OR replacement.

E181. The oligonucleotide-ligand conjugate of E181, wherein:

R ⁵ series selected from

.

E182. The oligonucleotide-ligand conjugate of E181, wherein:

R ⁵ series selected from

E183. Oligonucleotide-ligand conjugate of E181, wherein R ⁵ is

.

E184. An oligonucleotide-ligand conjugate for reducing CYP11B1 mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence that is complementary to a target sequence in CYP11B1 mRNA region and (ii) one or more target ligands, wherein the oligonucleotide-ligand conjugate is represented by formula IEIb or IEIc :

EIib

iEc

or a medically acceptable salt thereof; wherein

B is the nucleobase;

m ranges from 1 to 50;

X ¹ is -O- or -S-;

Y is hydrogen, or ;

R ³ is hydrogen or a suitable protecting group;

X ² is O or S;

X ³ is -O-, -S- or covalent bond;

Y ¹ is a linker attached to the 2' or 3' end of a nucleoside, nucleotide or oligonucleotide;

Y ² is hydrogen, a phosphoramidite analog, an internucleotide linker attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a linker attached to a solid support;

R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C(O)NR-, - NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR- or -P(S) OR substitution; and

R is hydrogen, a suitable protecting group, or an optionally substituted group selected from the group consisting of C _1-6 aliphatic, phenyl, and a heterogenous group having 1 to 2 independently selected from nitrogen, oxygen, and sulfur. 4 to 7 membered saturated or partially unsaturated heterocyclic rings of atoms and 5 to 6 membered heteroaromatic rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

E185. The oligonucleotide-ligand conjugate of E184, wherein:

R ⁵ series selected from

.

E186. Oligonucleotide-ligand conjugate of E185, wherein R ⁵ is

.

E187. Oligonucleotide-ligand conjugate of E185, wherein R ⁵ is

.

E188. The oligonucleotide-ligand conjugate of any one of E179 to E187, wherein the nucleotide sequence contains 1 to 10 target ligands.

E189. The oligonucleotide-ligand conjugate of any one of E179 to E188, wherein the nucleotide sequence contains 1, 2 or 3 target ligands.

E190. The oligonucleotide-ligand conjugate of any one of E179 to E189, wherein the cell associated with the adrenal cortex or adrenal gland is an epithelial cell.

E191. The oligonucleotide-ligand conjugate of any one of E179 to E189, wherein the oligonucleotide is single-stranded, optionally wherein the single-stranded oligonucleotide is 15 to 30 nucleotides long.

E192. The oligonucleotide-ligand conjugate of any one of E179 to E189, wherein the oligonucleotide is double-stranded.

E193. The oligonucleotide-ligand conjugate of E192, wherein the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the The sense strand and the antisense strand form a double helix region, wherein the antisense strand includes a complementary region to the target sequence, and wherein the complementary region is at least 15 contiguous nucleotides long.

E194. The oligonucleotide-ligand conjugate of E193, wherein the antisense strand is 19 to 27 nucleotides long.

E195. The oligonucleotide-ligand conjugate of E193 or E194, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long.

E196. The oligonucleotide-ligand conjugate of any one of E193 to E195, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long. .

E197. The oligonucleotide-ligand conjugate of any one of E193 to E196, wherein the double helix region is at least 19 nucleotides long.

E198. The oligonucleotide-ligand conjugate of any one of E193 to E197, wherein the double helix region is at least 21 nucleotides long, optionally wherein the double helix region is 20 nucleotides long.

E199. The oligonucleotide-ligand conjugate of any one of E193 to E198, the complementary region being at least 19 contiguous nucleotides long.

E200. The oligonucleotide-ligand conjugate of any one of E193 to E199, wherein the complementary region is at least 21 contiguous nucleotides long.

E201. The oligonucleotide-ligand conjugate of any one of E193 to E200, wherein the sense strand includes a stem loop as shown below at its 3' end: S1-L-S2, wherein S1 is complementary to S2 , and where L forms a 3 to 5 nucleotide long loop between S1 and S2.

E202. The oligonucleotide-ligand conjugate of E201, wherein L is a tetracyclic ring.

E203. The oligonucleotide-ligand conjugate of any one of E201 to E202, wherein L includes the sequence represented by GAAA.

E204. The oligonucleotide-ligand conjugate of any one of E201 to E203, wherein the one or more target ligands are bonded to the stem loop.

E205. The oligonucleotide-ligand conjugate of E204, wherein the one or more target ligands are conjugated to one or more nucleotides of L.

E206. The oligonucleotide-ligand conjugate of E205, wherein L includes 4 nucleotides at positions numbered 1 to 4 from 5' to 3', in which one or more target ligands Engage with position 2.

E207. The oligonucleotide-ligand conjugate of any one of E193 to E206, wherein the antisense strand includes a 3' overhang sequence of one or more nucleotides, optionally wherein the 3' overhang The sequence is 2 nucleotides long, optionally where the 3' overhang sequence is GG.

E208. The oligonucleotide-ligand conjugate of E179 to E207, wherein the oligonucleotide comprises at least one modified nucleotide.

E209. The oligonucleotide-ligand conjugate of E208, wherein the modified nucleotide comprises a 2'-modification.

E210 E.209 oligonucleotide-ligand conjugate, wherein the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O -Methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinic acid modifications.

E211. The oligonucleotide-ligand conjugate of any one of E208 to E210, wherein about 10 to 15%, 10%, 11%, 12%, 13%, 14% of the nucleotide of the sense strand % or 15% contains 2'-fluoro modifications.

E212. The oligonucleotide-ligand conjugate of any one of E208 to E211, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29% of the nucleotide of the antisense strand %, 30%, 31%, 32%, 33%, 34% or 35% contain a 2'-fluoro modification.

E213. The oligonucleotide-ligand conjugate of any one of E208 to E212, wherein about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% contained 2'-fluoro modifications.

E214. The oligonucleotide-ligand conjugate of any one of E208 to E213, wherein the sense strand includes 36 nucleotides from 5' to 3' at positions 1 to 36, wherein positions 8 to 11 Contains 2'-fluoro modification.

E215. The oligonucleotide-ligand conjugate of any one of E208 to E214, wherein the antisense strand comprises 22 nucleotides from 5' to 3' at positions 1 to 22, and wherein position 2 , 3, 4, 5, 7, 10 and 14 contain 2'-fluoro modifications.

E216. The oligonucleotide-ligand conjugate of any one of E208 to E215, wherein the remaining nucleotides comprise a 2'-O-methyl modification.

E217. The oligonucleotide-ligand conjugate of E215, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications.

E218. The oligonucleotide-ligand conjugate of E215 or E217, wherein positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand comprise 2'-O-methyl modifications.

E219. The oligonucleotide-ligand of any one of E208 to 218, wherein all nucleotides of the oligonucleotide are modified.

E220. The oligonucleotide-ligand conjugate of any one of E179 to E219, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

E221. The oligonucleotide-ligand conjugate of E220, wherein at least one modified internucleotide linkage is a phosphorothioate linkage.

E222. The oligonucleotide-ligand conjugate of E221, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

E223. The oligonucleotide-ligand conjugate of E221 or E222, wherein the antisense strand comprises 22 nucleotides from 3' to 5' at positions 1 to 22, and wherein the antisense strand comprises Phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22.

E224. The oligonucleotide-ligand conjugate of any one of E193 to E223, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog.

E225. The oligonucleotide-ligand conjugate of E224, wherein the phosphate analog is methoxyphosphonate, vinyl phosphonate or malonyl phosphonate.

E226. The oligonucleotide-ligand conjugate of any one of E185 to E225, wherein R ⁵ is

.

E227. The oligonucleotide-ligand conjugate of any one of E185 to E225, wherein R ⁵ is

.

E228. The oligonucleotide-ligand conjugate of any one of E185 to E225, wherein R ⁵ is

.

E229. A pharmaceutical composition comprising the oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E229, and a medically acceptable carrier, delivery agent or excipient.

E230. A kit comprising the oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228, a medically acceptable carrier if necessary, and instructions, which instructions include Instructions for administration to subjects with diseases, disorders, or conditions associated with expression of CYPB11.

E231. A kit of E230 containing instructions for administration to a subject who has received or is receiving an MC2R inhibitor.

E232. A method for treating a subject suffering from a disease, disorder or condition associated with expression of CYPB11, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or oligonucleotide of any one of E132 to E228 Acid-ligand conjugate or pharmaceutical composition of E229.

E233. The method of E232, wherein the disease, disorder or condition associated with expression of CYPB11 is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, Group consisting of congenital adrenal agenesis and familial Addison's disease.

E234. The method of E233, wherein the disease, disorder or condition associated with expression of CYPB11 is Cushing's syndrome.

E235. The method of any one of E232 to E234, wherein the oligonucleotide or pharmaceutical composition is administered together with a second composition or therapeutic agent.

E236. The method of E235, wherein the second composition or therapeutic agent is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production, or glucocorticoid effects on surrounding tissue, surgical procedures , radiation procedures, gene therapy, mifepristone, glucocorticoid receptor antagonists and competitive inhibitors of cortisol at this receptor, osilodrostat, metyrapone, MC2R inhibitors, pa Reitide or combinations thereof.

E237. The method of any one of E235 to E236, wherein the second composition or therapeutic agent targets MC2R.

E238. The method of any one of E232 to E237, wherein the oligonucleotide or oligonucleotide-ligand conjugate is administered together with at least 2 additional therapeutic agents.

E239. A method of treating Cushing's syndrome in a subject, comprising administering to the subject an oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228 or a pharmaceutical composition of E229, wherein The cells in the adrenal gland or adrenal cortex express CYPB11.

E240. A pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228, wherein the oligonucleotide reduces the expression, production or production of CYPB11. active.

E241. The use of an oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228 or a pharmaceutical composition of E229 in reducing CYPB11 expression in the adrenal gland.

E242. The oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228 for reducing CYPB11 expression in the adrenal gland of a subject.

E243. An oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E228 or a pharmaceutical composition of E229 for reducing CYPB11 expression in a subject suffering from a disease, disorder or condition associated with CYPB11 expression purpose.

E244. The use of E243, wherein the disease, disorder or condition associated with expression of CYPB11 is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, Group consisting of congenital adrenal agenesis and familial Addison's disease.

E245. The oligonucleotide or oligonucleotide-ligand conjugate of any one of E132 to E229, for use in reducing CYPB11 expression in a subject suffering from a disease, disorder or condition associated with CYPB11 expression.

E246. The oligonucleotide or oligonucleotide-ligand conjugate of E245, wherein the disease, disorder or condition associated with CYPB11 expression is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoids Deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal agenesis, and familial Addison's disease.

E247. A pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an MC2R inhibitor, and wherein the second therapeutic agent is an inhibitor of CYPB11 expression.

E248. A pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an oligonucleotide and is an inhibitor of MC2R expression, and wherein the second therapeutic agent is an inhibitor of CYPB11 expression .

E249. A pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an inhibitor of MC2R expression, and wherein the second therapeutic agent is an oligonucleotide and is an inhibitor of CYPB11 expression .

E250. A pharmaceutical composition comprising at least a first and a second therapeutic agent, wherein the first therapeutic agent is an oligonucleotide selected from any one of E1 to E113 and is an inhibitor of MC2R expression, and wherein the first therapeutic agent is an inhibitor of MC2R expression. The two therapeutic agents are oligonucleotides selected from any one of E132 to E228 and are inhibitors of CYPB11 expression.

E251. A combination product containing: (i) An oligonucleotide that inhibits MC2R comprising an antisense strand that is 15 to 30 nucleotides in length and includes a nucleotide sequence as set forth in any one of SEQ ID NO: 85 to 96; and 15 to a sense strand that is 40 nucleotides in length and includes the nucleotide sequence set forth in any of SEQ ID NO: 37 to 48, and (ii) An oligonucleotide that inhibits CYPB11, which contains an antisense strand that is 15 to 30 nucleotides long and includes a complementary region to a target sequence of CYP11B1, wherein the target sequence is 15 to 30 of SEQ ID NO: 226 contiguous nucleotides; and a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded helix region.

E252. Combination products of E251, wherein (i) and (ii) are formulated as an injectable suspension, gel, oil, pill, tablet, suppository, powder, capsule, mist, ointment, cream, patch, or Galenic form of means for prolonged and/or slow release.

E253. A combination product of any one of E251 to 252, which is used to treat diseases related to expression of MC2R or CYP11B1.

E254. A method of treating a disease associated with MC2R or CYP11B1 expression, comprising administering an oligonucleotide for reducing CYP11B1 expression to a patient who has received or is receiving an oligonucleotide for reducing MC2R expression.

E255. A method of treating a disease associated with MC2R or CYP11B1 expression, comprising administering an oligonucleotide for reducing CYP11B1 expression to a patient who has received or is receiving an oligonucleotide for reducing MC2R expression.

E256. A method of treating a disease associated with expression of MC2R or CYP11B1, comprising administering an oligonucleotide for reducing MC2R of any one of E1 to E113 to a patient who has received or is receiving an oligonucleotide for reducing the expression of CYP11B1. Nucleotide or oligonucleotide - ligand.

E257. The method of E256, wherein the oligonucleotide used to reduce the expression of CYP11B1 is an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E132 to E228.

E258. A method of treating a disease associated with MC2R or CYP11B1 expression, comprising administering any one of E132 to 228 for reducing CYP11B1 expression to a patient who has received or is receiving an oligonucleotide for reducing MC2R expression. oligonucleotide or oligonucleotide-ligand.

E259. The method of E258, wherein the oligonucleotide used to reduce MC2R expression is an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E1 to E113.

E260. The method of any one of E254 to E258, wherein the disease associated with expression of MC2R or CYP11B1 is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, and hereditary adrenocortical inertia to ACTH. Reactive, congenital adrenal hypoplasia, and familial Addison's disease.

E261. A method of treating a disease or disorder associated with cortisol synthesis and/or signaling pathways, comprising administering to a subject in need thereof an oligonucleotide for reducing MC2R expression, wherein the subject has received or is undergoing Receive oligonucleotides used to reduce the expression of CYP11B1.

