Compositions and methods for inhibiting adenylate cyclase 9 (AC9)

Abstract

Disclosed is a method for reducing serum low-density lipoprotein (LDL) levels in a subject, the method comprising inhibiting the expression or function of adenylate cyclase in the subject, wherein the inhibiting comprises administering to the subject an inhibitory nucleic acid molecule, such as an siRNA. TIFF2025528102000060.tif128128

Description

[Technical Field]

[0001] The present disclosure relates to inhibitory nucleic acid molecules and compositions, and methods, for reducing low-density lipoprotein (LDL) and increasing LDL receptor (LDLr) in a subject. [Background technology]

[0002] background Adenylate cyclase (AC), also known as adenyl cyclase and adenylyl cyclase, is a regulatory enzyme that regulates signaling pathways and physiological responses in cells by converting adenosine triphosphate (ATP) into the important second messenger 3',5'-cyclic AMP (cAMP). Adenylate cyclase type 9 (AC9) is an atypical member of the membrane-bound AC family that is weakly activated by forskolin (Ostrom et al. (2022) Physiol. Rev. 102:815-857 (Non-Patent Document 1)), is autoinhibited by its C-terminal cytoplasmic domain (C2b) (Palvoelgyi et al. (2018) Cell Signal. 51:266-275 (Non-Patent Document 2)), and is internalized into cells after stimulation by G protein-coupled receptors (GPCRs) (Lazar et al. (2020) eLife 9:e58039 (Non-Patent Document 3)). Like other ACs, AC9 can form heterodimers with AC5 and AC6 (Baldwin et al. (2019) Mol. Pharmacol. 9:349-360 (Non-Patent Document 4)). Notably, expression of full-length AC9 blocks endogenous GPCR-associated stimulation of AC and cAMP production, whereas C-terminally truncated AC9 does not (Palvoelgyi et al. (2018) Cell Signal. 51:266-275 (Non-Patent Document 2)).

[0003] Throughout the body, low-density lipoproteins (LDL) transport fat molecules to cells via the bloodstream. LDL receptors (LDLr) on the surface of receptor cells can bind to LDL and internalize it, thereby reducing LDL levels in the blood. Excess LDL in the blood is associated with increased cardiovascular complications such as coronary heart disease, accounting for as much as one-quarter of all deaths in developed countries (Goldstein et al. (2015) Cell 161:161-172 (Non-Patent Document 5)). Therefore, controlling circulating LDL by regulating LDLr is of therapeutic interest.

[0004] Several mechanisms are involved in the regulation of LDLr expression. Downregulation of LDLr protein can be mediated by inducible degrader of LDLr (IDOL), which stimulates proteasomal degradation of LDLr (Zelcer et al. (2009) Science 325:100-104 (Non-Patent Document 6)). In addition, lysosomal degradation of LDLr protein can be stimulated by proprotein convertase subtilisin / kexin type 9 (PCSK9) (Park et al. (2004) J Biol Chem. 48:50630-50638 (Non-Patent Document 7)). Conversely, transcriptional upregulation of LDLr can be mediated by sterol regulatory element-binding protein-2 (SREBP-2), which can translocate to the cell nucleus and stimulate LDLr gene expression when intracellular cholesterol is low (Goldstein et al. (2015) Cell 161:161-172 (Non-Patent Document 5)). Ac-cAMP and protein kinase A (PKA) can further influence LDLr upregulation. For example, phosphodiesterase (PDE) inhibitors can induce SREBP2 nuclear translocation through a PKA-dependent mechanism (Shimizu-Albergine et al. (2013) Proc Natl Acad Sci., 113:E5685-5693 (Non-Patent Document 8)), thereby increasing LDLr transcription. Furthermore, a functional cAMP response element (CRE) present in the LDLr promoter can stimulate LDLr transcription (Liu et al. (2000) J. Biol. Chem. 275:5214-5221 (Non-Patent Document 9)).

[0005] There remains a need for compositions and methods that can reduce LDL and / or increase LDLr in a subject. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Ostrom et al. (2022)Physiol.Rev.102:815-857 [Non-patent document 2] Palvoelgyi et al.(2018)Cell Signal.51:266-275 [Non-patent document 3] Lazar et al.(2020)eLife 9:e58039 [Non-patent document 4] Baldwin et al.(2019)Mol.Pharmacol.9:349-360 [Non-patent document 5] Goldstein et al.(2015)Cell 161:161-172 [Non-patent document 6] Zelcer et al.(2009)Science 325:100-104 [Non-Patent Document 7] Park et al.(2004)J Biol Chem.48:50630-50638 [Non-patent document 8] Shimizu-Albergine et al. (2013) Proc Natl Acad Sci., 113:E5685-5693 [Non-Patent Document 9] Liu et al.(2000)J Biol Chem.275:5214-5221 Summary of the Invention

[0007] The present invention provides compositions and methods for reducing low-density lipoprotein (LDL) in the serum of a subject. Additionally, the present invention provides compositions and methods for increasing LDL receptors (LDLr) in a subject.

[0008] In a first aspect, the present invention provides a method for reducing serum low-density lipoprotein (LDL) levels in a subject, comprising inhibiting adenylate cyclase expression or function in the subject, wherein the inhibiting comprises administering to the subject an inhibitory nucleic acid molecule.

[0009] In a second aspect, the present invention provides a method for increasing LDL receptor expression in a subject, comprising inhibiting adenylate cyclase expression or function in the subject, wherein the inhibition comprises administering to the subject an inhibitory nucleic acid molecule.

[0010] In some aspects, the adenylate cyclase is adenylate cyclase type 9 (AC9).

[0011] In some aspects, AC9 comprises the mRNA sequence of SEQ ID NO:16; and / or the DNA sequence of SEQ ID NO:17.

[0012] In some aspects, the inhibitory nucleic acid molecule is an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a double-stranded RNA (dsRNA), or a microRNA (miRNA).

[0013] In some aspects, the inhibitory nucleic acid molecule is an siRNA.

[0014] In some aspects, the siRNA comprises a sequence complementary to at least 15 consecutive nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

[0015] In some aspects, the siRNA comprises a sequence complementary to at least 19 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

[0016] In some aspects, the siRNA comprises a sequence complementary to at least 21 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

[0017] In some aspects, the siRNA comprises a sequence complementary to at least 25 contiguous nucleotides set forth in any one of SEQ ID NOs:16 and 17.

[0018] In some aspects, the siRNA molecule comprises a 3' overhang, for example, a single uracil overhang at one or more of the 3' ends of the siRNA; a double uracil overhang at one or more of the 3' ends of the siRNA; a single thymine overhang at one or more of the 3' ends of the siRNA; a double thymine overhang at one or more of the 3' ends of the siRNA; or a single cytosine and a single thymine overhang at one or more of the 3' ends of the siRNA.

[0019] In some aspects, the siRNA comprises the nucleotide sequence of any one or more of SEQ ID NOs: 1-10.

[0020] In some aspects, the siRNA comprises a sense strand comprising the sequence of SEQ ID NO:1 and an antisense strand comprising the sequence of SEQ ID NO:2. In some aspects, the siRNA comprises a sense strand comprising the sequence of SEQ ID NO:3 and an antisense strand comprising the sequence of SEQ ID NO:4. In some aspects, the siRNA comprises a sense strand comprising the sequence of SEQ ID NO:5 and an antisense strand comprising the sequence of SEQ ID NO:6. In some aspects, the siRNA comprises a sense strand comprising the sequence of SEQ ID NO:7 and an antisense strand comprising the sequence of SEQ ID NO:8. In some aspects, the siRNA comprises a sense strand comprising the sequence of SEQ ID NO:9 and an antisense strand comprising the sequence of SEQ ID NO:10.

[0021] In some aspects, the siRNA comprises non-natural or modified nucleosides or nucleotides.

[0022] In some aspects, the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate internucleoside (PS) linkages, and 2'-fluoro (2'-F) modified nucleosides.

[0023] In some aspects, the siRNA molecule targets any one of SEQ ID NOs: 11-15.

[0024] In some aspects, some methods of the above aspects further include administering to the subject a second therapeutic agent.

[0025] In some aspects, the second therapeutic agent is selected from the group consisting of statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ATP citrate lyase (ACL) inhibitors, lipoprotein(a) (Lp(a)) inhibitors, angiopoietin-like 3 (ANGPTL3) inhibitors, cholesteryl ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, apolipoprotein B (ApoB) inhibitors, bile acid-binding resins, and colchicine.

[0026] In some aspects, the statin is atorvastatin.

[0027] In some aspects, the PCSK9 inhibitor is an siRNA molecule or a monoclonal antibody that targets PCSK9.

[0028] In some aspects, the ACL inhibitor is bempedoic acid.

[0029] In some aspects, the Lp(a) inhibitor is an siRNA molecule that targets Lp(a).

[0030] In some aspects, the MTP inhibitor is lomitapide.