E262. A method of treating a disease or disorder associated with cortisol synthesis and/or signaling pathways, comprising administering to a subject in need thereof an oligonucleotide for reducing CYP11B1 expression, wherein the subject has received or is undergoing Receive oligonucleotides for reducing MC2R expression.

E263. A method of treating a disease or condition related to cortisol synthesis and/or signaling pathways, comprising administering any one of E1 to E113 to a subject who has received or is receiving an oligonucleotide for reducing MC2R expression. Oligonucleotides or oligonucleotide-ligand conjugates used to reduce CYP11B1 expression.

E264. The method of E263, wherein the oligonucleotide for reducing CYP11B1 expression is an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E132 to E228.

E265. A method of treating a disease or disorder associated with cortisol synthesis and/or signaling pathways, comprising administering any one of E132 to E228 to a subject who has received or is receiving an oligonucleotide for reducing CYP11B1 expression. Oligonucleotides used to reduce MC2R expression.

E266. The method of E265, wherein the oligonucleotide used to reduce MC2R expression is an oligonucleotide or an oligonucleotide-ligand conjugate of any one of E1 to E113.

E267. The method of any one of E261 to E266, wherein the disease or disorder related to cortisol synthesis and/or signaling pathways is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, genetic Adrenocortical unresponsiveness to ACTH, congenital adrenal hypoplasia, and familial Addison's disease. Example

Although the present disclosure has been described with reference to specific examples illustrated in the following examples, it will be understood by those skilled in the art that various changes may be made and may be made without departing from the true spirit and scope of the disclosure. Substituted by equivalents. Furthermore, the following examples are provided by way of illustration and are not intended to limit the scope of the present disclosure in any way. In addition, modifications may be made to suit the circumstances, materials, composition of matter, process, one or more process steps, purpose, spirit and scope of the disclosure. All such modifications are intended to be included within the scope of this disclosure. Standard techniques well known in the technical field to which the invention pertains or techniques specifically described below are used. Example 1 : General synthesis method for preparation of double-stranded RNAi oligonucleotides

The following examples are intended to illustrate the present disclosure and should not be construed as limiting it. Temperature is expressed in degrees Celsius (C). If not mentioned otherwise, all evaporations are performed under reduced pressure, preferably between about 15 mm Hg and 100 mm Hg (=20 to 133 mbar). Confirm the structure of final products, intermediates, and starting materials by standard analytical methods (e.g., microanalysis) and spectroscopic characteristics (e.g., MS, IR, NMR). The abbreviations used are those common in the technical fields to which their inventions belong.

All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents and catalysts used to synthesize the nucleic acids or analogs thereof of the present disclosure are commercially available or can be obtained by ordinary knowledge in the technical field to which the invention belongs. Produced by known organic synthesis methods ( Methods of Organic Synthesis, Thieme, Volume 21 (Houben-Weyl 4th Ed. 1952)). In addition, as shown in the following examples, the nucleic acids of the present disclosure or analogs thereof can be produced by organic synthesis methods known to those of ordinary skill in the art to which the invention belongs.

All reactions were performed under nitrogen or argon unless otherwise stated.

Proton NMR ( ¹ H NMR) was performed in deuterated solvents. In certain nucleic acids or analogs thereof disclosed herein, one or more ¹ H shifts overlap with residual protein solvent signals; these signals were not reported in the experiments provided below.

As described in the following examples , in certain illustrative embodiments, nucleic acids or analogs thereof are prepared according to the following general procedures. It will be understood that although general methods describe the synthesis of certain nucleic acids or analogs thereof of the present disclosure, the following general methods and other methods known to those of ordinary skill in the art to which the invention pertains are applicable to all nucleic acids as described herein. or analogs thereof, as well as subclasses and types of each of such nucleic acids or analogs thereof. Formic acid 2-(2-(((6aR,8R,9R,9aR)-8-(6- benzamide -9H- purin -9- yl )-2,2,4,4 -tetraisopropyl Tetrahydro -6H- furo [3,2-f][1,3,5,2,4] trioxosiloxane- 9- yl)oxy ) methoxy ) ethoxy ) ethoxy ) eth - 1 -Synthesis of ammonium ( 1-6)

A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was added with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (22.63 mL, 70.75 mmol) treatment. The resulting mixture was stirred at 25 °C for 3 h and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL) and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n-heptane (1:15, 320 mL) to give compound 1-2 as a white oily solid (37.20 g, 90%).

A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and _Ac2O (15 mL, 156.68 mmol). The mixture was stirred at 25 °C for 15 h. The reaction was diluted with EtOAc (100 mL) and quenched with _{saturated K2CO3} (50 mL). The aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were concentrated and recrystallized from ACN (30 mL) to provide compound 1-3 as a white solid (15.65 g, 38.4%).

A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25°C. The mixture was stirred to provide a clear solution, followed by treatment with 4Å molecular sieves (20.0 g), N -iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30°C until HPLC analysis showed >95% consumption of compound 1-3 . The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with saturated _NaHCO3 (2X100 mL), saturated _Na2SO3 (2X100 mL), and water (2X100 mL), and concentrated in vacuo to afford crude compound 1-4 as a yellow solid ( _26.34 g , 93.9%), which was used directly in the next step without further purification.

A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM/water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5°C. The mixture was stirred at 5 to 25 °C for 1 h. The organic layer was then separated, washed with water (100 mL) and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4Å molecular sieves (26.34 g) in four portions. The mixture was stirred for 1 h, concentrated, and recrystallized from a mixture of MTBE and DCM (5:1) to provide compound 1-6 (14.74 g, 62.9%) as a white solid: ¹ H NMR (400 MHz, d ₆ -DMSO) 8.73 (s, 1H), 8.58 (s, 1H), 8.15-8.02 (m, 2H), 7.65-7.60 (m, 1H), 7.59-7.51 (m, 2H), 6.52 (s, 2H) , 6.15(s, 1H), 5.08-4.90 (m, 3H), 4.83-4.78 (m, 1H), 4.15-3.90 (m, 3H), 3.79-3.65 (m, 2H), 2.98-2.85 (m, 6H), 1.20-0.95 (m, 28H). (2R,3R,4R,5R)-5-(6- benzamide- 9H- purin -9- yl )-2-(( bis (4- methyl Oxyphenyl )( phenyl ) methoxy ) methyl )-4-((2-(2-[ lipid ] -acylaminoethoxy ) ethoxy ) methoxy ) tetrahydrofuran - 3- yl Synthesis of (2- cyanoethyl ) diisopropylphosphoramidite (2-4a to 2-4e)

A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2-methyltetrahydrofuran was washed with ice _- cold aqueous _K2HPO4 (6%, 100 mL) and brine (20%, 2×100 mL). The organic layer was separated and treated with caproic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol) and DMAP (10.81 g, 147.52 mmol) at 0°C. The resulting mixture was warmed to 25 °C and stirred for 1 h. The solution was washed with water (2×100 mL), brine (100 mL), and concentrated in vacuo to provide a crude residue. Flash chromatography on silica gel (1:1 hexane/acetone) gave compound 2-1a as a white solid (34.95 g, 71.5%).

A mixture of compound 2-1a (34.95 g, 42.19 mmol) and TEA (9.28 mL, 126.58 mmol) in 80 mL THF was treated dropwise with triethylamine trihydrofluoride (20.61 mL, 126.58 mmol) at 10 °C. . The mixture was warmed to 25 °C and stirred for 2 h. The reaction was concentrated, dissolved in DCM (100 mL), and washed with saturated _NaHCO3 (5X20 mL) and brine (50 mL). The organic layer was concentrated in vacuo to provide crude compound 2-2a (24.72 g, 99%), which was used directly in the next step without further purification.

A solution of compound 2-2a (24.72 g, 42.18 mmol) in 50 mL DCM was treated with N -methylmorpholine (18.54 mL, 168.67 mmol) and DMTr-Cl (15.69 g, 46.38 mmol). The mixture was stirred at 25 °C for 2 h and quenched with saturated NaHCO ₃ (50 mL). The organic layer was separated, washed with water, and concentrated to provide a crude syrup. Flash chromatography on silica gel (1:1 hexane/acetone) gave compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid.

A solution of compound 2-3a (25.00 g, 28.17 mmol) in 50 mL of DCM was treated with N -methylmorpholine (3.10 mL, 28.17 mmol) and tetrazole (0.67 mL, 14.09 mmol) under nitrogen atmosphere. . Bis(diisopropylamino)chlorophosphine (9.02 g, 33.80 mmol) was added dropwise to the solution, and the resulting mixture was stirred at 25 °C for 4 h. The reaction was quenched with water (15 mL), and the aqueous layer was extracted with DCM (3×50 mL). The combined organic layers were washed with saturated NaHCO ₃ (50 mL) and concentrated to provide a crude solid which was recrystallized from a mixture of DCM/MTBE/n-hexane (1:4:40) to provide compound 2 as a white solid. -4a (25.52 g, 83.4%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.25 (s, 1H), 8.65-8.60 (m, 2 H), 8.09-8.02 (m, 2H), 7.71 ( s, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.85-6.79 (m, 4H), 6.23-6.20 (m, 1H), 5.23-5.14 (m, 1H), 4. 80-4.69 (m, 3H), 4.33-4.23 (m, 2H), 3.90-3.78 (m, 1H), 3.75 (s , 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.82-2.80 (m, 1H), 2.65-2.60 (m, 1H), 2.05-1.96 (m, 2H), 1.50-1.39 (m, 2H), 1.31-1.10 (m, 14H), 1.08-1.05 (m, 2 H), 0.85-0.79 (m, 3H ); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.43, 149.18.

Compounds 2-4b , 2-4c , 2-4d, and 2-4e were prepared using similar procedures to compound 2-4a described above. Compound 2-4b was obtained as a white solid (25.50 g, 85.4%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.23 (s, 1H), 8.65-8.60 (m, 2 H), 8.05-8.02 ( m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m , 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m , 1H), 2.68-2.62 (m, 1H), 2.05-1.97 (m, 2H), 1.50-1.38 (m, 2H), 1.31-1.10 (m, 18H), 1.08-1.05 (m, 2H), 0.85 -0.78 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.43, 149.19.

Compound 2-4c was obtained as an off-white solid (36.60 g, 66.3%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 ( m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.25-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m , 1H), 3.74 (s, 6H), 3.74-3.50 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m , 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.33-1.12 (m, 38H), 1.08-1.05 (m, 2 H), 0.86-0.80 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.42, 149.17.

Compound 2-4d was obtained as an off-white solid (26.60 g, 72.9%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 ( m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.33 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.22-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m , 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m , 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.08 (m, 38H), 1.08-1.05 (m, 2 H), 0.85-0.79 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.47, 149.22.

Compound 2-4e was obtained as a white solid (38.10 g, 54.0%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.21 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m , 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89 -6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.73 (s, 6H), 3.74-3.52 (m, 3H), 3.47-3.22 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.06 (m, 46H), 1.08-1.06 (m, 2 H), 0.85 -0.77 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.41, 149.15. Example 2. Synthesis reaction of GalXC RNAi oligonucleotide - lipid conjugate Figure 1. Single lipid (linear Synthesis of GalXC RNAi oligonucleotide-lipid conjugates conjugated to tetracyclic rings. Post-synthesis conjugation is achieved via amide coupling reactions. R ₁ COOH group represents fatty acids C8:0, C10:0, C11:0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:1, diyl C16:0 or diyl C18:1

Synthetic sense 1 and antisense 1 were prepared by solid phase synthesis. The meaning of joining is the synthesis of 1a to 1i .

The significance of synthetic joining by post-synthetic joining methods 1a . A solution of caprylic acid (0.58 mg, 4 umol) in DMA (0.75 mL) was treated with HATU (1.52 mg, 4 umol) in Eppendorf tube 1 at room temperature. In Ebende tube 2, a solution of oligosense 1 (10.00 mg, 0.8 umol) in H ₂ O (0.25 mL) was treated with DIPEA (1.39 uL, 8 umol). Add the solution in Ebende Tube 1 to Ebende Tube 2 and mix using a ThermoMixer at rt. After LC-MS analysis showed completion of the reaction, the reaction mixture was diluted with 5 mL of water and purified on a reverse phase XBridge C18 column using a 5 to 95% gradient of 100 mM TEAA in ACN and _H2O . Genevac was used to concentrate the product fraction under reduced pressure. Amicon® Ultra-15 Centrifugal (3K) was used to dialyze the combined residual solvents against water (1X), brine (1X), and water (3X). The Amicon membrane was washed with water (3 × 2 mL) and the combined solvents were lyophilized to provide conjugated sense 1a as an amorphous white solid (6.43 mg, 64% yield).

Joined senses 1b to 1i were prepared using similar procedures described for the synthesis of joined sense 1 a and were obtained in yields of 42% to 69%. Bonding of double helices 1a to 1j .

Dissolve conjugate 1a (10 mg, measured by weight) in 0.5 mL of deionized water to prepare a 20 mg/mL solution. Antisense 1 ( 10 mg, measured by OD) was dissolved in 0.5 mL of deionized water to prepare a 20 mg/mL solution, which was used to titrate the sense and quantitative duplex amounts of junction. Based on the calculation of molar amounts of both the sense and antisense of the conjugate, a portion of the desired antisense 1 was added to the conjugated sense 1a solution. The resulting mixture was stirred at 95 °C for 5 min and allowed to cool to rt. The adhesion progress was monitored by ion exchange HPLC. Based on the adhesion progress, antisense 1 was further added in certain proportions to complete adhesion with >95% purity. The solution was lyophilized to provide double helix 1a(C8) and the amount was calculated based on the molar amount of antisense molecule consumed in the binding.

Double helices 1b to 1i were prepared using the same procedure as described for the bonding of double helix 1a (C8) .

The following reaction schemes 1-2 depict the synthesis of nicked tetracyclic GalXC conjugates with a single lipid on the ring. Post-synthetic conjugation was achieved via a Cu-catalyzed alkyne-azide cycloaddition reaction.

Sense 1B and antisense 1B were prepared by solid phase synthesis. The meaning of joining 1j The synthesis.

A solution of oligomer (10.00 mg, 0.8 umol) in a 3:1 mixture of DMA/H ₂ O (0.5 mL) was treated with lipid linker azide (11.26 mg, 4 umol) in Ebende tube 1. . In Ebende tube 2, dissolve CuBr dimethyl sulfide (1.64 mg, 8 umol) in ACN (0.5 mL). Both solutions were degassed by bubbling _N2 through them for 10 min. A solution of CuBrSMe ₂ in ACN was then added to tube 1 and the resulting mixture was stirred at 40°C. After LC-MS analysis showed the reaction was complete, the reaction mixture was diluted with 0.5 M EDTA (2 mL) and an Amicon® Ultra-15 Centrifugal (3K) was used for dialysis against water (2X). Purification was performed on a reverse phase XBridge C18 column using a 5 to 95% gradient of 100 mM TEAA in ACN spiked in 30% IPA and _H2O . Genevac was used to concentrate the product fraction under reduced pressure. An Amicon® Ultra-15 Centrifugal (3K) was used to dialyze the combined residual solvents against water (1X), brine (1X), and water (3X). The Amicon membrane was washed with water (3 × 2 mL), and the combined solvents were lyophilized to provide the conjugated sense 1j as an amorphous white solid (6.90 mg, 57% yield).

Double helix 1j ( PEG2K- dicyl C18) was prepared using the same procedure described for the bonding of double helix 1a ( C8) .

The following Reaction Figures 1-3 depict the synthesis of a nicked tetracyclic GalXC conjugate with a dilipid on the ring using a post-synthetic conjugation approach. Sense 2 and antisense 2 were prepared by solid phase synthesis.

Conjugated senses 2a and 2b were prepared using a similar procedure as described for the synthesis of conjugated sense 1a , but using 10 eq of lipid, 10 eq of HATU, and 20 eq of DIPEA.

Double helices 2a (2XC11) and 2b (2XC22) were prepared using the same procedure described for the bonding of double helix 1a (C8) .

The following Reaction Figures 1-4 depict the synthesis of GalXC with a fully phosphorothioated stem-loop conjugated to a single lipid using a post-synthetic conjugation approach. Sense 3 and antisense 3 were prepared by solid phase synthesis.

The conjugated sense 3a was prepared using a similar procedure as described for the synthesis of conjugated sense 1a and was obtained in 65% yield.

Double helix 3a (PS-C22) was prepared using the same procedure described for the bonding of double helix 1a (C8) .

The following Reaction Figures 1-5 depict the synthesis of short sense GalXC conjugated to a single lipid using a post-synthetic conjugation approach. Sense 4 and antisense 4 were prepared by solid phase synthesis .

The conjugated sense 4a was prepared using a similar procedure as described for the synthesis of conjugated sense 1a and was obtained in 74% yield.

Double helix 4a (SS-C22) was prepared using the same procedure described for the bonding of double helix 1a (C8) .

Reaction Figures 1-6 below depict the synthesis of nicked tetracyclic GalXC partially conjugated to ternadamantane using a post-synthetic conjugation approach. Sense 5 and antisense 5 were prepared by solid phase synthesis.

Joined senses 5a and 5b were prepared using similar procedures as described for the synthesis of joined sense 1a and were obtained in 42% to 73% yields.

Double helix 5a (3X adamantane ) and double helix 5b (3X acetyl adamantane ) were prepared using the same procedure described for the bonding of double helix 1a (C8) .

The following Reaction Figures 1-7 depict examples of solid phase synthesis of nicked tetracyclic GalXC conjugated to lipid(s) on the ring. The meaning of joining is the synthesis of 6 .

Conjugated sense 6 was prepared by solid-phase synthesis using a commercial oligonucleotide synthesizer. Oligonuclear synthesis using 2'-modified nucleoside phosphoramidites (such as 2'-F or 2'-OMe) and fatty acid amide nucleoside phosphoramidites linked to 2'-diethoxymethanol glycosides. Oligonucleotide synthesis was performed on the solid support in the 3' to 5' direction using standard oligonucleotide synthesis protocols. In these efforts, 5-ethylthio-1H-tetrazole (ETT) was used as the activator of the coupling reaction. Use iodine solution for phosphite triester oxidation. 3-(Dimethylaminomethylene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) was used to form phosphorothioate linkages. The synthesized oligonucleotide was treated with concentrated ammonium aqueous solution for 10 h. Ammonia was removed from the suspension and solid support residue was removed by filtration. Crude oligonucleotides were treated with TEAA, analyzed, and purified by strong anion exchange high performance liquid chromatography (SAX-HPLC). Fractions were combined and dialyzed against water (3X), saline (1X), and water (3X) using an Amicon® Ultra-15 centrifuge (3K). Next, the remaining solvent is lyophilized to provide the desired conjugation significance6 .

Double helix 6 was prepared using the same procedure as described for the bonding of double helix 1a (C8) . Reaction Figure 8. Synthesis of a nicked tetracyclic GalXC bonded to an adamantane unit on the ring via a post-synthetic conjugation method. The Meaning of Joining The Synthesis of 7a and 7b

The ligated 7a and 7b are obtained using the same method or substantially similar method as the synthesis of the ligated 5 . Synthesis example of double helix 7a and 7b

Double helix 7a and double helix 7b are obtained using the same method or substantially similar method as the synthesis of double helix 5 .

Reaction Figure 9. Synthesis of a nicked tetracyclic GalXC bonded to an adamantane unit on the ring via a post-synthetic conjugation method. The Meaning of Joining The Synthesis of 8a and 8b

The ligation 8a and the ligation 8b are obtained using the same method or a substantially similar method for the synthesis of the ligation 5 . Synthesis example of double helices 8a and 8b

Double helices 8a and 8b were obtained using the same method or substantially similar method as the synthesis of double helix 5 .

The following Reaction Figures 1-10 depict the synthesis of short sense and short stem-loop GalXC conjugated to a single lipid using a post-synthetic conjugation approach. Synthesis of Yiyi 9a

Joined Sense 9a is obtained using the same method or substantially similar method as the synthesis of Joined Sense 5 . Synthesis example of double helix 9a

Double helix 9a is obtained using the same method or substantially similar method as that for the synthesis of double helix 5 .

Reactions 1-11 below depict the synthesis of GalXC conjugated to a single lipid at the 5' end using a post-synthetic conjugation approach. The Meaning of Joining 10a Synthesis

Joined meaning 10a is obtained using the same method or substantially similar method as the synthesis of joined meaning 5 . Synthesis example of double helix 10a

Double helix 10a is obtained using the same method or substantially similar method as the synthesis of double helix 5 .

The following reactions, Figures 1-12a and 1-12b , depict the synthesis of GalXC with a blunt end joined to a single lipid at the 3' or 5' end using a post-synthetic ligation approach. The synthesis of joint meaning 11a and 12a

Conjugated senses 11a and 12b are obtained using the same method or substantially similar method as the synthesis of conjugated sense 5 . Synthesis example of double helices 11a and 12a

Double helices 11a and 12a are obtained using the same method or substantially similar method as the synthesis of double helix 5 .

The conjugates double helix 8D and double helix 9D were obtained using the same or substantially similar methods for the synthesis of double helix 5 .

Later, the acyl chain is conjugated to a nucleic acid inhibitor molecule that targets the STAT3 gene, a gene expressed in the tissue of interest. A follower strand with a 2'-amine linker [ademA] was used for solid phase post-ligation. Different types of lipids were conjugated using the same chemistry to generate a series of conjugates ( Figures 1A to 1B ). SAR studies were performed to identify lipid conjugates that can be used to deliver payloads to tissues of interest to mediate target attenuation. Example 3 : Inhibition of MC2R expression in vivo by RNAi oligonucleotides

Melanocortin 2 receptor (MC2R) is a melanocortin receptor activated by adrenocorticotropic hormone (ACTH). Altered MC2R manifestations and diseases (such as Cushing's disease, Cushing's syndrome, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal hypoplasia, and familial Addison's disease) Related.

In vivo sequence screening in mice was performed to identify tool compounds active on MC2R. To identify potent sequences, computer-based algorithms were used to in silico generate MC2R target sequences suitable for determining inhibition of overall MC2R expression by the RNAi pathway. The algorithm provides RNAi oligonucleotide guide sequences that are complementary to mouse or three species (mouse, macaque, human). To characterize the ability of RNAi oligonucleotides to attenuate MC2R in vivo, a hydrodynamic injection (HDI) mouse model was used. The nucleotide sequences of the 17 selected dsRNAs were used to generate corresponding double-stranded RNAi oligonucleotides containing structures conjugated to a nicked tetracyclic GalNAc (referred to herein as "GalNAc-conjugated MC2R oligonucleotides"). acid"), this structure has a 36-nucleotide follower strand and a 22-nucleotide leader strand. Furthermore, the nucleotide sequences comprising the follower and leader strands of MC2R oligonucleotides conjugated to GalNAc have different patterns of modified nucleotides and phosphorothioate linkages (see, e.g., Figure 1A for this Schematic representation of the general structure and chemical modification pattern of the screened MC2R oligonucleotides conjugated to GalNAc). Each of the three adenosine nucleotides containing the four rings is joined to the GalNAc moiety (CAS#: 14131-60-3).

The pattern of modified nucleotide and phosphorothioate linkages is as follows: Crossed with: (Description of modifiers: Table 1 ). Table 1. Description of modifiers symbol Modification/keying mX Nucleotides modified with 2'- O -methyl fx 2'-fluoro modified nucleotides -S- Phosphorothioate linkage - Phosphodiester linkage [MePhosphonate-4O-mX] Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl ademA-GalNAc GalNAc-modified adenine nucleotides

The oligonucleotides in Table 2 were evaluated in mice engineered to transiently express mouse MC2R mRNA in hepatocytes of mouse liver (HDI mode). Briefly, 6- to 8-week-old female CD-1 mice (n = 4 to 5) were administered subcutaneously with a dose of 3 mg/kg of GalNAc-conjugated MC2R oligonucleotide formulated in PBS. Only control mice (n = 5) were administered PBS. Three days later (72 hours), mice were given hydrodynamic injection (HDI) (25 μg) encoding the complete mouse MC2R gene (SEQ ID NO: 227) under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. The DNA plasmid. One day after the introduction of DNA plasmids, liver samples from HDI mice were collected. Total RNA from these HDI mice was analyzed by qRT-PCR to determine MC2R mRNA levels. Measuring mRNA levels of mouse mRNA. A Taqman probe against Mc2R (Mm00434865_s1) was used to measure transfection efficiency, and values were normalized to Ppib (Taqman probe: Mm00478295_m1).

This screen provided 14 active hits against mouse MC2R mRNA ( Figure 2 ). The three most robust sequences were selected for further characterization in a dose-response study ( Figures 3A to 3B ) using the same HDI in vivo as described above at doses of 0.3, 1, and 3 mg/kg. Filter.

These three sequences, GalXC-MC2R-791, GalXC-MC2R-1185, and GalXC-MC2R-1287, were used to generate molecules conjugated to C22 lipids (see Reaction Figures 1-5). The modification pattern of the oligonucleotide conjugated to the C22 lipid is as follows: Crossed with: (Description of modifiers: Table 3 ) Table 3. Description of modifiers symbol Modification/keying mX Nucleotides modified with 2'- O -methyl fx 2'-fluoro modified nucleotides -S- Phosphorothioate linkage - Phosphodiester linkage [MePhosphonate-4O-mX] Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl ademA-C22 Adenine modified with a 22-carbon chain

MC2R oligonucleotides conjugated to C22 lipids were used in dose-response studies to evaluate their ability to attenuate endogenous MC2R target mRNA in mouse adrenal glands ( Table 4 ).

Briefly, 6- to 8-week-old female CD-1 mice (n = 5) were administered subcutaneously with C22-conjugated MC2R oligonucleotides in PBS at doses of 3.75, 7.5, 15, and 30 mg/kg. glycosides. Only control mice (n = 5) were administered PBS. Fourteen days after dosing, adrenal tissue samples from the mice were collected. Total RNA from these adrenal samples was analyzed by qRT-PCR to determine MC2R mRNA levels. Measuring mRNA levels of mouse mRNA. A Taqman probe against Mc2R (Mm00434865_s1) was used to measure transfection efficiency, and values were normalized to Ppib (Taqman probe: Mm00478295_m1). Dose-dependent target attenuation was observed in all lipid conjugates tested ( Figure 4A ). This finding was confirmed by RNAscope performed on adrenal tissue samples collected from mice treated with 30 mg/kg of MC2R oligonucleotide conjugated to C22 lipids ( Fig. 4B ). Specifically, RNAscope® 2.5 HD Reagent Kit-RED (ACD Bio, Newark, CA, Cat. No.: 322350) and RNAscope® LS 2.5 Probe-Mm-Mc2r (Cat. No.: 318898) were used to prepare RNAscope samples from formalin-fixed , Slides of paraffin-embedded adrenal tissue. Slides were pretreated using standard pretreatment conditions (15 min target extraction, 30 min protease boost), and the assay was performed according to the manufacturer's protocol. RNAscope-stained slides were viewed using the HALO® Image Analysis Platform (Indica Labs, Albuquerque, NM) for histological quantification. Various ROIs (regions of interest) were placed in the globular zone (ZG) and fascicular zone (ZF) of each tissue sample, and software was used to detect the total number of spots, which is a visual signal representing a single mRNA transcript. Quantification using RNAscope and HALO showed a 10% reduction in Mc2r in ZG and a 35 to 40% reduction in Mc2r in ZF. A decrease in Mc2r was observed in ZG, more so in ZF. Example 4 : RNAi oligonucleotide inhibition of CYP11B1 expression in vivo

Cytochrome P450 family 11 subfamily B member 1 (CYP11B1) is a gene encoding steroid 11β-hydroxylase. Similar to MC2R, alterations in CYP11B1 are associated with disease (eg, Cushing's syndrome, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal hypoplasia, familial Addison's disease) related.