[0031] In some aspects, the ApoB inhibitor is mipomersen.

[0032] In a third aspect, the present invention provides an siRNA molecule comprising a sense strand comprising the sequence of SEQ ID NO:3 and an antisense strand comprising the sequence of SEQ ID NO:4; a sense strand comprising the sequence of SEQ ID NO:5 and an antisense strand comprising the sequence of SEQ ID NO:6; a sense strand comprising the sequence of SEQ ID NO:7 and an antisense strand comprising the sequence of SEQ ID NO:8; or a sense strand comprising the sequence of SEQ ID NO:9 and an antisense strand comprising the sequence of SEQ ID NO:10.

[0033] In some aspects, the siRNA comprises non-natural or modified nucleosides or nucleotides.

[0034] In some aspects, the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate internucleoside (PS) linkages, and 2'-fluoro (2'-F) modified nucleosides.

[0035] In some aspects, the siRNA molecule targets any one of SEQ ID NOs: 11-15. [Brief explanation of the drawings]

[0036] The accompanying drawings are included to illustrate aspects of the present disclosure and to provide a further understanding of its practice.

[0037] [Figure 1-1]Figure 1A shows Western blots of AC9, LDLr, and ABCA1 protein levels after siRNA-mediated knockdown of AC9 in HepG2 cells compared with the siScramble control. Actin is shown as a loading control. Figure 1B is a graph showing quantification of AC9, LDLr, and ABCA1 protein levels (left bars for AC9, LDLr, and ABCA1, respectively) after siRNA-mediated knockdown of AC9 in HepG2 cells relative to the siScramble control (right bars for AC9, LDLr, and ABCA1, respectively). Briefly, densitometry data from the Western blot in Figure 1A were calculated for AC9, LDLr, and ABCA1 and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of the siScramble control. Error bars represent the mean ± standard deviation. n = 4–6. Paired t-test: *=p≦0.05; **=p≦0.01; and ***=p≦0.001 versus siScramble. [Figure 1-2]Figure 1C is a graph showing quantification of 3H-CE-LDL association after siRNA-mediated knockdown of AC9 in HepG2 cells compared with siScramble control. Briefly, human LDL labeled with 3H-cholesteryl oleate (CE) was incubated with HepG2 cells at 20 μg protein / ml for 4 hours. Finally, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter and normalized to cellular protein estimated by Lowry assay. Error bars represent mean ± standard deviation. n = 5. Paired t-test: * = p ≤ 0.05 vs. siScramble. Figure 1D is a graph showing quantification of 3H-CE-LDL cholesterol efflux after siRNA-mediated knockdown of AC9 in HepG2 cells. Briefly, transfected HepG2 cells were loaded with 3H-cholesterol for 24 hours and allowed to equilibrate for 18 hours. Cholesterol efflux (4 h) to apoA-I at 10 μg / ml was then performed. This dose of apoA-I is considered to be saturating for the ABCA1 transporter. Finally, the medium was collected, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated in a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the medium by the radioactivity measured in the cells and medium. Error bars represent the mean ± standard deviation. n = 4. Paired t-test: * = p ≤ 0.05 vs. siScramble. [Figure 2]Figure 2A is a graph showing quantification of AC9, LDLr, SREBP2, and ABCA1 protein levels by untargeted relative proteomics after siRNA-mediated knockdown of AC9 in HepG2 cells compared to siScramble control. Error bars represent mean ± standard deviation. n = 4. Paired t-test: * = p < 0.05; and *** = p < 0.001 vs. siScramble. Figure 2B is a graph showing quantification of PRKAR1A, PRKAR1B, and AKAP12 protein levels by untargeted relative proteomics after siRNA-mediated knockdown of AC9 in HepG2 cells compared to siScramble control. Error bars represent mean ± standard deviation. n = 4. Paired t-test: * = p < 0.05; and *** = p < 0.001 vs. siScramble. [Figure 3] This graph shows the quantification of 3H-CE-LDL association in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 5 μM atorvastatin (Ato) 24 hours before cholesterol association assay. Briefly, 3H-cholesteryl oleate (CE)-labeled human LDL was incubated with HepG2 cells at 20 μg protein / ml for 4 hours. Finally, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter and normalized to cellular protein estimated by Lowry assay. For both control and Ato, the left bar represents siScramble, and the right bar represents siAC9. These results demonstrate the additive effect of AC9 siRNA and atorvastatin on LDL cholesterol uptake by the LDLr. Error bars represent the mean ± standard deviation. n=5. Repeated measures ANOVA with uncorrected Fisher's LSD: *=p≦0.05 vs. siScramble; a=p≦0.05 vs. control. [Figure 4]Figure 1 shows the quantification of LDLr protein in HepG2 cells treated with 300 μM exogenous cAMP, 5 μM atorvastatin (Ato), or both cAMP and Ato. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of the control without Ato. For both without Ato and with Ato, the left bar is control and the right bar is cAMP. These results demonstrate the combined effect of exogenous cAMP and atorvastatin to increase LDLr protein. Error bars represent the mean ± standard deviation. n=5. Paired t-test: *=p≦0.05 vs. control; a=p≦0.05 vs. "without Ato." [Figure 5-1]Figure 5A is a graph showing quantification of LDLr protein expression in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (a PKA inhibitor) 24 hours before cellular protein extraction. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of the siScramble control. For both siScramble and siAC9, the left bar is control and the right bar is H89. These results indicate that the increase in LDLr caused by AC9 siRNA is primarily PKA-dependent. Error bars represent the mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05 vs. siScramble-control; b = p ≤ 0.05 vs. siAC9-control. Figure 5B is a graph showing quantification of ABCA1 protein expression in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (a PKA inhibitor) 24 h prior to cellular protein extraction. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of the siScramble control. For both siScrmable and siAC9, the left bar is the control and the right bar is H89. These results indicate that the increase in ABCA1 due to AC9 siRNA is primarily PKA-dependent. Error bars represent the mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05 vs. siScramble-control. [Figure 5-2]Figure 5C is a graph showing the quantification of H-CE-LDL association in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (a PKA inhibitor) 24 h prior to the cholesterol association assay. Briefly, H-cholesteryl oleate (CE)-labeled human LDL was incubated with HepG2 cells at 20 μg protein / ml for 4 h. This dose of H-CE-LDL is considered to be saturating with respect to the LDLr transporter. Finally, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter and normalized to cellular protein estimated by Lowry assay. For both siScramble and siAC9, the left bar represents the control, and the right bar represents H89. These results indicate that the increase in LDL uptake caused by AC9 siRNA is primarily PKA-dependent. Error bars represent the mean ± standard deviation. n = 3. Paired t-test: a = p ≤ 0.05 vs. siScramble-control. Figure 5D is a graph showing the quantification of cholesterol efflux in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (a PKA inhibitor) 24 h prior to the cholesterol efflux assay. Briefly, treated HepG2 cells were loaded with 3H-cholesterol, equilibrated, and used for cholesterol efflux (4 h) to apoA-I at 10 μg / ml. For both siScramble and siAC9, the left bar represents the control, and the right bar represents H89. This dose of apoA-I is considered to be saturating for the ABCA1 transporter. Finally, the medium was collected, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the medium by the radioactivity measured in the cells and medium. Error bars represent the mean ± standard deviation. n = 4. Paired t-test: a = p < 0.05 vs. siScramble-control. [Figure 6]Figure 1 shows the quantification of AC9, SREBP2, PCSK9, and LDLr mRNA expression by quantitative PCR in HepG2 cells 48 hours after transfection with AC9 siRNA (siAC9) compared to the siScramble control. These results indicate that AC9 siRNA increases mRNA expression of SREBP2 and LDLR, but not PCSK9. For AC9, SREBP2, LDLr, and PCSK9, the left bar is siScramble and the right bar is siAC9. Error bars represent the mean ± standard deviation. n = 6. Paired t-test: * = p ≤ 0.05 and *** = p ≤ 0.001 versus the siScramble control. [Figure 7] This graph shows the quantification of SREBP2 transcriptional activity in HepG2 cells 24 and 48 hours after treatment with AC9 siRNA (siAC9) compared with the siScramble control. Briefly, SREBP-2 transcriptional activity was estimated using a kit in which SREBP-2 contained in nuclear extracts specifically binds to immobilized SREBP response elements and is detected by adding a specific primary antibody against SREBP-2. For each time point, the left bar represents siScramble, and the right bar represents siAC9. These results show that AC9 siRNA increases SREBP2 transcriptional activity. Error bars represent the mean ± standard deviation. n = 5. Paired t-test: * = p ≤ 0.05 and ** = p ≤ 0.01 versus siScramble. [Figure 8]Figure 1 shows the quantification of LDLr protein expression in HepG2 cells treated with AC9 siRNA (siAC9), SREBP2 siRNA (siSREBP2), or both siAC9 and siSREBP2 relative to the siScramble control. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of the siScramble control. These results demonstrate that SREBP2 siRNA can block the increase in LDLr protein caused by AC9 siRNA. Error bars represent the mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05 for siScramble; b = p ≤ 0.05 for siAC9. [Figure 9]Figure 9A is a graph showing the quantification of cholesterol efflux to apoA1 in HepG2 cells treated with AC9 siRNA (siAC9), SREBP2 siRNA (siSREBP2), or both siAC9 and siSREBP2; siScramble served as a control. Briefly, transfected HepG2 cells were loaded with 3H-cholesterol, equilibrated, and used for cholesterol efflux to apoA1 at 10 μg / ml for 4 h. This dose of apoA1 is considered to be saturating for the ABCA1 transporter. Finally, the medium was collected, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the medium by the radioactivity measured in the cells and medium. These results indicate that SREBP2 is involved in the effect of AC9 siRNA on ABCA1-mediated cholesterol efflux. Error bars represent the mean ± standard deviation. n = 6. Repeated-measures ANOVA with uncorrected Fisher's LSD: a = p ≤ 0.05 for siScramble; b = p ≤ 0.05 for siAC9. Figure 9B shows a graph showing the quantification of cholesterol efflux to apoA1 in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 5 μM GSK-2033 (an LXR inhibitor) 24 h prior to the cholesterol efflux assay; siScramble served as a control. Briefly, transfected HepG2 cells were loaded with 3H-cholesterol, equilibrated, and used for cholesterol efflux to apoA1 at 10 μg / ml (4 h). This dose of apoA1 is considered to be saturating for the ABCA1 transporter. Finally, the medium was collected, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter. Percent cholesterol efflux was calculated by dividing the radioactivity in the medium by the radioactivity measured in the cells and medium. For both control and GSK-2033, the left bar is siScramble and the right bar is siAC9.These results indicate that LXR is involved in the effect of AC9 siRNA on ABCA1-mediated cholesterol efflux. Error bars represent the mean ± standard deviation. n = 6. Repeated measures ANOVA with uncorrected Fisher's LSD: a = p ≤ 0.05 vs. siScramble-control; b = p ≤ 0.05 vs. siAC9-control. [Figure 10]Figure 10A is a graph showing quantification of ABCA1 protein expression in HepG2 cells treated with AC9 siRNA (siAC9), LDLr siRNA (siLDLr), or both siAC9 and siLDLr relative to the siScramble control. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data for the actin loading control. Protein expression is expressed as a percentage of siScramble. These results show that LDLr siRNA blocks the increase in ABCA1 protein caused by AC9 siRNA. Error bars represent the mean ± standard deviation. n = 4. Repeated measures ANOVA with uncorrected Fisher's LSD: a = p < 0.05 for siScramble; b = p < 0.05 for siAC9. Figure 10B is a graph showing quantification of cholesterol efflux to apoA-I in HepG2 cells treated with AC9 siRNA (siAC9), LDLr siRNA (siLDLr), or both siAC9 and siLDLr; siScramble served as a control. Briefly, transfected HepG2 cells were loaded with 3H-cholesterol, equilibrated, and used for cholesterol efflux to apoA-I at 10 μg / ml for 4 h. This dose of apoA-I is considered to be saturating for the ABCA1 transporter. Finally, the medium was collected, cells were solubilized in 0.2 N NaOH, and radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the medium by the radioactivity measured in the cells and medium. These results demonstrate that LDLr siRNA blocks the increase in cholesterol efflux caused by AC9 siRNA. Error bars represent the mean ± standard deviation. n = 13. Repeated measures ANOVA with uncorrected Fisher's LSD: a = p ≤ 0.05 for siScramble; b = p ≤ 0.05 for siAC9. [Figure 11]This diagram shows the mechanism of AC9 knockdown effects on LDLr and ABCA1 expression and function. Briefly, reduced AC9 expression on proteins leads to elevated cellular cAMP levels due to the elimination of AC9's known inhibitory properties on other family members. This, in turn, leads to PKA activation and target phosphorylation. One of these is SREBP-2, whose activity is increased through increased expression and PKA-mediated phosphorylation. SREBP-2 is a key transcription factor responsible for increased LDLr mRNA expression. This leads to higher LDLr protein levels, which are associated with enhanced LDL particle uptake. When LDL particles are degraded in lysosomes, cellular cholesterol is concentrated, leading to the activation of LXR activity. While both SREBP-2 and LXR can increase ABCA1 mRNA expression, PKA activation is associated with ABCA1 phosphorylation. Overall, this leads to higher ACBA1 protein levels and cholesterol efflux to apoA-I as a donor, a homeostatic physiological response to higher levels of cellular cholesterol. Therefore, AC9 knockdown may result in higher LDL particle uptake, which may lead to lower LDL-cholesterol levels in the blood. DETAILED DESCRIPTION OF THE INVENTION

[0038] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or incidental definitions. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. The use of "or" means "and / or" unless expressly stated otherwise. The use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0039] As used herein, the term "about," when applied to one or more values, unless otherwise specified or apparent from the context, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated reference value (except where such number would exceed 100% of the possible values).

[0040] As used herein, "administration" refers to providing or giving a therapeutic agent to a subject by any effective route. Exemplary routes of administration are described herein below.

[0041] As used herein, the terms "administered in combination" or "administration in combination" mean that two or more agents are administered to a subject simultaneously or within such intervals that there may be an overlap in the effect of each agent on the patient. In some embodiments, the agents are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the administration of the agents is spaced sufficiently close to each other so that a combined (e.g., synergistic) effect is achieved.

[0042] As used herein, the term "auxiliary moiety" refers to any moiety that can be conjugated to a nucleic acid molecule, including, but not limited to, small molecules, peptides, carbohydrates, neutral organic polymers, positively charged polymers, therapeutic agents, targeting moieties, endosomal escape moieties, and any combination thereof. In some embodiments, an "auxiliary moiety" is linked to an inhibitory nucleic acid molecule disclosed herein by forming one or more covalent or non-covalent bonds with one or more conjugate groups attached to a phosphate bond, a phosphorothioate bond, the 5' position of a nucleotide sugar, or any portion of a nucleobase. Those skilled in the art will readily recognize suitable attachment points for a particular auxiliary moiety to a nucleic acid molecule.

[0043] As used herein, "delivery vehicle" refers to any substance (eg, molecules, peptides, conjugates, and constructs) that at least partially facilitates the in vivo delivery of a nucleic acid molecule to a target cell.

[0044] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of the compositions described herein refer to an amount sufficient to produce a beneficial or desired result when administered to a subject; therefore, "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of reducing low-density lipoprotein (LDL), the term refers to the amount of the composition sufficient to achieve this therapeutic response compared to the response obtained without administering the composition. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors (e.g., a given drug, pharmaceutical composition, route of administration, type of disease or disorder, subject identity (e.g., age, sex, weight), or host being treated, and the like), but can nevertheless be routinely determined by one of ordinary skill in the art.

[0045] As used herein, a "formulation" includes at least an inhibitory nucleic acid molecule and a delivery vehicle.

[0046] As used herein, the term "in vitro" refers to events that take place in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.) rather than within a living organism (e.g., an animal, plant, or microorganism).

[0047] As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0048] As used herein, the term "inhibitory nucleic acid molecule" refers to a nucleic acid molecule that has sufficient complementarity to bind to a target nucleic acid molecule and inhibit the expression of the protein encoded by the target nucleic acid molecule. Exemplary inhibitory nucleic acid molecules are antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), double-stranded RNAs (dsRNAs), and microRNAs (miRNAs). An inhibitory nucleic acid molecule can reduce target protein expression by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more). In one embodiment, the target nucleic acid molecule encodes AC9.

[0049] As used herein, "modified" refers to a change in the state or structure of a nucleic acid molecule as described herein. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one aspect, the inhibitory nucleic acid molecules of the invention are modified by the introduction of non-natural nucleosides and / or nucleotides. In another aspect, the inhibitory nucleic acid molecules of the invention are modified by the conjugation of auxiliary moieties.

[0050] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent (optionally combined with one or more pharmaceutically acceptable excipients, diluents, and / or carriers) that is administered to a subject, such as a mammal (e.g., a human), to prevent, treat, or control a particular disease or condition that is affecting or may affect the subject.

[0051] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0052] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the full length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an example, the percent sequence identity of a given nucleic acid or amino acid sequence A to a given nucleic acid or amino acid sequence B, the percent sequence identity between a given nucleic acid or amino acid sequence A and a given nucleic acid or amino acid sequence B, or the percent sequence identity of a given nucleic acid or amino acid sequence A against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored by a sequence alignment program (e.g., BLAST) as identical matches in A and B in the program's alignment, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0053] As used herein, the term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0054] As used herein, "treatment" and "treating," in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results (e.g., clinical results). Beneficial or desired results may include, but are not limited to, the following, whether detectable or undetectable: alleviation or amelioration of one or more symptoms or conditions; reduction in the extent of the disease or condition; a stabilized (i.e., not worsening) state of the disease, disorder, or condition; prevention of the spread of the disease or condition; delay or slowing of the progression of the disease or condition; recovery or palliation of the disease or condition; and remission (partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0055] As used herein, the term "vector" refers to a replicon (e.g., a plasmid, phage, viral construct, or cosmid) to which another nucleic acid (e.g., DNA or RNA) segment may be attached. Vectors are used to transduce and express nucleic acid segments in cells.