In vivo sequence screening in mice was performed to identify tool compounds active on CYP11B1. To identify potent sequences, computer-based algorithms were used to in silico generate CYP11B1 target sequences suitable for measuring inhibition of total CYP11B1 expression by the RNAi pathway. The algorithm provides RNAi oligonucleotide guide sequences that are complementary to mouse or all three species (mouse, macaque, human). To characterize the ability of RNAi oligonucleotides to attenuate MC2R in vivo, the HDI mouse model was used. The nucleotide sequences of the 17 selected dsRNAs were used to generate corresponding double-stranded RNAi oligonucleotides containing structures conjugated to a nicked tetracyclic GalNAc (referred to herein as "GalNAc-conjugated CYP11B1 oligonucleotides"). acid"), this structure has a 36-nucleotide follower strand and a 22-nucleotide leader strand. Furthermore, the nucleotide sequences comprising the follower and leader strands of the CYP11B1 oligonucleotide conjugated to GalNAc have different patterns of modified nucleotides and phosphorothioate linkages (see, e.g., Figure 1A for this Schematic representation of the general structure and chemical modification pattern of the screened CYP11B1 oligonucleotides that conjugate GalNAc). Each of the three adenosine nucleotides containing the four rings is conjugated to a GalNAc moiety (CAS#: 14131-60-3) (as shown in Example 3 ).

The oligonucleotides in Table 5 were evaluated in mice engineered to transiently express mouse CYP11B1 mRNA in hepatocytes of mouse liver (HDI mode).

Briefly, 6- to 8-week-old female CD-1 mice (n = 4 to 5) were administered subcutaneously with a dose of 3 mg/kg of GalNac-conjugated CYPB11B1 oligonucleotide formulated in PBS. Only control mice (n = 5) were administered PBS. Three days later (72 hours), mice were given hydrodynamic injection (HDI) of the complete mouse CYPB11B1 gene (SEQ ID NO: 225) (25 μg) under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. of DNA plasmids. One day after the introduction of DNA plasmids, liver samples from HDI mice were collected. Total RNA from these HDI mice was analyzed by qRT-PCR to determine CYPB11B1 mRNA levels. Measuring mRNA levels of mouse mRNA. Transfection efficiency was measured using a Taqman probe for CYP11B1 (Taqman Probe: Mn01204952_m1), and values were normalized to Ppib (Taqman Probe: Mm00478295_m1) using the NeoR gene included on the DNA plasmid.

This screen provided 12 actives against CYP11B1 mRNA ( Figure 5 ). The five most robust sequences, GalXC-CYP11B1-0633, GalXC-CYP11B1-0843, GalXC-CYP11B1-1188, GalXC-CYP11B1-1224, and GalXC-CYP11B1-0854, were selected for use in the dose-response study ( Figure 6 ). To further characterize, this dose-response study used the same HDI in vivo screen as described above at doses of 0.3, 1, and 3 mg/kg.

Next, two of these sequences, GalXC-CYP11B1-0633 and GalXC-CYP11B1-0843, were used to generate molecules conjugated to C22 lipids using the same modification pattern as described in Example 3 . These C22 lipid-conjugated CYP11B1 oligonucleotides were used in a dose-response study to evaluate their ability to attenuate endogenous CYP11B1 target mRNA in mouse adrenal glands ( Table 6 ).

Briefly, 6- to 8-week-old female CD-1 mice (n = 5) were administered subcutaneously with C22-conjugated CYP11B1 oligonucleotides in PBS at doses of 3.75, 7.5, 15, and 30 mg/kg. glycosides. Only control mice (n = 5) were administered PBS. Fourteen days after dosing, adrenal tissue samples from the mice were collected. Total RNA from these adrenal samples was analyzed by qRT-PCR to determine CYP11B1 mRNA levels. To measure attenuation of CYP11B1 mRNA in the adrenal gland, total RNA isolated from one adrenal gland of each mouse was used. Multiplex analysis was performed using the ThermoFisher Scientific TaqMan™ Gene Expression Assay (Mm01204952_m1; Cat. No.: 4331182) targeting the Cyp11b1 gene and PPIB (Mm00478295_m1; Cat. No.: 4331182) as a housekeeping gene. Dose-dependent target attenuation was observed in all lipid conjugates tested ( Figure 7 ).

To measure the duration of target attenuation, 6- to 8-week-old female CD-1 mice (n = 5) were again dosed subcutaneously with a dose of 15 mg/kg of C22-conjugated CYP11B1 oligonucleotide in PBS. (GalXC-CYP11B1-0633-C22 or GalXC-CYP11B1-0843-C22) or MC2R oligonucleotide conjugated to C22 (GalXC-MCR2-1185-C22). Only control mice (n = 5) were administered PBS. Fourteen (14) and 28 days after dosing, adrenal tissue samples from mice were collected. Total RNA from these adrenal samples was analyzed by qRT-PCR to determine mouse CYP11B1 or MC2R mRNA levels. Figure 8A shows that GalXC-CYP11B1-0633-C22 and GalXC-CYP11B1-0843-C22 caused attenuation of CYP11B1 mRNA on days 14 and 28, and the mRNA expression began to rebound on day 28. A similar effect was noted for GalXC-MC2R-1185 ( Fig. 8B ). Example 5 : Optimization of a mouse model of Cushing's disease and inhibition of MC2R to alleviate characteristics of Cushing's disease

To evaluate the activity of oligonucleotides conjugated to GalXC-MC2R-C22 and GalXC-CYP11B1-C22 lipids as single agents and in combination against endpoints related to Cushing's disease, a mouse model of Cushing's disease was optimized (Leung CK. et al., Virchows Arch A. Pathol Anat Histol. 1982 Aug; 396(3):303-12). In this model, the presence of ACTH-secreting mouse pituitary tumors, which were implanted subcutaneously under optimal conditions, resulted in a 4-fold increase in plasma corticosterone, mimicking the effects of Cushing's disease.

Briefly, AtT-20 mouse pituitary tumor cells were obtained from ATCC (ATCC® CCL-89™, Virginia, USA) and cultured using sterile cell culture methods. Cells were maintained in complete growth medium containing F-12K medium containing 2.5% fetal calf serum to a final concentration of 2.5% and horse serum to a final concentration of 15%. Cells were stored at 37°C in a humidified atmosphere of 95% air and 5% _CO2 until implanted in nude mice.

A total of 15 female nude mice were obtained in this study and randomly assigned to 3 groups (initial test, PBS and GalXC-MC2R-C22), n=5. After a 72-hour adaptation period, the PBS and GalXC-MC2R-C22 groups were Mice were injected with 1 x 10 ⁶ cells/mL ArT-20 cells suspended in Matrigel® Matrix (Fisher Scientific, CB-40234, Corning®, New York). The initial trial group did not receive tumor cell injection or treatment. Anesthetize mice using 3% isoflurane in an induction chamber with 100% oxygen before injection. Once successful induction of anesthesia is confirmed, inject cells subcutaneously as a 0.2 mL bolus to the right side above the scapula to prevent obstruction of movement.

On the day of cell implantation (day 0), each group was subjected to the treatments indicated below. As an experimental control, mice were administered PBS. The GalXC-MC2R-C22 group received subcutaneous injection of GalXC-MC2R-1185-C22 at 15 mg/kg. On days 7 and 21, additional treatments were administered to the PBS and GalXC-MC2R-C22 groups.

After cell implantation, the tumor volume (mm ³ ) of each mouse was measured on days 14, 21, and 35. To obtain tumor volume, the length (mm) and width (mm) of each tumor were measured using digital calipers. Specify the longer axis as the length and the shorter axis as the width. Next, use the formula [V = ((W ² ) x L ) / 2] to calculate the final volume. The progression of tumor volume in all groups is shown in Figure 9 .

At the end time point (day 35), all mice were euthanized. Mouse/rat ACTH ELISA set (ab263880, Abcam Inc, Massachusetts, USA) and corticosteroid ELISA set (ab108821, Abcam Inc, Massachusetts, USA) were used to quantify ACTH in plasma samples collected from all groups. and plasma levels of corticosterone. Prepare reagents and standards according to manufacturer's protocols. Plasma analysis on days 14, 21, and 35 showed that by day 35, ACTH in tumor-bearing mice increased 22-fold compared to baseline levels ( Figure 10 ). Plasma ACTH levels correlated with tumor volume, and animals treated with GalXC-MC2R-C22 showed higher ACTH levels on days 21 and 35, suggesting that the KD of the ACTH receptor affects the plasma clearance of ACTH.

Plasma corticosterone levels in tumor-bearing mice increased 4-fold and 8-fold on days 21 and 35, respectively ( Figures 11A to 11B ). At day 21, GalXC-MC2R-C22 treatment reduced plasma corticosterone by approximately 40% two weeks after the first 15 mg/kg dose. An approximately 70% reduction in corticosterone levels was detected four weeks after two doses of GalXC-MC2R-C22 on Day 35 (the endpoint time point). These measurements showed that plasma corticosterone levels were close to normal on day 35. Without wishing to be bound by theory, an additional dosing cycle may be required to achieve normal range. Reported normal cortisol levels in mice range from 30 to 650 ng/mL.

Melanocortin 2 receptor accessory protein (MRAP) functions to transport MC2R from the endoplasmic reticulum to the cell surface, and the protein functions in a feedback loop for expression (Webb and Clark, MOL ENDOCRINOL . March 1, 2010; 24( 3): 475–484). To measure attenuation of mouse MC2R and Mrap mRNA in the adrenal gland (Mrap is transcriptionally upregulated in response to tumors; much higher than MC2R), total RNA isolated from one adrenal gland of each mouse on day 35 of the study was used to assess qRT -PCR representation of MC2R and Mrap mRNA. The ThermoFisher Scientific TaqMan™ Gene Expression Assay for MC2R (Mm01267187_m1; Cat. No.: 4351372) and the ThermoFisher Scientific TaqMan™ Gene Expression Assay for Mrap (Mm00547149_m1) were multiplexed with PPIB (Mm00478295_m1; Cat. No.: 4331182) as a housekeeping gene.

Figures 12A to 12B show that treatment with GalXC-MC2R-C22 resulted in 74% attenuation of MC2R mRNA and 40% attenuation of the downstream pathway gene Mrap .

At the completion of the study, mouse adrenal glands were dissected, fixed in 10% neutral buffered formalin for 24 hours, and prepared for histological evaluation. Sections were stained with H&E to visualize adrenal compartmentalization and cellular organization. Changes in adrenal gland morphology were analyzed and graded by a blinded pathologist to assess zone fasciculata (ZF) vacuolization, zone glomeruli (ZG) extrusion, and adrenal gland size. Due to the hypertrophy and proliferation of zF cells, the adrenal glands of tumor-bearing mice were composed of significantly enlarged cortical layers ( Figure 22 ). These observations are similar to clinical findings in humans.

The size of each adrenal gland was measured in both major and minor axes. Tumor-bearing mice had enlarged and structurally disorganized adrenal cortex compared with control mice. As shown in Figures 13A - 13B , larger adrenal glands were identified in tumor-bearing mice as measured in both long and short axis. GalXC-MC2R-C22-treated mice had smaller adrenal glands compared to PBS animals, and adrenal gland size decreased toward baseline after GalXC-MCR2-C22 treatment. Furthermore, as shown in Figures 14A - 14B , the adrenal cortex of Cushing's disease mice was significantly thicker than that of control mice. This may be the result of increased corticosterone synthesis and subsequent cellular hypertrophy. Mice administered GalXC-MC2R-C22 showed reduced adrenal cortex thickness, indicating reduced cortisol synthesis activity.

ZF vacuolation measures the foamy lipid vesicles present in normal fascicular cells. In tumor-bearing mice, cells bearing lipid vesicles became dense eosinophils. A significant reduction in ZF vacuolization and ZG extrusion was observed in a Cushing's disease mouse model.

GalXC-MC2R-C22 treatment showed a trend of ZF vacuolization and ZG extrusion reversal ( Figures 15A to 15B ). Cell morphology in the ZG showed extrusion into the outer capsule in all tumor-bearing mice, and treatment partially but not completely rescued this abnormality.

Developmental studies in this model demonstrate the ability to replicate some of the pathological findings of Cushing's disease, as well as the ability of GalXC-MC2R-C22 oligonucleotides to mitigate some of these pathologies. Example 6 : RNAi oligonucleotides that inhibit expression of MC2R and CYP11B1 alone or in combination in a mouse model of Cushing's disease

A mouse model of Cushing's disease was generated using AtT-20 mouse pituitary tumor cells as described in Example 5 .

A total of 45 female nude mice were obtained for this study and randomly assigned to 7 groups (A to G as defined in Table 7 ), n = 6. After a 72-hour adaptation period, mice in groups B to G were injected 1 x 10 ⁶ cells/mL ArT-20 cells suspended in Matrigel® Matrix (Fisher Scientific, CB-40234, Corning®, New York USA). Group A was designated as trial naïve and had not received tumor cell injections or treatment. Anesthetize mice using 3% isoflurane in an induction chamber with 100% oxygen before injection. Once successful induction of anesthesia is confirmed, inject cells subcutaneously as a 0.2 mL bolus to the right side above the scapula to prevent obstruction of movement.

On the day of cell implantation (Day 0), groups B to G were administered the treatments shown in Table 7. On day 14, whole blood was collected from all mice via submandibular blood sampling, and samples were placed in EDTA-containing tubes for plasma analysis. Mice were anesthetized before blood collection to prevent induced stress and cortisol spikes.

After cell implantation, the tumor volume (mm ³ ) of each mouse was measured on days 7, 14, and 28. To obtain tumor volume, the length (mm) and width (mm) of each tumor were measured using digital calipers. Specify the longer axis as the length and the shorter axis as the width. Next, use the formula [V = ((W ² ) x L ) / 2] to calculate the final volume. The progression of tumor volume in all groups is shown in Figure 16 .

At the end point (day 28), all mice were euthanized, and their adrenal glands were dissected and weighed. As shown in Figure 17 , the average adrenal gland weight in the naïve group increased twofold from 13.8 mg to 25.7 mg compared with the PBS group. The MC2R oligonucleotide experimental group had an average value of 14.2 mg, while the CYP11B1 oligonucleotide experimental group showed no effect on adrenal weight ( Figure 17 ). The combined treatment group showed a decrease in adrenal weight at 16 mg for Combination 1 and 13.2 mg for Combination 2.