[0056] Detailed Description Described herein are compositions (e.g., inhibitory nucleic acid molecules) and methods for reducing low-density lipoprotein (LDL) in the serum of a subject. Additionally, the present invention provides compositions (e.g., inhibitory nucleic acid molecules) and methods for increasing LDL receptor (LDLr) expression in a subject.

[0057] The inhibitory nucleic acid molecules described herein (e.g., small interfering RNA (siRNA), double-stranded RNA (dsRNA), antisense oligonucleotide (ASO), microRNA (miRNA), or short hairpin RNA (shRNA)) or compositions thereof can be used in methods for reducing the expression of adenylate cyclase 9 (AC9). Advantageously, the methods of the present disclosure provide an effective mechanism for reducing LDL and / or increasing LDLr in a subject. In doing so, the methods are useful for reducing LDL concentrations in the blood (e.g., serum) of a subject.

[0058] inhibitory nucleic acid molecule Representative inhibitory nucleic acid molecules of the present disclosure are siRNA, dsRNA, ASO, miRNA, and shRNA, although any nucleic acid molecule capable of reducing AC9 mRNA and / or protein expression is contemplated for use in the methods described herein. In some instances, the inhibitory nucleic acid molecules of the present disclosure may be referred to as RNA inhibitory (RNAi) molecules.

[0059] With respect to any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, shRNA, or other inhibitory nucleic acid molecules capable of reducing expression of a target gene), the inhibitory nucleic acid molecule comprises at least some sequence complementarity to the nucleotide sequence of SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 16 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 17 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 18 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 19 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 20 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 21 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 22 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 23 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 24 contiguous nucleotides set forth in SEQ ID NO:16.In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 25 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 26 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 27 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 28 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 29 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 30 contiguous nucleotides set forth in SEQ ID NO:16.

[0060] In some embodiments, the inhibitory nucleic acid is an siRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is a dsRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an ASO targeting AC9. In some embodiments, the inhibitory nucleic acid is an miRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an shRNA targeting AC9. Each of these modalities is further described below.

[0061] Small interfering RNA (siRNA) The siRNA of the present disclosure is a single-stranded (ss) or double-stranded (ds) nucleic acid molecule, which is made of DNA, RNA, or both DNA and RNA (for example, chimera), and is complementary to the target gene of interest and prevents the translation of target mRNA into protein.When siRNA molecule enters cells, it is incorporated into RNA-induced silencing complex (RISC).When siRNA is hybridized to target mRNA, RISC complex cuts target mRNA, thereby inactivating target mRNA, resulting in the reduction of target mRNA level and protein level.

[0062] In some embodiments, the siRNA of the present disclosure may comprise a nucleotide sequence of about 10 to about 30 nucleotides in length (e.g., 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or 31 nucleotides in length).

[0063] In some embodiments, the siRNA of the present disclosure may comprise a nucleotide sequence that is 10 to 30 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0064] It is within the scope of this disclosure that any length known in the art and any previously unknown length may be utilized in the present invention.

[0065] In some embodiments, the siRNA comprises an antisense strand. In some embodiments, the length of the antisense strand of an siRNA molecule of the present disclosure is 10 to 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 17 nucleotides. In some embodiments, the antisense strand is 18 nucleotides. In some embodiments, the antisense strand is 19 nucleotides. In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides.In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0066] In some embodiments, the siRNA comprises a sense strand. In some embodiments, the sense strand of an siRNA molecule of the present disclosure is 10 to 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 to 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

[0067] In some embodiments, the sense strand and antisense strand of the siRNA molecule of the present disclosure are completely complementary.In some embodiments, the sense strand and antisense strand of the siRNA molecule of the present disclosure are completely complementary as long as their lengths overlap each other.Depending on the sequence of the first and second strand, complementarity does not need to be complete or perfect, which means that the first and second strands are not 100% base-paired due to mismatch.One or more mismatches can exist in ds siRNA without affecting the ability of siRNA to reduce the expression of target gene of interest.

[0068] The nucleotide sequence of the siRNA of the present disclosure can comprise sufficient complementarity to a part of the target gene of interest (for example, AC9 mRNA), so that the siRNA can hybridize with the target gene of interest.In some embodiments, the siRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (for example, AC9 mRNA) or a part thereof.In some embodiments, the siRNA is complementary to the target gene of interest (for example, AC9 mRNA) or a part thereof.

[0069] In some embodiments, the nucleotide sequence of the siRNA may comprise sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of AC9). In some embodiments, the nucleotide sequence of the siRNA may comprise sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the siRNA of the present disclosure may comprise sufficient complementarity to a pre-mRNA transcript or mRNA transcript encoding AC9. The target sequence of interest may be any one of SEQ ID NOs: 11-15 (see, e.g., Table 2). The target gene of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (see, e.g., Table 3).

[0070] In some embodiments, the siRNA described herein has a 3' overhang of 0 to 7 nucleotides or a 5' overhang of 0 to 4 nucleotides. In some embodiments, the siRNA molecule has a single uracil (e.g., U) overhang at each 3' end of the siRNA. In some embodiments, the siRNA molecule has a double uracil (e.g., UU) overhang at each 3' end of the siRNA. In some embodiments, the siRNA molecule has a single thymine (e.g., T) overhang at each 3' end of the siRNA. In some embodiments, the siRNA molecule has a double thymine (e.g., TT) overhang at each 3' end of the siRNA. In some embodiments, the siRNA molecule has a cytosine and a thymine (e.g., CT) overhang at each 3' end of the siRNA.

[0071] Various siRNAs may be combined to reduce the protein expression of a target gene of interest (e.g., AC9). The methods of the present invention may use a combination of two siRNAs, for example, two, three, four, or five different siRNAs targeting the same gene of interest (e.g., AC9 or a mutant thereof). In some embodiments, the siRNA sequence may comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one or more of SEQ ID NOs: 1-10 (see, e.g., Table 1), or a complementary sequence thereof. In some embodiments, the siRNA sequence may comprise any one or more of SEQ ID NOs: 1-10 (see, e.g., Table 1), or a complementary sequence thereof.

[0072] In some embodiments, the siRNA comprises at least 15 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises at least 16 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises at least 17 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises at least 18 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises at least 19 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises at least 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, e.g., Table 1). In some embodiments, the siRNA comprises 21 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-10 (see, eg, Table 1).

[0073] [Table 1]

[0074] In some embodiments, the siRNA of the present disclosure may target the nucleotide sequence of any one of SEQ ID NOs:11-15 (see, e.g., Table 2), or a complementary sequence thereof, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% variants thereof.

[0075] [Table 2]

[0076] In some embodiments, the siRNA comprises a sequence complementary to at least 15 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 16 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 17 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 18 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 19 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 20 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 21 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 22 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 23 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 24 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 25 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 26 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 27 contiguous nucleotides set forth in SEQ ID NO:16. In some embodiments, the siRNA comprises a sequence complementary to at least 28 contiguous nucleotides set forth in SEQ ID NO:16.In some embodiments, the siRNA comprises a sequence complementary to at least 29 contiguous nucleotides set forth in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence complementary to at least 30 contiguous nucleotides set forth in SEQ ID NO: 16. The nucleotide sequence of SEQ ID NO: 16 is shown in Table 3.

[0077] In some embodiments, the siRNA comprises a sequence complementary to at least 15 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 17 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 17 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 18 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 19 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 20 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 21 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 22 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 23 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 24 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 25 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 26 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 27 contiguous nucleotides set forth in SEQ ID NO:17. In some embodiments, the siRNA comprises a sequence complementary to at least 28 contiguous nucleotides set forth in SEQ ID NO:17.In some embodiments, the siRNA comprises a sequence complementary to at least 29 contiguous nucleotides set forth in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence complementary to at least 30 contiguous nucleotides set forth in SEQ ID NO: 17. The nucleotide sequence of SEQ ID NO: 17 is shown in Table 3.