To measure attenuation of MC2R and CYP11B1 mRNA in the adrenal gland, MC2R and CYP11B1 mRNA expression was assessed by qRT-PCR using total RNA isolated from one adrenal gland of each mouse. Combine the ThermoFisher Scientific TaqMan™ Gene Expression Assay for MC2R (Mm01267187_m1; Cat. No.: 4351372) and the ThermoFisher Scientific TaqMan™ Gene Expression Assay for CYP11B1 (Mm01204952_m1; Cat. No.: 4331182) with PPIB as a housekeeping gene (Mm00478295_m1; Cat. No.: 4331182) for multiplex analysis.

Figure 18 shows that treatment with GalXC-CYP11B1-0633-C22 and GalXC-CYP11B1-0843-C22 resulted in an 80% and 81% attenuation of CYP11B1 mRNA in the adrenal gland, respectively. Combination 1 resulted in a 73% attenuation of CYP11B1 mRNA, while combination 2 resulted in a 67% attenuation of CYP11B1 mRNA. Likewise, treatment with GalXC-MC2R-1185-C22 resulted in a 71% attenuation of MC2R mRNA, while combinations 1 and 2 resulted in a 72% and 69% attenuation, respectively ( Figure 19 ). Both CYP11B1 and MC2R are expressed relative to gene expression in the experimental control/PBS group.

To assess the activity of the oligonucleotides tested in this study, the Mouse/Rat ACTH ELISA Kit (ab263880, Abcam Inc, Massachusetts, USA) and the Corticosterone ELISA Kit (ab108821, Abcam Inc, Massachusetts, USA) were used. USA) to quantify plasma levels of ACTH and corticosterone in plasma samples collected from all experimental groups. Prepare reagents and standards according to manufacturer's protocols. Analysis of plasma on day 14 ( Fig. 20A ) showed a 3-fold increase in ACTH over baseline levels in tumor mice and a 22-fold increase on day 28 ( Fig. 20B ). All experimental groups showed increased ACTH levels relative to baseline on days 14 and 28, which correlated with tumor volume measurements.

Plasma corticosterone levels in tumor mice increased 2-fold on day 14 ( Fig. 21A ) and 33-fold on day 28 ( Fig. 21B ). At the endpoint time point, GalXC-MC2R-C22 treatment reduced plasma corticosterone by approximately 60%, while GalXC-CYP11B1-C22 treatment reduced plasma corticosterone by 40 to 50%. Combination treatment was most effective in reducing plasma corticosterone levels, with combination 1 and combination 2 reducing it by 80% and 83%, respectively ( Figure 21B ).

At the completion of the study, mouse adrenal glands were dissected, fixed in 10% neutral buffered formalin for 24 hours, and prepared for histological evaluation. Sections were stained with H&E to visualize adrenal compartmentalization and cellular organization. Compared with control mice, tumor-bearing mice had enlarged and structurally disorganized adrenal cortex ( Fig. 22 ). Adrenal morphological changes were analyzed and graded by a blinded pathologist.

The size of each adrenal gland was measured in both major and minor axes. As shown in Figures 23A - 23B , GalXC-MC2R-C22, Combination 1, and Combination 2 treatments significantly reduced adrenal gland size by 23%, 24%, and 28%, respectively. Adrenocortical measurements are reported as the sum of two zones ZG and ZF. As shown in Figures 23C to 23D , tumor-bearing mice displayed large adrenocortices, with GalXC-MC2R-C22, Combination 1, and Combination 2 treatments showing significant reductions toward baseline of 22%, 11%, and 28%, respectively. This is based on measurements of the long axis. Short-axis adrenocortical measurements also showed similar reductions in the GalXC-MC2R-C22 and combination experimental groups.

Cell morphology in the ZG showed extrusion into the outer capsule in all tumor-bearing mice, and this abnormality was not completely rescued. Pathological evaluation showed that treatment with GalXC-MC2R-C22 and both combinations partially rescued ZG compression ( Figure 23E ).

In summary, animals receiving MC2R alone or in combination had smaller adrenal gland sizes compared to PBS-treated animals. They also show reduced adrenal cortex thickness, indicating reduced cortisol synthetic activity. These studies support the potential of combined treatments to slow down Cushing's disease. Example 7 : Inhibition of MC2R expression in vivo by RNAi oligonucleotides

Sequence screening was performed in mice to identify tool compounds active on MC2R. To identify potent sequences, computer-based algorithms were used to in silico generate MC2R target sequences suitable for determining inhibition of overall MC2R expression by RNAi pathways in humans and non-human primates. To characterize the ability of RNAi oligonucleotides to attenuate MC2R in vivo, a hydrodynamic injection (HDI) mouse model was used. The nucleotide sequences of the 12 selected dsRNAs were used to generate corresponding double-stranded RNAi oligonucleotides containing structures conjugated to a nicked tetracyclic GalNAc (referred to herein as "GalNAc-conjugated MC2R oligonucleotides"). acid"), this structure has a 36-nucleotide follower strand and a 22-nucleotide leader strand. Furthermore, the nucleotide sequences comprising the follower and leader strands of MC2R oligonucleotides conjugated to GalNAc have different patterns of modified nucleotides and phosphorothioate linkages as described in Example 3 (e.g. , see Figure 1A for a schematic representation of the general structure and chemical modification pattern of GalNAc-conjugated MC2R oligonucleotides used in this screen).

The oligonucleotides in Table 8 were evaluated in mice engineered to transiently express mouse MC2R mRNA in hepatocytes of mouse liver (HDI mode). Briefly, 6- to 8-week-old female CD-1 mice (n = 4 to 5) were administered subcutaneously with a dose of 3 mg/kg of GalNAc-conjugated MC2R oligonucleotide formulated in PBS. Only control mice (n = 5) were administered PBS. Three days later (72 hours), mice were hydrodynamically injected (HDI) encoding the complete mouse MC2R gene (SEQ ID NO: 228) (25 μg) under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. ) DNA plasmid. One day after the introduction of DNA plasmids, liver samples from HDI mice were collected. Total RNA from these HDI mice was analyzed by qRT-PCR to determine MC2R mRNA levels. Measures mRNA levels of human mRNA. Values were normalized for transfection efficiency using the NeoR gene included on the DNA plasmid.

The screen provided activity against human MC2R mRNA ( Figure 24 ). Using the same HDI in vivo screen as described above at doses of 0.3, 1 and 3 mg/kg, the three most robust sequences were selected for further characterization in dose response studies ( Figure 25 ). MC2R-205 and MC2R-374 showed reduced intensity at low doses, while MC2R-442 showed reduced intensity at higher doses. In conclusion, oligonucleotides targeting human MC2R are able to potently reduce MC2R in vivo. Example 8 : Inhibition of MC2R expression in vivo by RNAi oligonucleotides

Sequence screening was performed in vitro to identify additional compounds active against MC2R. To identify potent sequences, computer-based algorithms were used to in silico generate MC2R target sequences suitable for determining inhibition of total MC2R expression by the RNAi pathway. The algorithm provides RNAi oligonucleotide leader sequences that are complementary to human (Hs), human and macaque (Hs/Mf), or three species (human, macaque, and mouse; Hs/Mf/Mm). To characterize the ability of RNAi oligonucleotides to attenuate MC2R in vivo, 138 GalNAc-conjugated MC2R oligonucleotides with a 36-nt follower strand and a 22-nt leader were stably transfected. Human MC2R ORF plasmids (CHO-K1/MC2/Gα15; catalog number: M00336; lot number: R10151905-3) were expressed in CHO (Chinese Hamster Ovary) cells ( Table 10 ). Furthermore, the nucleotide sequences comprising the follower and leader strands have different patterns of modified nucleotides and phosphorothioate linkages. Each of the three nucleotides that comprise the four loops is joined to the GalNAc moiety (CAS#14131-60-3). The modification mode of each chain is as shown in the figure below: Crossed with: (Description of modifiers: Table 9 ). symbol Modify / Link Symbol description 1 mX Nucleotides modified with 2'- O -methyl fx 2'-fluoro modified nucleotides -S- Phosphorothioate linkage - Phosphodiester linkage [MePhosphonate-4O-mX] Nucleotide modified with 5'-methoxyphosphonate-4'-oxy group ademA-GalNAc GalNAc attached to adenine nucleotide Symbol description 2 [mXs] 2'- O -methyl modified nucleotides with phosphorothioate linkages to adjacent nucleotides [fXs] 2'-fluoro modified nucleotides with phosphorothioate linkages to adjacent nucleotides [mX] 2'-O-methyl modified nucleotides with phosphodiester linkages to adjacent nucleotides [fX] 2'-fluoro modified nucleotides with phosphodiester linkages to adjacent nucleotides

Twenty-eight hours after transfection, the amount of remaining human MC2R mRNA from transfected cells was determined using a TAQMAN® ^- based qPCR assay. Two qPCR assays (3' assay and 5' assay) were used to determine MC2R mRNA levels. As shown in Table 10 and Figure 26 , % remaining RNA was determined with each primer and normalized to human NeoR-F168 (probe-labeled neomycin resistance gene) and human NeoR-F-172. Oligonucleotides that resulted in more than 50% attenuation of MC2R mRNA compared to mock-transfected cells were considered "active" and selected for further in vivo testing. Example 9 : Inhibition of MC2R expression in vivo by RNAi oligonucleotides

The in vitro screening assay in Example 8 demonstrates the ability of MC2R -targeting oligonucleotides to attenuate target mRNA. To confirm the ability of RNAi oligonucleotides to attenuate MC2R in vivo, the HDI mouse model was used as described in Example 3 to evaluate the GalNAc-engaged MC2R oligos in Table 11 targeting the coding region and 3'UTR of MC2R mRNA. Nucleotides. A MC2R oligonucleotide conjugated to C22 lipids (MC2R-205; containing the C22 modification pattern described in Example 3 ) was used as a baseline control, and mRNA levels in PBS-treated animals were normalized to NeoR-F172.

The results in Figures 27A - 27B show that GalNAc-conjugated MC2R oligonucleotides designed to target human, monkey and/or mouse MC2R mRNA by targeting the coding sequence of MC2R mRNA ( Figure 27A ) or 3' UTR region ( Fig. 27B ), while inhibiting human MC2R mRNA expression. A decrease in human MC2R mRNA expression was observed in liver samples from HDI mice treated with MC2R oligonucleotides conjugated to GalNAc relative to control HDI mice treated with PBS alone.

The most robust sequences from the coding sequence region and the 3' UTR region were selected for a thorough comparison of efficacy ( Figure 28 ), and among those sequences, the most robust ones were selected for further use in dose response studies Characterization ( Figure 29 ). Using the same HDI in vivo screen as described above, MC2R oligonucleotides conjugated to GalNAc were administered at doses of 0.75 and 1.5 mg/kg. At the dose of 1.5 mg/kg, MC2R-120, MC2R-1319, MC2R-1324 and MC2R-1325 showed strong inhibitory effects. In summary, oligonucleotides targeting human MC2R are able to potently reduce MC2R in vivo. Example 10: MC2R inhibition in non-human primates

To assess the ability of lipid-conjugated oligonucleotides to inhibit MC2R mRNA in vivo, non-human primates (NHP) will be administered lipid-conjugated MC2R oligonucleotides. Specifically, NHPs were administered MC2R oligonucleotides (oligonucleotides provided in Table 12 ) containing the 5' terminal C22 lipid of the formula: Crossed with: (Description of modifier symbols: Table 3 ).

Following administration of MC2R-120, MC2R-205, MC2R-1319 and MC2R-1324 oligonucleotides, adrenal glands will be harvested and MC2R mRNA measured as described above.

[ Figure 1A ] Provides the structure of a double-stranded RNAi oligonucleotide molecule with chemical modifications in which GalNAc (top) or lipid (bottom) is conjugated to the molecule to create an oligonucleotide-ligand conjugate.

[ Fig. 1B ] Provides the structure of an exemplary lipid tail suitable for conjugation to an RNAi oligonucleotide molecule.

[ Figure 2 ] provides a graph depicting the percentage (%) of mouse MC2R mRNA remaining in liver samples from hydrodynamic injection (HDI) mode of CD-1 mice. Three days after subcutaneous dosing of the indicated GalNAc-conjugated MC2R oligonucleotides, mice were injected via HDI with plasmids encoding mouse MC2R , and 1 day later relative to administration of phosphate-buffered saline (PBS ) percentage (%) of mouse MC2R mRNA in mice. Arrows indicate MC2R oligonucleotides selected for further study that conjugated to GalNAc.

[ Figures 3A to 3B ] Provided are graphs depicting the dose response ( Figure 3A ) and _ED50 ( Figure 3B ) of 3 MC2R -targeting oligonucleotides selected based on the inhibitory efficacy shown in Figure 2 . As described in Figure 2 , the response rates from mice administered the indicated GalNAc-conjugated MC2R oligos were measured at different doses (0.3, 1, or 3 mg/kg) relative to mice administered phosphate buffered saline (PBS). Percentage (%) of mouse MC2R mRNA remaining in mouse liver samples.

[ Figure 4A ] provides a graph depicting the dose response of three MC2R -targeting oligonucleotides conjugated to C22 lipids as depicted in Figure 1A . Measurements at 14 days postdose, relative to mice administered phosphate buffered saline (PBS), at different doses (3.75, 7.5, 15, and 30 mg/kg) from subcutaneous administration of the indicated C22 lipid-conjugated The percentage (%) of mouse MC2R mRNA remaining in mouse adrenal tissue samples using MC2R oligonucleotides.

[ Figure 4B ] Provides a graph depicting adrenal tissue from mice treated with the indicated MC2R oligonucleotides conjugated to C22 lipids relative to mice administered phosphate buffered saline (PBS) as measured by RNA Scope An image of the level or amount of MC2R protein in a sample. ZF marks the fascicular zone, while ZG marks the globular zone.

[ Figure 5 ] provides a graph depicting the percentage (%) of mouse CYP11B1 mRNA remaining in liver samples from hydrodynamic injection (HDI) mode of CD-1 mice. Three days after subcutaneous dosing of the indicated GalNAc-conjugated CYP11B1 oligonucleotides, mice were injected via HDI with plasmids encoding mouse CYP11B1 , and 1 day later relative to administration of phosphate-buffered saline (PBS) ) of mouse CYP11B1 mRNA in mice. The arrow indicates the CYP11B1 oligonucleotide selected for further study that conjugated to GalNAc.

[ Figure 6 ] provides a graph depicting the dose response of five CYP11B1-targeting oligonucleotides conjugated to GalNAc as depicted in Figure 1A . As described in Figure 5 , measurements were performed relative to untreated mice or mice treated with phosphate-buffered saline (PBS) at different doses (0.3, 1, or 3 mg/kg) from doses administered as indicated. Percentage (%) of mouse CYP11B1 mRNA remaining in liver samples from mice with GalNAc-conjugated CYP11B1 oligonucleotides.

[ Figure 7 ] provides a graph depicting the dose response of 2 CYP11B1 oligonucleotides conjugated to C22 lipids. Measurements at 14 days postdose, relative to mice administered phosphate buffered saline (PBS), at different doses (3.75, 7.5, 15, and 30 mg/kg) from mice administered the indicated C22 lipid-conjugated Graph of the percentage (%) of mouse CYP11B1 mRNA remaining in mouse adrenal tissue samples using CYP11B1 oligonucleotides.

[ Figures 8A to 8B ] Provided are graphs depicting the effects of the indicated CYP11B1 oligonucleotides conjugated to C22 lipids or conjugated to C22 lipids at 14 and 28 days post-dose, relative to mice administered phosphate-buffered saline. Plot of percentage (%) of mouse CYP11B1 mRNA ( Figure 8A ) and mouse MC2R mRNA ( Figure 8B ) remaining in adrenal tissue samples of CD-1 mice conjugated to MC2R oligonucleotides.

[ Figure 9 ] Provides a graph depicting MC2R oligos conjugated to C22 lipids relative to untreated tumor-free mice (naïve) or tumor-bearing mice administered phosphate buffered saline (PBS). Tumor volume of AtT-20 tumors in tumor-bearing mice treated with nucleotide (GalXC-MC2R-1185-C22) over time. AtT-20 tumors were implanted into mice as a model for Cushing's syndrome.

[ Figure 10 ] Provides a graph depicting oligonucleotides from MC2R conjugated to C22 lipids relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS) Plot of plasma adrenocorticotropic hormone (ACTH) levels over time in AtT-20 tumor-bearing mice treated with acid (GalXC-MC2R-1185-C22).

[ Figures 11A to 11B ] Provides a depiction of MC2R oligonucleotides conjugated to C22 lipids relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS). Plasma corticosterone levels over time in AtT-20 tumor-bearing mice treated with GalXC-MC2R-1185-C22. Figure 11A depicts corticosterone levels relative to mice administered PBS. Figure 11B depicts absolute corticosterone levels.

[ Figures 12A to 12B ] Provides a depiction of oligonucleotides from MC2R conjugated to C22 lipids relative to untreated tumor-free mice (naive) or mice administered phosphate buffered saline (PBS) Graph showing the percentage (%) of remaining MC2R mRNA ( Figure 12A ) and Mrap mRNA ( Figure 12B ) in adrenal tissue samples of AtT-20 tumor-bearing mice 35 days after acid (GalXC-MC2R-1185-C22) treatment .

[ Figures 13A to 13B ] provide a graph depicting the effect of MC2R oligos conjugated to C22 lipids relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS). Graph showing the size of the long adrenal axis ( Fig. 13A ) and the short adrenal axis ( Fig. 13B ) of AtT-20 tumor-bearing mice 35 days after nucleotide (GalXC-MC2R-1185-C22) treatment.

[ Figures 14A to 14B ] provide a graph depicting the effect of MC2R oligos conjugated to C22 lipids relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS). Graph showing the size of the long cortical axis ( Fig. 14A ) and the short cortical axis ( Fig. 14B ) of AtT-20 tumor-bearing mice 35 days after nucleotide (GalXC-MC2R-1185-C22) treatment.

[ Figs . 15A to 15B ] Provides a graph depicting the effect of MC2R oligos conjugated to C22 lipids relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS). Picture of the size of zF adrenal gland vacuolation ( Fig. 15A ) and zG extrusion ( Fig. 15B ) in AtT-20 tumor-bearing mice 35 days after nucleotide (GalXC-MC2R-1185-C22) treatment.

[ Figure 16 ] Provides a graph depicting MC2R conjugated to C22 lipids (GalXC-MC2R) relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS) Plot of weekly tumor volume in AtT-20 tumor-bearing mice treated with -1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or the combination. 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figure 17 ] Provides a graph depicting MC2R conjugated to C22 lipids (GalXC-MC2R) relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS) Total adrenal gland weight of AtT-20 tumor-bearing mice 28 days after treatment with -1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or combination (treated at 15 mg/kg on days 0 and 14) Figure. 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figure 18 ] Provides a graph depicting MC2R conjugated to C22 lipids (GalXC-MC2R) relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS) -1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or combination treatment in AtT-20 tumor-bearing mice 28 days after treatment (as described in Figure 17 ). . Combination 1 represents the combination of GalXC-CYP11B1-0633-C22 and GalXC-MC2R-1185-C22, while combination 2 represents the combination of GalXC-CYP11B1-0843-C22 and GalXC-MC2R-1185-C22.

[ Figure 19 ] Provides a graph depicting MC2R conjugated to C22 lipids (GalXC-MC2R) relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS) -1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or combination treatment in AtT-20 tumor-bearing mice 28 days after treatment ( as described in Figure 17 ). . 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figs . 20A to 20B ] provides a graph depicting the effects of tumor-free mice (naive) on days 14 ( Fig. 20A ) and 28 ( Fig. 20B ) post-treatment on days 14 (Fig. 20A) and 28 (Fig. 20B). Tumor-bearing mice in saline (PBS), AtT-20 tumor-bearing mice treated with MC2R conjugated to C22 lipid (GalXC-MC2R-1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or a combination (such as Plot of plasma ACTH percentage (described in Figure 17 ). 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figures 21A to 21B ] provide a graph depicting the effects of tumor-free mice on days 14 ( Figure 21A ) and 28 ( Figure 21B ) after treatment relative to untreated tumor-free mice (naive) or administered phosphate buffered physiological Tumor-bearing mice in saline (PBS), AtT-20 tumor-bearing mice treated with MC2R conjugated to C22 lipid (GalXC-MC2R-1185-C22), CYP11B1 oligonucleotide conjugated to C22 lipid, or a combination (such as Plot of plasma corticosterone percentage (%) as described in Figure 17 ). 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figure 22 ] Provides a graph depicting C22 lipid conjugation at 28 days after treatment, relative to untreated tumor-free mice (naive) or tumor-bearing mice administered phosphate buffered saline (PBS). Diagram of the adrenal structure of AtT-20 tumor-bearing mice treated with MC2R (GalXC-MC2R-1185-C22), CYP11B1 oligonucleotides conjugated to C22 lipids, or the combination ( as described in Figure 17 ). 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with GalXC-MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with GalXC-MC2R-1185-C22 combination (combination 2).

[ Figures 23A to 23E ] provide graphs depicting adrenal measurements at day 28 post-treatment relative to untreated tumor-free mice or phosphate-buffered saline (PBS)-treated tumor-bearing mice, as indicated The short axis of the adrenal gland (Fig. 23A), the long axis of the adrenal gland ( Fig . 23A ) of AtT-20 tumor-bearing mice ( as described in Fig . 17 ) treated with MC2R conjugated to C22 lipid, CYP11B1 oligonucleotide conjugated to C22 lipid, or combination Figure 23B ), cortical short axis ( Figure 23C ), cortical long axis ( Figure 23D ), and zG extrusion ( Figure 23E ) . 1-represents GalXC-CYP11B1-0633-C22 alone or in combination with MC2R-1185-C22 (combination 1), while 2-represents GalXC-CYP11B1-0843-C22 alone or in combination with MC2R-1185-C22 (combination 1) 2).

[ Figure 24 ] provides a graph depicting the percentage (%) of human MC2R mRNA remaining in liver samples from the hydrodynamic injection (HDI) model of CD-1 mice. Three days after subcutaneous dosing of the indicated GalNAc-conjugated MC2R oligonucleotides, mice were injected via HDI with plasmids encoding human MC2R and measured 1 day later relative to administration of phosphate buffered saline (PBS) Percent (%) of human MC2R mRNA in mice.

[ Figure 25 ] provides a graph depicting the dose response of 3 oligonucleotides targeting human MC2R selected based on the inhibitory efficacy shown in Figure 24 . As shown in Figure 24 , the response rates from mice administered the indicated GalNAc-conjugated MC2R oligos at different doses (0.3, 1, or 3 mg/kg) were measured relative to mice administered phosphate buffered saline (PBS). Percentage (%) of human MC2R mRNA remaining in mouse liver samples.

[ Fig. 26 ] Provides a graph depicting the percentage (%) of human MC2R mRNA remaining in CHO cells stably expressing the human MC2R ORF administered 1 nM of the GalNAc-conjugated MC2R oligonucleotide in Table 10 . MC2R mRNA was measured 1 day after administration. The location of the human MC2R gene is shown (CDS = coding sequence; UTR = untranslated region).

[ Figures 27A - 27B ] provide graphs depicting the percentage (%) of human MC2R mRNA remaining in liver samples from the hydrodynamic injection (HDI) model of CD-1 mice. Mice were injected via HDI three days after subcutaneous administration of designated GalNAc-conjugated MC2R oligonucleotides targeting the 5'UTR or coding sequence ( Fig. 27A ) or the 3'UTR region ( Fig. 27B ) of MC2R mRNA. Plasmids encoding human MC2R were generated, and the percentage (%) of human MC2R mRNA relative to mice administered phosphate buffered saline (PBS) was measured after 1 day. MC2R-205 is a reference oligonucleotide conjugated to human/monkey C22 lipids. Hs/Mf = targets human and monkey mRNA; Hs unique = targets human mRNA; Hs/Mf/Mm = targets human, monkey, and mouse mRNA.

[ Figure 28 ] provides a graph depicting the percentage (%) of human MC2R mRNA remaining in liver samples from the hydrodynamic injection (HDI) model of CD-1 mice. Three days after subcutaneous administration of designated GalNAc-conjugated MC2R oligonucleotides targeting the 5'UTR or coding sequence or 3'UTR region of MC2R mRNA, mice were injected via HDI with plasmids encoding human MC2R , and The percentage (%) of human MC2R mRNA relative to mice administered phosphate buffered saline (PBS) was measured after 1 day. MC2R-205 is a reference oligonucleotide conjugated to human/monkey C22 lipids. Hs/Mf = targets human and monkey mRNA; Hs unique = targets human mRNA.

[ Figure 29 ] Provides a graph depicting the dose response of eight selected oligonucleotides targeting human MC2R based on the inhibitory efficacy shown in Figure 28 . As shown in Figure 28 , measurements from mice administered the indicated MC2R oligonucleotides conjugated to GalNAc at different doses (0.75 or 1.5 mg/kg) were measured relative to mice administered phosphate buffered saline (PBS). Percentage (%) of human MC2R mRNA remaining in acidic mouse liver samples. MC2R-205 is a reference oligonucleotide conjugated to human/monkey C22 lipids. Hs/Mf = targets human and monkey mRNA; Hs unique = targets human mRNA.

[ Figure 30 ] provides a schematic diagram of the sequences and modification patterns of MC2R-120, MC2R-205, MC2R-1319 and MC2R-1324.

[ Figures 31A to 34B ] The respective molecular structures of MC2R-120 ( Figures 31A to 31B ), MC2R-205 ( Figures 32A to 32B ), MC2R-1319 ( Figures 33A to 33B ), and MC2R-1324 ( Figure 34A ) are provided ( Figure 34B) . Each molecular structure is separate, with A-share being a continuous strand connected to point A, and B-share being a continuous strand connected to point B.