[0078] [Table 3] TIFF2025528102000005.tif221170TIFF2025528102000006.tif223170TIFF2025528102000007.tif227170TIFF2025528102000008.tif227170TIFF2025528102000009.tif222170TIFF2025528102000010.tif226170TIFF2025528102000011.tif224170TIFF2025528102000012.tif222170TIFF2025528102000013.tif222170TIFF2025528102000014.tif224170TIFF2025528102000015.tif225170TIFF2025528102000016.tif226170TIFF2025528102000017.tif225170TIFF2025528102000018.tif225170TIFF2025528102000019.tif224170TIFF2025528102000020.tif221170TIFF2025528102000021.tif227170TIFF2025528102000022.tif233170TIFF2025528102000023.tif226170TIFF2025528102000024.tif222170TIFF2025528102000025.tif223170TIFF2025528102000026.tif224170TIFF2025528102000027.tif220170TIFF2025528102000028.tif226170TIFF2025528102000029.tif227170TIFF2025528102000030.tif222170TIFF2025528102000031.tif227170TIFF2025528102000032.tif225170TIFF2025528102000033.tif224170TIFF2025528102000034.tif223170TIFF2025528102000035.tif223170TIFF2025528102000036.tif224170TIFF2025528102000037.tif224170TIFF2025528102000038.tif224170TIFF2025528102000039.tif224170TIFF2025528 102000040.tif226170TIFF2025528102000041.tif227170TIFF2025528102000042.tif222170T IFF2025528102000043.tif224170TIFF2025528102000044.tif221170TIFF2025528102000045. tif223170TIFF2025528102000046.tif223170TIFF2025528102000047.tif226170TIFF2025528 102000048.tif223170TIFF2025528102000049.tif223170TIFF2025528102000050.tif224170T IFF2025528102000051.tif220170TIFF2025528102000052.tif224170TIFF2025528102000053. tif229170TIFF2025528102000054.tif225170TIFF2025528102000055.tif221170TIFF2025528 102000056.tif221170TIFF2025528102000057.tif222170TIFF2025528102000058.tif178170.

[0079] double-stranded RNA (dsRNA) The dsRNA of the present disclosure is a ds nucleic acid molecule made of DNA, RNA, or both DNA and RNA (for example, chimera), which is complementary to the target gene of interest and prevents the translation of target mRNA into protein.Usually, dsRNA is longer than siRNA, and is processed in cells to form siRNA molecules.Then, siRNA is incorporated into RNA-induced silencing complex (RISC).When siRNA is hybridized with target mRNA, RISC complex cuts target mRNA, thereby inactivating target mRNA, resulting in the reduction of target mRNA level and protein level.

[0080] In some embodiments, dsRNAs of the present disclosure can comprise a sense strand and an antisense strand, each of which can be about 25 to about 5000 nucleotides in length, or more nucleotides in length (e.g., 20, about 25, 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 100, about 105, about 110, about 11 5, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 380, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500 1000, about 1600, about 1700, about 1800, about 1900, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, about 5000, about 6000, about 7000, about 8000, about 9000, or about 10000 nucleotides in length).

[0081] In some embodiments, dsRNAs of the disclosure can comprise a sense strand and an antisense strand, each of which can be 25 to 5000 nucleotides in length, or more (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 380, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600 600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length).

[0082] It is within the scope of this disclosure that any length known in the art and any previously unknown length may be utilized in the present invention.

[0083] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the present disclosure are completely complementary.In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the present disclosure are completely complementary as long as their lengths overlap each other.Depending on the sequence of the first and second strand, complementarity does not need to be complete or perfect, which means that the first and second strands are not 100% base-paired due to mismatch.One or more mismatches can exist in ds dsRNA without affecting the ability of dsRNA to reduce the expression of target gene of interest.

[0084] The nucleotide sequence of dsRNA of the present disclosure can comprise sufficient complementarity to a part of the target gene of interest (for example, AC9 mRNA), so that this dsRNA can hybridize with this target gene of interest.In some embodiments, this dsRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (for example, AC9 mRNA) or a part thereof.In some embodiments, this dsRNA is complementary to the target gene of interest (for example, AC9 mRNA) or a part thereof.

[0085] In some embodiments, the nucleotide sequence of the dsRNA may comprise sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of AC9). In some embodiments, the nucleotide sequence of the dsRNA may comprise sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the dsRNA of the present disclosure may comprise sufficient complementarity to a pre-mRNA transcript or mRNA transcript encoding AC9. The target sequence of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (see, e.g., Table 3).

[0086] Various dsRNAs can be combined to reduce the protein expression of target gene of interest (for example, AC9).In the method of the present invention, the combination of two dsRNAs can be used, for example, two different dsRNAs, three different dsRNAs, four different dsRNAs, or five different dsRNAs can be used that target the same gene of interest (for example, AC9 or its mutant).

[0087] Antisense oligonucleotides (ASOs) The ASO of the present disclosure is a single (ss) nucleic acid molecule made of DNA, RNA, or both DNA and RNA (e.g., chimera) that is complementary to a target gene of interest and prevents translation of the target mRNA into protein. Upon hybridization to the target mRNA, RNase H degrades the mRNA by hydrolysis, resulting in a decrease in the target mRNA and protein levels.

[0088] In some embodiments, the ASOs of the present disclosure may comprise a nucleotide sequence that is 12 to 50 nucleotides in length (e.g., 11, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length).

[0089] In some embodiments, the ASOs of the present disclosure may comprise a nucleotide sequence that is 12 to 50 nucleotides in length (e.g., 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0090] It is within the scope of this disclosure that any length known in the art and any previously unknown length may be utilized in the present invention.

[0091] The nucleotide sequence of the ASO may contain sufficient complementarity to a portion of the target gene of interest (e.g., AC9 mRNA) so that the ASO can hybridize to the target gene of interest. In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof. In some embodiments, the ASO is complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof.

[0092] In some embodiments, the nucleotide sequence of the ASO may comprise sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of AC9). In some embodiments, the nucleotide sequence of the ASO may comprise sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, an ASO of the present disclosure may comprise sufficient complementarity to a pre-mRNA transcript or mRNA transcript encoding AC9. The target gene of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (see, e.g., Table 3).

[0093] Various ASOs can be combined to reduce the protein expression of a target gene of interest (for example, AC9). In the method of the present invention, a combination of two ASOs can be used, for example, two, three, four, or five different ASOs that target the same gene of interest (for example, AC9 or its mutants).

[0094] MicroRNA (miRNA) The miRNA of the present disclosure is a single-stranded (ss) nucleic acid molecule made of DNA, RNA, or both DNA and RNA (for example, chimera), which is complementary to the target gene of interest and prevents the translation of target mRNA into protein.When miRNA molecule enters cells, it is incorporated into RNA-induced silencing complex (RISC).When miRNA hybridizes to target mRNA, RISC complex cuts target mRNA, thereby inactivating target mRNA, resulting in a decrease in target mRNA level and protein level.

[0095] In some embodiments, miRNAs of the present disclosure can comprise a nucleotide sequence between 6 and 30 nucleotides in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length).

[0096] In some embodiments, miRNAs of the present disclosure can comprise a nucleotide sequence between 6 and 30 nucleotides in length (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0097] The nucleotide sequence of the miRNA may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9 mRNA) so that the miRNA can hybridize to the target gene of interest. In some embodiments, the miRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof. In some embodiments, the miRNA is complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof.

[0098] In some embodiments, the nucleotide sequence of the miRNA may comprise sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of AC9). In some embodiments, the nucleotide sequence of the miRNA may comprise sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the miRNA of the present disclosure may comprise sufficient complementarity to a pre-mRNA transcript or mRNA transcript encoding AC9. The target sequence of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (see, e.g., Table 3).

[0099] Various miRNAs can be combined to reduce the protein expression of a target gene of interest (e.g., AC9). The method of the present invention can use a combination of two or more miRNAs, for example, two, three, four, or five different miRNAs that target the same gene of interest (e.g., AC9 or its variant).

[0100] Short hairpin RNA (shRNA) The shRNA of the present disclosure is a ss or ds nucleic acid molecule made of DNA, RNA, or both DNA and RNA (for example, chimera), which is complementary to the target gene of interest and prevents the translation of target mRNA into protein.When shRNA molecule enters cells, it is incorporated into RNA-induced silencing complex (RISC).When shRNA hybridizes to target mRNA, RISC complex cuts target mRNA, thereby inactivating target mRNA, resulting in the reduction of target mRNA level and protein level.

[0101] In some embodiments, shRNAs of the present disclosure can comprise a nucleotide sequence that is 60 to 100 nucleotides in length (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 nucleotides in length).

[0102] In some embodiments, shRNAs of the present disclosure can comprise a nucleotide sequence that is 60 to 100 nucleotides in length (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length).

[0103] The shRNAs of the present disclosure comprise variable hairpin loop structures and stem sequences. In some embodiments, the stem sequence can be 10 to 50 nucleotides in length (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length). In some embodiments, the hairpin size is 4 to 50 nucleotides in length, but the loop size can be larger without significantly affecting silencing activity. The shRNA molecules of the present disclosure can contain mismatches without reducing efficacy, for example, GU mismatches between the two strands of the shRNA stem. In some embodiments, shRNAs are designed to contain one or several GU pairings in the hairpin stem to stabilize the hairpin, for example, during propagation in bacteria.