TW202334414A_111137911_SEQL.xml

Claims (162)

An oligonucleotide for reducing the expression of melanocortin 2 receptor (MC2R), the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length. A strand, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has a complementary region to the target sequence of MC2R as shown in any one of SEQ ID NOs: 201 to 212. An oligonucleotide for reducing the expression of melanocortin 2 receptor (MC2R), the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides long and a sense strand of 15 to 40 nucleotides long A strand, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has a complementary region to the target sequence of MC2R as shown in any one of SEQ ID NOs: 229 to 420. An oligonucleotide for reducing the expression of melanocortin 2 receptor (MC2R), the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length. strand, wherein the sense strand and the antisense strand form a double helix region, wherein the antisense strand has complementarity to an MC2R target sequence as set forth in any one of SEQ ID NOs: 201 to 212 and 229 to 240 district. The oligonucleotide of any one of claims 1 to 3, wherein the complementary region is completely complementary to the target sequence of MC2R. The oligonucleotide of any one of claims 1 to 4, wherein the antisense strand is 19 to 27 nucleotides long. The oligonucleotide of any one of claims 1 to 5, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long. The oligonucleotide of any one of claims 1 to 3, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleotides long. The oligonucleotide of any one of claims 1 to 7, wherein the double helix region is at least 19 nucleotides long. The oligonucleotide of any one of claims 1 to 8, wherein the double helix region is at least 20 nucleotides long, optionally wherein the double helix region is 21 nucleotides long. The oligonucleotide of any one of claims 1 to 9, wherein the complementary region is at least 19 contiguous nucleotides. The oligonucleotide of any one of claims 1 to 10, wherein the complementary region is at least 21 contiguous nucleotides long. The oligonucleotide of any one of claims 1 and 3 to 11, wherein the antisense strand comprises the sequence shown in any one of SEQ ID NO: 37 to 48. The oligonucleotide of any one of claims 1 and 3 to 12, wherein the sense strand comprises the sequence shown in any one of SEQ ID NO: 85 to 96. The oligonucleotide of any one of claims 2 to 11, wherein the antisense strand comprises the sequence shown in any one of SEQ ID NO: 805 to 996. The oligonucleotide of any one of claims 2 to 11 and 14, wherein the sense strand comprises a sequence as shown in any one of SEQ ID NO: 613 to 804. The oligonucleotide of any one of claims 1 to 15, wherein the sense strand includes a stem loop at its 3' end as shown below: S1-L-S2, where S1 is complementary to S2, and where L A 3 to 5 nucleotide long loop is formed between S1 and S2. An oligonucleotide for reducing the performance of MC2R, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and has the same characteristics as SEQ ID NO: 201 to the complementary region of the target sequence of MC2R shown in any one of 212, wherein the sense strand includes at its 3' end a stem-loop shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a double helix region of at least 19 nucleotides long. An oligonucleotide for reducing the performance of MC2R, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and has the same characteristics as SEQ ID NO: 229 to the complementary region of the target sequence of MC2R shown in any one of 420, wherein the sense strand includes a stem loop at its 3' end as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a double helix region of at least 19 nucleotides long. An oligonucleotide for reducing the performance of MC2R, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides long and has the same characteristics as SEQ ID NO: 201 to the complementary region of the target sequence of MC2R shown in any one of 212 and 229 to 420, wherein the sense strand includes a stem loop at its 3' end as shown below: S1-L-S2, wherein S1 and S2 is complementary, and wherein L forms a loop between S1 and S2 that is 3 to 5 nucleotides long, and wherein the antisense strand and the sense strand form a double helix region of at least 19 nucleotides long. The oligonucleotide of any one of claims 17 to 19, wherein the complementary region is 19 nucleotides long and is completely complementary to the target sequence. The oligonucleotide of any one of claims 16 to 20, wherein L is a tetracyclic ring. The oligonucleotide of any one of claims 16 to 21, wherein L is 4 nucleotides long. The oligonucleotide of any one of claims 16 to 22, wherein L includes the sequence represented by GAAA. The oligonucleotide of any one of claims 1 to 23, wherein (i) the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long, or (ii) the antisense strand The sense strand is 22 nucleotides long and the sense strand is 36 nucleotides long. The oligonucleotide of claim 24, wherein the antisense strand and the sense strand form a double-stranded helix region that is 25 nucleotides long or 20 nucleotides long. The oligonucleotide of any one of claims 1 to 25, wherein the antisense strand includes a 3' overhang sequence of one or more nucleotides, optionally wherein the 3' overhang sequence is 2 nucleotides long, optionally wherein the 3' overhang sequence is GG. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide. The oligonucleotide of claim 27, wherein the modified nucleotide comprises a 2'-modification. The oligonucleotide of claim 28, wherein the 2'-modification is selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl and modification of 2'-deoxy-2'-fluoro-β-d-arabinic acid. The oligonucleotide of any one of claims 26 to 29, wherein the sense strand is about 10 to 15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotide Contains 2'-fluoro modification. Such as the oligonucleotide of any one of claims 26 to 30, wherein the antisense strand is about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30% of the nucleotide , 31%, 32%, 33%, 34% or 35% contain 2'-fluorine modification. The oligonucleotide of any one of claims 26 to 31, wherein the oligonucleotide contains about 25 to 35%, 25%, 26%, 27%, 28%, 29%, 30% of the nucleotide. %, 31%, 32%, 33%, 34% or 35% contain a 2'-fluoro modification. The oligonucleotide of any one of claims 26 to 32, wherein the sense strand includes 36 nucleotides from 5' to 3 at positions 1 to 36, wherein positions 8 to 11 include 2'- Fluorine modification. The oligonucleotide of any one of claims 26 to 33, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, and positions 2, 3, and 4 thereof , 5, 7, 10 and 14 contain 2'-fluoro modifications. The oligonucleotide of any one of claims 29 to 34, wherein the remaining nucleotides comprise a 2'-O-methyl modification. The oligonucleotide of claim 33, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications. The oligonucleotide of claim 34 or 36, wherein positions 1, 6, 8, 9, 11 to 13 and 15 to 22 of the antisense strand comprise 2'-O-methyl modifications. The oligonucleotide of any one of claims 26 to 37, wherein all nucleotides of the oligonucleotide are modified. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified inter-nucleotide linkage. The oligonucleotide of claim 39, wherein the at least one modified inter-nucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 40, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand. The oligonucleotide of claim 40 or 41, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand includes positions 1 and 2, Phosphorothioate linkages between 2 and 3, 20 and 21, and 21 and 22. The oligonucleotide of any one of the preceding claims, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog. The oligonucleotide of claim 43, wherein the phosphate analog is methoxyphosphonate, vinyl phosphonate or malonyl phosphonate. The oligonucleotide of any one of the preceding claims, wherein at least one nucleotide of the oligonucleotide is bound to one or more target ligands. The oligonucleotide of claim 45, wherein the nucleotide is bonded to more than one target ligand, wherein the target ligands are the same or different. The oligonucleotide of claim 45 or 46, wherein the one or more target ligands are selected from carbohydrates, amino sugars, cholesterol, polypeptides or lipids. The oligonucleotide of claim 45 or 46, wherein the one or more target ligands are saturated or unsaturated fatty acid moieties. The oligonucleotide of claim 45 or 46, wherein the target ligand is a saturated fatty acid moiety ranging in size from C10 to C24. The oligonucleotide of claim 49, wherein the target ligand is a C16 saturated fatty acid moiety. The oligonucleotide of claim 49, wherein the target ligand is a C18 saturated fatty acid moiety. The oligonucleotide of claim 49, wherein the target ligand is a C22 saturated fatty acid moiety. The oligonucleotide of claim 45 or 46, wherein the target ligand comprises an N-acetylgalactosamine (GalNAc) moiety. The oligonucleotide of claim 53, wherein the GalNAc part is a monovalent GalNAc part, a divalent GalNAc part, a trivalent GalNAc part or a tetravalent GalNAc part. The oligonucleotide of any one of claims 16 to 54, wherein up to 4 nucleotides of L of the stem loop are each conjugated to a monovalent GalNAc moiety. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17, and 19 to 55, wherein the sense strand and the antisense strand comprise nucleotides selected from the group consisting of: sequence: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively. The oligonucleotide of any one of claims 1 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; (l) SEQ ID NO: 48 and 96 respectively; (m) SEQ ID NO: 632 and 824 respectively; (n) SEQ ID NO: 765 and 957 respectively; (o) SEQ ID NOs: 770 and 962 respectively; and (p) SEQ ID NO: 38 and 86 respectively. The oligonucleotide of any one of claims 1 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) SEQ ID NO: 38 and 86 respectively. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 85 nucleotide sequence. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 38, and the antisense strand comprises SEQ Nucleotide sequence of ID NO: 86. The oligonucleotide of any one of claims 2 to 11, 14 to 14 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 248, and the antisense strand comprises SEQ ID NO : 440 nucleotide sequence. The oligonucleotide of any one of claims 2 to 11, 14 to 14 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 381, and the antisense strand comprises SEQ ID NO :573 nucleotide sequence. The oligonucleotide of any one of claims 2 to 11, 14 to 14 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 386, and the antisense strand comprises SEQ ID NO :578 nucleotide sequence. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 203, and the antisense strand comprises SEQ ID NO: 215 nucleotide sequence. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NO: 176 to 187. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NO: 174 to 175. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17, 19 to 55 and 66, wherein the antisense strand comprises the nucleotide of any one of SEQ ID NO: 188 to 199 sequence. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17, and 19 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the following: (a) SEQ ID NO: 186 and 188 respectively; (b) SEQ ID NO: 187 and 189 respectively; (c) SEQ ID NO: 176 and 190 respectively; (d) SEQ ID NO: 177 and 191 respectively; (e) SEQ ID NO: 178 and 192 respectively; (f) SEQ ID NO: 179 and 193 respectively; (g) SEQ ID NO: 180 and 194 respectively; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 184 and 198 respectively; and (l) SEQ ID NO: 185 and 199 respectively. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 186, and the antisense strand comprises SEQ ID NO: 186 Nucleotide sequence of ID NO: 188. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 187, and the antisense strand comprises SEQ ID NO:189 nucleotide sequence. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17, and 19 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the following: (a) SEQ ID NO: 174 and 188 respectively; and (b) SEQ ID NO: 175 and 189 respectively. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 174, and the antisense strand comprises SEQ Nucleotide sequence of ID NO: 188. The oligonucleotide of any one of claims 1, 3 to 13, 16 to 17 and 19 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 175, and the antisense strand comprises SEQ ID NO: 189 nucleotide sequence. The oligonucleotide of any one of claims 1 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the following: (a) SEQ ID NO: 186 and 188 respectively; (b) SEQ ID NO: 187 and 189 respectively; (c) SEQ ID NO: 176 and 190 respectively; (d) SEQ ID NO: 177 and 191 respectively; (e) SEQ ID NO: 178 and 192 respectively; (f) SEQ ID NO: 179 and 193 respectively; (g) SEQ ID NO: 180 and 194 respectively; (h) SEQ ID NO: 181 and 195 respectively; (i) SEQ ID NO: 182 and 196 respectively; (j) SEQ ID NO: 183 and 197 respectively; (k) SEQ ID NO: 184 and 198 respectively; (l) SEQ ID NO: 185 and 199 respectively; (m) are SEQ ID NO: 997 and 1001 respectively; (n) SEQ ID NO: 998 and 1002 respectively; (o) SEQ ID NO: 999 and 1003 respectively; and (p) are SEQ ID NO: 1000 and 1004 respectively. The oligonucleotide of any one of claims 2 to 11, 14 to 16, and 18 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the following: (a) SEQ ID NO: 997 and 1001 respectively; (b) SEQ ID NO: 998 and 1002 respectively; (c) SEQ ID NO: 999 and 1003 respectively; and (d) SEQ ID NO: 1000 and 1004 respectively. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 997, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1001. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 998, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1002. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 999, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1003. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1000, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1004. The oligonucleotide of any one of claims 2 to 11, 14 to 16, and 18 to 55, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the following: (a) SEQ ID NO: 1005 and 1001 respectively; (b) SEQ ID NO: 1006 and 1002 respectively; (c) SEQ ID NO: 1007 and 1003 respectively; and (d) SEQ ID NO: 1008 and 189 respectively. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1005, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1001. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1006, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1002. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1007, and the antisense strand comprises SEQ ID NO : The nucleotide sequence of 1003. The oligonucleotide of any one of claims 2 to 11, 14 to 16 and 18 to 55, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1008, and the antisense strand comprises SEQ ID NO : 189 nucleotide sequence. An oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising a complementary region to a target sequence in MC2R mRNA; (ii) one or more target ligands, wherein the oligonucleotide-ligand conjugate is represented by Formula Ia : Or its medically acceptable salt, wherein: B is a nucleobase; R ¹ and R ² are independently hydrogen, halogen, ^RA , -CN, -S(O)R, -S(O) ₂ R , -Si(OR) ₂ R, -Si(OR)R ₂ or -SiR ₃ ; or R ¹ and R ² on the same carbon together with their intervening atoms form 0 to 3 independently selected from nitrogen, oxygen and a 3 to 7-membered saturated or partially unsaturated ring of sulfur heteroatoms; each R ^A is independently an optionally substituted group selected from C _1-6 aliphatic, phenyl, 4 to 7 membered saturated or partially unsaturated heterocycles having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur to a 6-membered heteroaromatic ring; each R is independently hydrogen, a suitable protecting group or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaryls having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur Ring; or two R groups on the same atom together with their intervening atoms form a 4 to 7 membered saturated, partially unsaturated or heteroaryl having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, silicon and sulfur ring; each target ligand is a lipid-ligating moiety (LC); and wherein LC is independently a lipid-ligating moiety comprising a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain 0 to 10 methylene units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O- , -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR- substitution; Each -Cy- is independently a bivalent ring that is optionally substituted, The divalent ring system is selected from the group consisting of a phenylene group, an 8- to 10-membered bicyclic aryl group, a 4- to 7-membered saturated or partially unsaturated carbocyclylenyl group, and a 4- to 11-membered saturated or partially unsaturated spirocyclic ring. Carbocyclyl, 8 to 10-membered bicyclic saturated or partially unsaturated carbocyclyl, 4 to 7-membered saturated or partially unsaturated carbocyclyl with 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. Heterocyclyl, a 4 to 11-membered saturated or partially unsaturated spirocyclic heterocyclyl group having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, having 1 to 2 heteroatoms independently selected from nitrogen, 8 to 10 membered bicyclic saturated or partially unsaturated heterocyclyl groups with heteroatoms of oxygen and sulfur, 5 to 6 membered heteroaryl groups with 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or An 8- to 10-membered bicyclic heteroaryl group having 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or Branched C _1-50 hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR-, -P(S)OR-, -V ¹ CR ² W ¹ -or Substitution; m is 1 to 50; X ¹ , V ¹ and W ¹ are independently -C(R) ₂ -, -OR, -O-, -S-, -Se- or -NR-; Y is hydrogen , Suitable hydroxyl protecting group, or ; R ³ is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group, which group is selected from C _1-6 aliphatic, phenyl, having 1 to 2 independently selected from X ² is ^{O ,} S or _NR ; The linking group; Y ² is hydrogen, a suitable protecting group, a phosphoramidite analogue, an internucleotide linking group attached to the 5' end of a nucleoside, nucleotide or oligonucleotide, or a solid support attached and Z is -O-, -S-, -NR- or -CR ₂ -. Such as the oligonucleotide-ligand conjugate of claim 85, which is represented by formula II-b or II-c : Or a medically acceptable salt thereof, wherein: L ¹ is a covalent bond, a monovalent or divalent saturated or unsaturated, straight or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene The base units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O) -, -S(O) ₂ -, -P(O)OR-, -P(S)OR-or Replacement; R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene units independently -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O ) ₂ -, -P(O)OR- or -P(S)OR substitution. The oligonucleotide-ligand conjugate of claim 86, wherein: R ⁵ is selected from . The oligonucleotide-ligand conjugate of claim 86, wherein ^R5 is selected from . The oligonucleotide-ligand conjugate of claim 86, wherein ^R5 is . An oligonucleotide-ligand conjugate for reducing MC2R mRNA expression in cells of the adrenal cortex or adrenal gland, comprising (i) a nucleotide sequence comprising a complementary region to a target sequence in MC2R mRNA; (ii) one or more target ligands, wherein the oligonucleotide-ligand conjugate is represented by formula II-Ib or II-Ic : or its medically acceptable salt; wherein B is a nucleobase; m is 1 to 50; X ¹ is -O- or -S-; Y is hydrogen, or ; ^{R 3} is hydrogen or a suitable protecting group; ^{X 2} is O or S; A linker at the 2'- or 3'end; Y ² is hydrogen, a phosphoramidite analog, an internucleotide linker or an attachment at the 5' end of an attached nucleoside, nucleotide or oligonucleotide The linking group of the solid support; R ⁵ is a saturated or unsaturated, linear or branched C _1-50 hydrocarbon chain, in which the 0 to 10 methylene units of the hydrocarbon chain are independently passed through -O-, -C (O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) ₂ -, -P(O)OR - or -P(S)OR substitution; and R is hydrogen, a suitable protecting group, or an optionally substituted group selected from C _1-6 aliphatic, phenyl, having 1 to 2 4 to 7 membered saturated or partially unsaturated heterocycles having heteroatoms independently selected from nitrogen, oxygen and sulfur and 5 to 6 membered heteroaryls having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur ring. The oligonucleotide-ligand conjugate of claim 90, wherein ^R5 is selected from . The oligonucleotide-ligand conjugate of any one of claims 90 to 91, wherein ^R5 is . The oligonucleotide-ligand conjugate of any one of claims 85 to 92, wherein the nucleotide sequence contains 1 to 10 target ligands. The oligonucleotide-ligand conjugate of any one of claims 85 to 93, wherein the nucleotide sequence contains 1, 2 or 3 target ligands. The oligonucleotide-ligand conjugate of any one of claims 85 to 94, wherein the adrenal cortex or the cell in the adrenal gland is an epithelial cell. The oligonucleotide-ligand conjugate of any one of claims 85 to 95, wherein the oligonucleotide is single-stranded, optionally wherein the single-stranded oligonucleotide has 15 to 30 nuclei. Long nucleotide. The oligonucleotide-ligand conjugate of any one of claims 85 to 96, wherein the oligonucleotide is double-stranded. The oligonucleotide-ligand conjugate of claim 97, wherein the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein The sense strand and the antisense strand form a double helix region, wherein the antisense strand includes a complementary region to the target sequence, and wherein the complementary region is at least 15 contiguous nucleotides long. The oligonucleotide-ligand conjugate of claim 98, wherein the antisense strand is 19 to 27 nucleotides long. The oligonucleotide-ligand conjugate of claim 98 or 99, wherein the antisense strand is 21 to 27 nucleotides long, optionally wherein the antisense strand is 22 nucleotides long. The oligonucleotide-ligand conjugate of any one of claims 98 to 100, wherein the sense strand is 19 to 40 nucleotides long, optionally wherein the sense strand is 36 nucleosides Sour and long. The oligonucleotide-ligand conjugate of any one of claims 98 to 101, wherein the double helix region is at least 19 nucleotides long. The oligonucleotide-ligand conjugate of any one of claims 98 to 102, wherein the double helix region is at least 20 nucleotides long, optionally wherein the double helix region is 21 nucleotides long Long nucleotide. The oligonucleotide-ligand conjugate of any one of claims 98 to 103, wherein the complementary region is at least 19 contiguous nucleotides long. The oligonucleotide-ligand conjugate of any one of claims 98 to 104, wherein the complementary region is at least 21 contiguous nucleotides long. The oligonucleotide-ligand conjugate of any one of claims 98 to 105, wherein the sense strand includes a stem loop shown below at its 3' end: S1-L-S2, wherein S1 and S2 is complementary, and where L forms a 3 to 5 nucleotide long loop between S1 and S2. The oligonucleotide-ligand conjugate of claim 106, wherein L is a tetracyclic ring. The oligonucleotide-ligand conjugate of any one of claims 106 to 107, wherein L includes a sequence represented by GAAA. The oligonucleotide-ligand conjugate of any one of claims 106 to 108, wherein the one or more target ligands are bonded to the stem loop. The oligonucleotide-ligand conjugate of claim 109, wherein one or more target ligands are conjugated to one or more nucleotides of L. The oligonucleotide-ligand conjugate of claim 110, wherein L includes 4 nucleotides at positions numbered 1 to 4 from 5' to 3', wherein one or more targets are coordinated The base is engaged with position 2. The oligonucleotide-ligand conjugate of any one of claims 98 to 108, wherein the one or more target ligands are conjugated to the 5' end nucleotide of the sense strand. The oligonucleotide-ligand conjugate of any one of claims 98 to 111, wherein the antisense strand comprises one or more nucleotide-long 3' overhang sequences, optionally wherein the 3' The overhang sequence is 2 nucleotides long, optionally where the 3' overhang sequence is GG. The oligonucleotide-ligand conjugate of claims 85 to 113, wherein the oligonucleotide comprises at least one modified nucleotide. The oligonucleotide-ligand conjugate of claim 114, wherein the modified nucleotide comprises a 2'-modification. The oligonucleotide-ligand conjugate of claim 114, wherein the 2'-modification is selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O -Methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinic acid modifications. The oligonucleotide-ligand conjugate of any one of claims 114 to 116, wherein about 10 to 15%, 10%, 11%, 12%, 13% of the nucleotide of the sense strand , 14% or 15% contain 2'-fluoro modification. The oligonucleotide-ligand conjugate of any one of claims 114 to 117, wherein the antisense strand is about 25 to 35%, 25%, 26%, 27%, 28% of the nucleotide , 29%, 30%, 31%, 32%, 33%, 34% or 35% contain 2'-fluorine modification. The oligonucleotide-ligand conjugate of any one of claims 114 to 118, wherein about 25 to 35%, 25%, 26%, 27%, 28% of the nucleotide of the oligonucleotide %, 29%, 30%, 31%, 32%, 33%, 34% or 35% contain a 2'-fluoro modification. The oligonucleotide-ligand conjugate of any one of claims 114 to 119, wherein the sense strand includes 36 nucleotides from 5' to 3' at positions 1 to 36, wherein positions 8 to 11 contain 2'-fluoro modifications. The oligonucleotide-ligand conjugate of any one of claims 114 to 120, wherein the antisense strand comprises 22 nucleotides from 5' to 3' at positions 1 to 22, and wherein Positions 2, 3, 4, 5, 7, 10 and 14 contain 2'-fluoro modifications. The oligonucleotide-ligand conjugate of any one of claims 114 to 121, wherein the remaining nucleotides comprise 2'-O-methyl modification. The oligonucleotide-ligand conjugate of claim 120, wherein positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand comprise 2'-O-methyl modifications. The oligonucleotide-ligand conjugate of claim 121 or 123, wherein positions 1, 6, 8, 9, 11 to 13 and 15 to 22 of the antisense strand comprise 2'-O-methyl modifications. The oligonucleotide-ligand of any one of claims 114 to 124, wherein all nucleotides of the oligonucleotide are modified. The oligonucleotide-ligand conjugate of any one of claims 85 to 125, wherein the oligonucleotide comprises at least one modified inter-nucleotide linkage. The oligonucleotide-ligand conjugate of claim 126, wherein the at least one modified inter-nucleotide linkage is a phosphorothioate linkage. The oligonucleotide-ligand conjugate of claim 127, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand. The oligonucleotide-ligand conjugate of claim 127 or 128, wherein the antisense strand comprises 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the antisense strand comprises Phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22. The oligonucleotide-ligand conjugate of any one of claims 127 to 129, wherein the antisense strand includes 22 nucleotides from 3' to 5' at positions 1 to 22, wherein the The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22. The oligonucleotide-ligand conjugate of any one of claims 98 to 130, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog. The oligonucleotide-ligand conjugate of claim 131, wherein the phosphate analog is methoxyphosphonate, vinylphosphonate or malonylphosphonate. The oligonucleotide-ligand conjugate of any one of claims 90 to 132, wherein ^R5 is . The oligonucleotide-ligand conjugate of any one of claims 90 to 132, wherein ^R5 is . The oligonucleotide-ligand conjugate of any one of claims 90 to 132, wherein ^R5 is . The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; and (l) SEQ ID NO: 48 and 96 respectively. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 37 and 85 respectively; (b) SEQ ID NO: 38 and 86 respectively; (c) SEQ ID NO: 39 and 87 respectively; (d) SEQ ID NO: 40 and 88 respectively; (e) SEQ ID NO: 41 and 89 respectively; (f) SEQ ID NO: 42 and 90 respectively; (g) SEQ ID NO: 43 and 91 respectively; (h) SEQ ID NO: 44 and 92 respectively; (i) SEQ ID NO: 45 and 93 respectively; (j) SEQ ID NO: 46 and 94 respectively; (k) SEQ ID NO: 47 and 95 respectively; (l) SEQ ID NO: 48 and 96 respectively; (m) SEQ ID NO: 632 and 824 respectively; (n) SEQ ID NO: 765 and 957 respectively; (o) SEQ ID NOs: 770 and 962 respectively; and (p) SEQ ID NO: 38 and 86 respectively. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 632 and 824 respectively; (b) SEQ ID NO: 765 and 957 respectively; (c) SEQ ID NO: 770 and 962 respectively; and (d) SEQ ID NO: 38 and 86 respectively. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 174, and the antisense strand comprises SEQ ID NO: 188 the nucleotide sequence. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 175, and the antisense strand comprises SEQ ID NO: 189 the nucleotide sequence. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1005, and the antisense strand comprises SEQ ID NO: 1001 the nucleotide sequence. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1006, and the antisense strand comprises SEQ ID NO: 1002 the nucleotide sequence. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1007, and the antisense strand comprises SEQ ID NO: 1003 the nucleotide sequence. The oligonucleotide-ligand conjugate of any one of claims 98 to 135, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1008, and the antisense strand comprises SEQ ID NO: 189 the nucleotide sequence. A pharmaceutical composition comprising the oligonucleotide or oligonucleotide-ligand conjugate of any one of the preceding claims and a medically acceptable carrier, delivery agent or excipient. A kit comprising the oligonucleotide or oligonucleotide-ligand conjugate of any one of claims 1 to 144, a medically acceptable carrier if necessary, and instructions, the instructions Instructions for administration to a subject suffering from a disease, disorder or condition associated with expression of MC2R are included. A method of treating a subject suffering from a disease, disorder or condition associated with expression of MC2R, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or oligonucleotide of any one of claims 1 to 144 An acid-ligand conjugate or a pharmaceutical composition according to claim 145. The method of claim 147, wherein the disease, disorder or condition associated with manifestation of MC2R is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH , congenital adrenal insufficiency, and familial Addison's disease. The method of claim 147, wherein the disease, disorder or condition associated with MC2R expression is Cushing's syndrome. The method of claim 147, wherein the disease, disorder or condition associated with MC2R expression is congenital adrenal insufficiency. The method of any one of claims 147 to 149, wherein the oligonucleotide or pharmaceutical composition is administered together with a second composition or therapeutic agent. The method of claim 151, wherein the second composition or therapeutic agent is selected from the group consisting of: inhibitors of pituitary ACTH secretion, adrenal cortisol production or glucocorticoid effects on surrounding tissue, surgical procedures, radiation procedures, gene therapy, mifepristone, glucocorticoid receptor antagonists and competitive inhibitors of cortisol at this receptor, osilodrostat, metyrapone, CYP11B1 inhibitors, Pasireotide or combinations thereof. The method of any one of claims 147 to 152, wherein the oligonucleotide or pharmaceutical composition is administered together with at least 2 additional therapeutic agents. A method of treating Cushing's syndrome in a subject, comprising administering to the subject an oligonucleotide or oligonucleotide-ligand conjugate as in any one of claims 1 to 144 or an oligonucleotide-ligand conjugate as in claim 145 Pharmaceutical compositions, wherein the cells in the adrenal gland or adrenal cortex express MC2R. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide or oligonucleotide-ligand conjugate of any one of claims 1 to 144, wherein the oligonucleotide or oligonucleotide- Ligand conjugates reduce the performance, production or activity of MC2R. Use of an oligonucleotide or oligonucleotide-ligand conjugate according to any one of claims 1 to 144 or a pharmaceutical composition according to claim 145 for reducing MC2R expression in the adrenal gland. The oligonucleotide or oligonucleotide-ligand conjugate of any one of claims 1 to 144, for use in reducing MC2R expression in the adrenal gland of a subject. An oligonucleotide or oligonucleotide-ligand conjugate according to any one of claims 1 to 144 or a pharmaceutical composition according to claim 145 for reducing the risk of suffering from a disease, disorder or condition associated with MC2R expression The purpose of the MC2R representation of the object. The use of claim 158, wherein the disease, disorder or condition associated with manifestation of MC2R is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoid deficiency, hereditary adrenocortical unresponsiveness to ACTH , congenital adrenal insufficiency, and familial Addison's disease. The oligonucleotide or oligonucleotide-ligand conjugate of any one of claims 1 to 144 for reducing MC2R expression in a subject suffering from a disease, disorder or condition associated with MC2R expression . The oligonucleotide or oligonucleotide-ligand conjugate of claim 160, wherein the disease, disorder or condition associated with MC2R expression is selected from the group consisting of Cushing's syndrome, Cushing's disease, familial glucocorticoids The group consisting of hypochromia, hereditary adrenocortical unresponsiveness to ACTH, congenital adrenal hypoplasia, and familial Addison's disease. The oligonucleotide or oligonucleotide-ligand conjugate of claim 161, wherein the disease, disorder or condition associated with MC2R expression is congenital adrenal insufficiency.

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