[0104] The nucleotide sequence of the shRNA can comprise sufficient complementarity to a portion of the target gene of interest (e.g., AC9 mRNA) so that the shRNA can hybridize with the target gene of interest. In some embodiments, the shRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof. In some embodiments, the shRNA is complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof.

[0105] In some embodiments, the nucleotide sequence of the shRNA may comprise sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of AC9). In some embodiments, the nucleotide sequence of the shRNA may comprise sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the shRNA of the present disclosure may comprise sufficient complementarity to a pre-mRNA transcript or mRNA transcript encoding AC9. The target sequence of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (see, e.g., Table 3).

[0106] Various shRNAs can be combined to reduce the protein expression of target gene of interest (for example, AC9).In the method of the present invention, the combination of two or more shRNAs can be used, for example, two different shRNAs, three different shRNAs, four different shRNAs, or five different shRNAs can be used that target the same gene of interest (for example, AC9 or its mutant).

[0107] Modifications to inhibitory nucleic acid molecules It is contemplated that any of the inhibitory nucleic acid molecules disclosed herein can be used in the methods disclosed herein in unmodified or modified form. Unmodified inhibitory nucleic acid molecules contain nucleobases that include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below.

[0108] Modifications can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

[0109] Modifications can be achieved, for example, by incorporating one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative internucleoside linkages, which are further described below. Typically, these types of modifications are introduced to optimize the efficacy or biophysical properties of the molecule (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, reduced immunogenicity, and / or targeting to a particular location or cell type).

[0110] Modification can further be achieved by covalently or non-covalently conjugating a moiety (e.g., a targeting moiety, a hydrophobic moiety, a cell-penetrating peptide, or a polymer) to the 5' and / or 3' ends of the inhibitory nucleic acid molecule, as described in more detail below.

[0111] Nucleoside Modifications Modifications of the inhibitory nucleic acid molecules described herein include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkyl derivatives of pyrimidine bases. nyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. The inhibitory nucleic acid molecule may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles (e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone). Further modifications of the inhibitory nucleic acid molecules described herein may include the nucleobases disclosed in U.S. Patent No. 3,687,808; Kroschwitz, JI, ed., The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302.

[0112] sugar modification Modifications of the inhibitory nucleic acid molecules described herein may also include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (also known as 2'-O-CH2CHOCH3, 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CHOCH2N(CH3)2. Other possible 2'-modifications that may modify the inhibitory nucleic acid molecules described herein include OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or all possible orientations of O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Other potential sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluoro (F). The 2'-sugar substituent can be in the arabino (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions on the interfering RNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0113] Internucleoside linkage modifications Modifications of the inhibitory nucleic acid molecules described herein may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates, e.g., 3'-alkylenephosphonates, 5'-alkylenephosphonates, phosphinates, phosphoramidates, e.g., 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphototriesters, selenophosphates, and boranophosphates having normal 3'-5'-linkages, 2'-5'-linked analogs thereof, and those with inverted polarity, where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0114] Conjugates Any of the inhibitory nucleic acid molecules described herein can be modified by the addition of an auxiliary moiety, such as a cell-penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety, which can be present as a 5'-terminal modification (e.g., covalently attached to the 5'-terminal nucleoside), a 3'-terminal modification (e.g., covalently attached to the 3'-terminal nucleoside), or an internucleoside linkage (e.g., covalently attached to a phosphate or phosphorothioate in the internucleoside linkage).

[0115] CPPs are known in the art (e.g., TAT or ARg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2, 43-51). Specific examples of CPPs are provided in WO 2011157713, which is incorporated herein by reference in its entirety.

[0116] The inhibitory nucleic acid molecules of the present disclosure can include covalently attached neutral polymeric auxiliary moieties, including poly(C1-6 alkylene oxides), such as poly(ethylene glycol) and poly(propylene glycol), and copolymers thereof, such as diblock and triblock copolymers.

[0117] Inhibitory nucleic acid molecules containing a hydrophobic moiety may exhibit superior cellular uptake compared to inhibitory nucleic acid molecules lacking the hydrophobic moiety. The hydrophobic moiety may be a monovalent group such as a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyllithocholic acid, or oleoylcholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrin, texaphyrin, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas Red, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen covalently linked to the nucleic acid backbone (e.g., at the 5' end) of the inhibitory nucleic acid molecule.

[0118] Targeting moieties are selected based on their ability to target the oligonucleotides of the invention to a desired or selected cell population that expresses a binding partner (e.g., either the corresponding receptor or ligand) corresponding to the selected targeting moiety. For example, oligonucleotides of the invention can be targeted to hepatocytes that express the asialoglycoprotein receptor (ASGP-R) by selecting a targeting moiety that includes N-acetylgalactosamine (GalNAc).

[0119] The targeting moiety can include one or more ligands (e.g., 1-9 ligands, 1-6 ligands, 1-3 ligands, 3 ligands, or 1 ligand). The ligands can target cells expressing the asialoglycoprotein receptor (ASGP-R), IgA receptor, HDL receptor, LDL receptor, or transferrin receptor. Non-limiting examples of the ligands include N-acetylgalactosamine (e.g., tri-branched N-acetylgalactosamine), glycyrrhetinic acid, glycyrrhizin, lactobionic acid, lactoferrin, IgA, or bile acids (e.g., lithocholyl taurine or taurocholic acid).

[0120] The ligand can be a small molecule, such as a small molecule that targets cells expressing the asialoglycoprotein receptor (ASGP-R). A non-limiting example of a small molecule that targets the asialoglycoprotein receptor is N-acetylgalactosamine. Alternatively, the ligand can be an antibody or an antigen-binding fragment or engineered derivative thereof, such as an Fcab or a fusion protein (e.g., scFv).

[0121] Preparation of inhibitory nucleic acid molecules The inhibitory nucleic acid molecules of the present disclosure can be prepared using techniques and methods known in the art for oligonucleotide synthesis. For example, the inhibitory nucleic acid molecules of the present disclosure can be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of: (1) deblocking a 5'-protected nucleotide to produce a 5'-deblocked nucleotide; (2) coupling the 5'-deblocked nucleotide with a 5'-protected nucleoside phosphoramidite to produce a nucleoside linked via a phosphite; (3) repeating steps (1) and (2) one or more times as necessary; (4) capping the 5'-terminus; and (5) oxidizing or sulfurizing the internucleoside phosphite. Reagents and reaction conditions useful for oligonucleotide synthesis are known in the art.

[0122] The inhibitory nucleic acid molecules disclosed herein may be linked to a solid support as a result of solid-phase synthesis. Cleavable solid supports that can be used are known in the art. Non-limiting examples of such solid supports include, for example, controlled pore glass or macroporous polystyrene, which are linked to the chain via a cleavable linker (e.g., a succinate-based linker) known in the art (e.g., UnyLinker™). The nucleic acid linked to the solid support can be removed from the solid support by cleaving the linker connecting the nucleic acid to the solid support.

[0123] composition The inhibitory nucleic acid molecules described herein can be formulated into various compositions (e.g., pharmaceutical compositions) for administration to a subject in a biologically compatible form suitable for in vivo administration. For example, the inhibitory nucleic acid molecules described herein (e.g., siRNA molecules of SEQ ID NOs: 1-10, or variants thereof) can be administered in a suitable diluent, carrier, or excipient, and can further include a preservative, e.g., to prevent microbial growth. Conventional procedures and ingredients for the selection and preparation of suitable compositions can be found, for example, in Remington, J.P., The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22. nd ed, and The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0124] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal (e.g., non-human animals, e.g., non-human mammals). Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and / or perform such modifications with no more than routine experimentation, if at all. Subjects to which administration of pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates and mammals.

[0125] Compositions comprising the inhibitory nucleic acids described herein may further comprise a second therapeutic agent (e.g., a nucleic acid molecule, polypeptide, or drug to be expressed in the cell). For example, the second therapeutic agent may be a blood pressure medication, an anti-inflammatory agent (e.g., a steroid or colchicine), or an immunosuppressant. In some aspects, the second therapeutic agent is a statin. Non-limiting examples of second therapeutic agents are statins (e.g., atorvastatin), proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors (e.g., siRNA or monoclonal antibodies targeting PCSK9), ATP citrate lyase (ACL) inhibitors (e.g., bempedoic acid), lipoprotein(a) (Lp(a)) inhibitors (e.g., siRNA targeting Lp(a)), angiopoietin-like 3 (ANGPTL3) inhibitors (e.g., siRNA targeting ANGPTL3), cholesteryl ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors (e.g., lomitapide), apolipoprotein B (ApoB) inhibitors (e.g., mipomersen), bile acid-binding resins, and anti-inflammatory drugs (e.g., colchicine).

[0126] In some embodiments, a second therapeutic agent (e.g., a statin) is administered in combination with the inhibitory nucleic acid molecule of the present disclosure. In some embodiments, the subject is administered the statin orally. In some embodiments, the subject is administered the statin daily.

[0127] Treatment method The present disclosure provides methods for reducing LDL in serum of a subject. In some embodiments, the methods include administering to the subject an inhibitory nucleic acid molecule described herein, where the inhibitory nucleic acid molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject an siRNA molecule described herein (e.g., SEQ ID NO: 1-10, or a variant thereof), where the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject a dsRNA molecule described herein, where the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject an ASO molecule described herein, where the ASO molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method includes administering to the subject an miRNA molecule described herein, wherein the miRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method includes administering to the subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0128] The present disclosure provides methods of increasing LDLr in a subject. In some embodiments, the methods include administering to the subject an inhibitory nucleic acid molecule described herein, where the inhibitory nucleic acid molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject an siRNA molecule described herein (e.g., SEQ ID NO: 1-10, or a variant thereof), where the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject a dsRNA molecule described herein, where the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the methods include administering to the subject an ASO molecule described herein, where the ASO molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method includes administering to the subject an miRNA molecule described herein, wherein the miRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method includes administering to the subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0129] Any of these methods may administer a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA).

[0130] Delivery Vehicle The inhibitory nucleic acid molecules of the present disclosure can be delivered to a subject (e.g., a human) using any suitable delivery vehicle. For example, the delivery vehicle for any of the inhibitory nucleic acid molecules described herein can be a vector, a plasmid, or a nanoparticle (e.g., a micelle, liposome, exosome, or lipid nanoparticle (LNP)).

[0131] The inhibitory nucleic acid molecule of the present disclosure and its composition can be delivered to a subject via a vector (for example, a viral vector).Any suitable viral vector system can be used, such as adenovirus (for example, Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdovirus (for example, vesicular stomatitis virus), retrovirus, adeno-associated vector, poxvirus, herpesvirus vector, and Sindbis virus vector.

[0132] The inhibitory nucleic acid molecules and compositions thereof of the present disclosure may be delivered to a subject via liposomes. Liposomes are artificially prepared vesicles that may be composed primarily of lipid bilayers and may be used as delivery vehicles for administering the inhibitory nucleic acids and compositions thereof described herein. Liposomes may be of various sizes, including, but not limited to, multilamellar vesicles (MLVs), which may be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which may be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which may be 50-500 nm in diameter. Liposome designs may include, but are not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may have a low or high pH to improve delivery of pharmaceutical compositions.

[0133] The inhibitory nucleic acid molecules and compositions thereof of the present disclosure can be delivered to a subject via exosomes. Exosomes produced from cells can be collected from cell culture medium by any suitable method. Typically, exosome preparations can be prepared from cell culture or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, using standard methods, exosomes can be prepared by differential centrifugation, i.e., low-speed (less than 20,000 g) centrifugation to pellet larger particles, followed by high-speed (more than 100,000 g) centrifugation to pellet exosomes, size filtration using an appropriate filter (e.g., a 0.22 micron filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination of these methods.

[0134] The inhibitory nucleic acid molecules and compositions thereof of the present disclosure can be delivered to a subject via LNPs. For example, the inhibitory nucleic acid molecules (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA) can be formulated into lipid nanoparticles such as those described in International Publication No. 2012170930 (incorporated herein by reference in its entirety). As a non-limiting example, the LNP formulation can include a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxyl-propyl-3-amine (PEG-c-DOMG), distearoyl-rac-glycerol (DSG), and / or dimethylaminobutanoate (DMA). As a non-limiting example, a lipid molar ratio of 1-5% of PEG-c-DOMG compared to the cationic lipid, DSPC, and cholesterol can be used. In another embodiment, PEG-c-DOMG can be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, for example, but not limited to, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane (DLin-DMA), C12-200, and N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA).

[0135] Exemplary commercially available reagents useful for lipid-based delivery of inhibitory nucleic acid molecules include, but are not limited to, TransIT-TKO™ (Mirus, catalog number MIR 2150), Transmessenger™ (Qiagen, catalog number 301525), Oligofectamine™ and Lipofectamine™ (Invitrogen, catalog numbers MIR 12252-011 and 13778-075), siPORT™ (Ambion, catalog number 1631), and DharmaFECT™ (Fisher Scientific, catalog number T-2001-01).

[0136] Dosage The actual dosage of the composition of the present disclosure administered to a subject may be determined by physical and physiological factors, such as the patient's weight, the severity of the condition, prior or concurrent therapeutic interventions, idiopathic diseases, and the route of administration. Depending on the dosage (e.g., mg / kg) and the route of administration, the preferred dosage and / or the number of administrations of an effective amount may vary depending on the subject's response. The physician responsible for administration will, in any event, determine the concentration of the active ingredient in the composition and the appropriate dose for each individual subject. Administration may be any suitable number of times per day, and for as long as necessary. The subject may be an adult or pediatric human, with or without coexisting conditions.

[0137] Administration route The compositions utilized in the methods described herein may be administered to a subject by any suitable route of administration. For example, compositions comprising the inhibitory nucleic acids of the present disclosure may be administered intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, injection, infusion, continuous infusion, by localized perfusion bathing the target cells directly, by catheter, by lavage, in a cream, or in a lipid composition.

[0138] In some embodiments, the compositions utilized in the methods described herein may be administered to a subject intravenously, hi some embodiments, the compositions utilized in the methods described herein may be administered to a subject subcutaneously. [Example]

[0139] Example 1. Effect of siRNA targeting of human AC9 in vitro The following examples describe the materials and methods utilized to obtain the results described herein.

[0140] Materials and Methods cell culture HepG2 (hepatocellular carcinoma) cells were grown in Eagle's minimum essential medium (EMEM). This medium was supplemented with 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin. Cells were cultured at 37°C in 5% CO2 and harvested weekly using trypsin-EDTA. For experiments, cells were trypsinized, seeded, and cultured for at least 3 days before assay.

[0141] siRNA transfection HepG2 cells were transfected with ADCY9, SREBP2, LDLr, or scrambled siRNA in the presence of Lipofectamine RNAiMAX in Opti-MEM for 72 hours unless otherwise stated.

[0142] Cellular cholesterol efflux Cells were treated with 2 μCi / ml [1,2- 3Cells were labeled in EMEM containing [H]cholesterol + 1% FBS. Cells were then equilibrated with EMEM containing 0.2% BSA with or without H89 or GSK-2033 for 18 hours at 37°C. Efflux assays were performed in the absence or presence of 10 μg / ml apoA-I. At the end of the incubation, the medium was collected and the cells were solubilized. The medium and cells were counted for radioactivity in a β-counter. The percentage of efflux was calculated by subtracting the radioactive counts in the medium in the absence of cholesterol acceptor from the radioactive counts in the presence of the acceptor and then dividing by the sum of the radioactive counts in the medium and the cellular fraction.

[0143] Lipoprotein isolation and radiolabeling Lipoproteins were isolated from human plasma obtained from BioIVT (Westbury, NY). Prior to isolation, the plasma was adjusted to 0.01% ethylenediaminetetraacetic acid (EDTA), 0.02% sodium azide, and 10 μM phenylmethylsulfonyl fluoride (PMSF). Human LDL (d = 1.025–1.063 g / ml) was prepared by ultracentrifugation as described by Brissette et al. (doi: 10.1042 / bj3180841). LDL was purified by cleavage of 1,2-[[(2-methyl- ... 3 LDL was labeled with [H]cholesteryl oleate. The labeled LDL was then reisolated by ultracentrifugation. The specific activity of the labeled LDL in CE ranged from 7,000 to 12,000 cpm / μg protein.

[0144] Lipoprotein cell association assay [ 3Cell association of [H]CE-lipoproteins (20 μg protein / ml) was measured in 12-well plates over 4 hours at 37°C. Cells were washed twice with 1 ml of phosphate-buffered saline (PBS) and incubated in a total volume of 250 μl containing 125 μl (2×) of culture medium, 4% bovine serum albumin, pH 7.4 (total binding). Nonspecific association was assessed by the addition of 1.5 mg protein / ml of unlabeled lipoproteins. At the end of the incubation, the cells were washed twice with 1 ml of PBS containing 0.2% BSA (PBS-BSA), followed by two washes with 1 ml of PBS. Cells were then solubilized in 1.5 ml of 0.2 N NaOH. Radioactivity counts in the homogenates were obtained in a beta counter. CE( 3 To compare the association of lipoproteins labeled in HCl, the association data were estimated as micrograms of protein per milligram of cellular protein (apparent uptake). 3 The specific activity of [H]CE-lipoprotein was expressed as counts per microgram of lipoprotein protein. Specific association was calculated by subtracting nonspecific association from total association.

[0145] Immunoblotting For Western blotting, cells were washed with cold PBS, then scraped and lysed in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, and protease inhibitor cocktail). Lysates were microcentrifuged for 20 min at 4°C, and the supernatants were assayed for cellular protein. Proteins (30–50 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting onto PVDF membranes.

[0146] Quantification of mRNA expression by reverse transcription-quantitative PCR Total RNA from HepG2 cells was extracted using the RNeasy isolation kit according to the manufacturer's protocol. cDNA was synthesized using components from the High-Capacity cDNA Reverse Transcription Kit and MultiScribe reverse transcriptase according to the manufacturer's instructions. RNA was quantified using the Quant-it RiboGreen RNA Assay Kit according to the manufacturer's instructions, and RNA quality was assessed using the Agilent RNA 6000 Nano Kit for the Bioanalyzer 2100 System. Primers were designed using Beacon designer software v.8 and obtained from IDT. Reference genes for normalization, PPIA and TBP, were selected using Bio-Rad CFX Maestro software using the GeNorm method. qPCR was performed using a SYBR-Green reaction mix. qPCR conditions consisted of an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of amplification, each consisting of 15 seconds at 95°C and 60 seconds at 60°C. Results were analyzed by the delta-delta Ct method using Bio-Rad CFX Maestro software.

[0147] SREBP-2 transcriptional activity SREBP-2 transcriptional activity was estimated using a kit in which SREBP-2 contained in nuclear extracts obtained from transfected HepG2 cells is specifically bound to immobilized SREBP response element oligonucleotides according to the kit supplier's protocol and detected by the addition of a specific primary antibody against SREBP-2.

[0148] Tandem Mass Tag (TMT) Proteomics Analysis HepG2 cells were transfected and harvested 3 days later for untargeted proteomic analysis. Briefly, eight tandem mass tags (TMTs) were used to label two different conditions (siScramble and siAC9) in four different assays. Once labeled, all samples were combined and analyzed in a single liquid chromatography-mass spectrometry (LC-MS) experiment.

[0149] Other methods Protein content was determined by the method of Lowry using BSA as a standard.

[0150] result The protein expression of adenylate cyclase type 9 (AC9), low-density lipoprotein receptor (LDLr), and ATP-binding cassette subfamily A member 1 (ABCA1) was analyzed in HepG2 cells after siRNA-mediated knockdown (KD) of AC9 (Figure 1A). The protein expression of AC9 was significantly decreased, whereas the protein expression of LDLr and ABCA1 was significantly increased (Figure 1B). 3 H-CE-LDL association (Fig. 1C) and 3 Quantification of H-CE-LDL cholesterol efflux (Fig. 1D) showed a significant increase after siRNA-mediated KD of AC9.

[0151] The protein expression of AC9, LDLr, sterol regulatory element-binding protein-2 (SREBP-2), and ABCA1 was analyzed by untargeted relative proteomics in HepG2 cells after siRNA-mediated knockdown of AC9. The peptide levels of AC9 were significantly decreased, whereas those of LDLr and SREBP2 were significantly increased (Figure 2A).

[0152] Protein expression of protein kinase c-AMP-dependent type I regulatory alpha (PRKAR1A), protein kinase c-AMP-dependent type I regulatory beta (PRKAR1B), and A-kinase anchoring protein 12 (AKAP12) was analyzed by untargeted relative proteomics in HepG2 cells after siRNA-mediated knockdown of AC9. Peptide levels of PRKAR1A, PRKAR1B, and AKAP12 were significantly increased (Figure 2B).

[0153] In HepG2 cells after siRNA-mediated knockdown of AC9 (siAC9) with or without co-treatment with 5 μM atorvastatin (Ato), 3 H-CE-LDL association was analyzed. Co-treatment with siAC9 and Ato resulted in an additive effect on LDL cholesterol uptake by the LDLr (FIG. 3).

[0154] LDLr expression was analyzed in HepG2 cells after treatment with 300 μM exogenous cAMP (to mimic the effect of AC9 knockdown), 5 μM Ato, or both cAMP and Ato. The combined effect of exogenous cAMP and atorvastatin increased LDLr protein expression (Figure 4).

[0155] LDLr and ABCA1 expression was analyzed in HepG2 cells after siRNA-mediated knockdown of AC9 with or without co-treatment with 2 μM H89 (a PKA inhibitor). siRNA-mediated knockdown of AC9 resulted in increased LDLr and ABCA1 expression, which was reversed upon co-treatment with H89, a PKA inhibitor (Figure 5A-B). Furthermore, in HepG2 cells treated with siAC9 and H89, 3 Although a decrease in H-CE-LDL association was observed, no cholesterol efflux was observed (Figure 5C-D). Collectively, these results indicate that the increases in LDLr and ABCA1 observed upon AC9 KD are primarily PKA-dependent.

[0156] Gene expression of AC9, SREBP2, proprotein convertase subtilisin / kexin type 9 (PCSK9), and LDLr was analyzed in HepG2 cells after siRNA-mediated knockdown of AC9 (Figure 6). These results show that AC9 siRNA increases mRNA expression of SREBP2 and LDLR, but not PCSK9.

[0157] SREBP2 transcriptional activity was analyzed in HepG2 cells 24 and 48 hours after siRNA-mediated KD of AC9. These results show that AC9 siRNA increases SREBP2 transcriptional activity (Figure 7).

[0158] LDLr protein expression was analyzed in HepG2 cells after siRNA-mediated KD of AC9, SREBP2, or both AC9 and SREBP2 (Figure 8). KD of AC9 resulted in increased LDLr protein expression. These results indicate that SREBP2 siRNA can block siAC9-mediated increase in LDLr protein.

[0159] Cholesterol efflux to apoA1 in HepG2 cells was analyzed after siRNA-mediated knockdown of AC9, SREBP2, or both AC9 and SREBP2, revealing a significant increase in cholesterol efflux only upon KD of AC9 (Figure 9A). Next, cholesterol efflux was examined in HepG2 cells after siRNA-mediated knockdown of AC9 with or without co-treatment with 5 μM GSK-2033 (an LXR inhibitor). The LXR inhibitor was able to significantly reduce the siAC9-mediated increase in ABCA1-mediated cholesterol efflux (Figure 9B). These results indicate that LXR is involved in the effect of AC9 siRNA on ABCA1-mediated cholesterol efflux.

[0160] ABCA1 protein expression was analyzed in HepG2 cells after siRNA-mediated knockdown of AC9, LDLr, or both AC9 and LDLr (Figure 10A). Only knockdown of AC9 alone increased ABCA1 expression. Cholesterol efflux also increased upon knockdown of AC9 (Figure 10B). Cotreatment with AC9 and LDLr siRNA also increased cholesterol efflux, but at a significantly lower level than AC9 siRNA alone. These results indicate that LDLr siRNA partially blocks siAC9-mediated increase in ABCA1 protein.

[0161] The above results demonstrate the effect of AC9 KD on the expression and function of LDLr and ABCA1 (see, eg, FIG. 11).

[0162] We analyzed the protein expression of AC9 and LDLr in HepG2 cells transfected with various siRNA sequences targeting ADCY9 mRNA at various exons. The protein expression of AC9 was significantly reduced, whereas the protein expression of LDLr was significantly increased by all siRNA sequences used (Table 4).

[0163] [Table 4]

[0164] Table 4 shows the protein levels of AC9 and LDLr after transfection of HepG2 cells with various siRNA sequences targeting ADCY9 mRNA at various exons compared to the siScramble control. Results were obtained by Western blotting, and actin was used as a loading control. Protein expression is expressed as a percentage of the siScramble control. n=8. Paired t-test: *=p≦0.05; **=p≦0.01; and ***=p≦0.001 vs. siScramble.

[0165] Other Aspects All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0166] While the invention has been described in relation to particular embodiments thereof, it will be understood that it is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations in accordance with principles, within known or customary practice in the art to which this invention pertains, including such departures from the invention as may be applicable to the essential features hereinabove described, and in accordance with the scope of the claims.

[0167] Other embodiments are within the scope of the claims.

Claims

1. A method for reducing serum low-density lipoprotein (LDL) levels in a subject, comprising inhibiting the expression or function of adenylate cyclase in the subject, wherein said inhibiting comprises administering an inhibitory nucleic acid molecule to the subject.

2. A method for increasing LDL receptor expression in a subject, comprising inhibiting the expression or function of adenylate cyclase in said subject, said inhibiting comprising administering to said subject an inhibitory nucleic acid molecule.

3. 3. The method of claim 1, wherein the adenylate cyclase is adenylate cyclase type 9 (AC9).

4. The AC9 is (i) the mRNA sequence of SEQ ID NO: 16; and / or (ii) the DNA sequence of SEQ ID NO: 17 The method of claim 3, comprising:

5. 5. The method of any one of claims 1 to 4, wherein the inhibitory nucleic acid molecule is an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a double-stranded RNA (dsRNA), or a microRNA (miRNA).

6. The method of claim 4 , wherein the inhibitory nucleic acid molecule is an siRNA.

7. 6. The method of claim 5, wherein the siRNA comprises a sequence complementary to at least 15 consecutive nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

8. 7. The method of claim 6, wherein the siRNA comprises a sequence complementary to at least 19 consecutive nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

9. 8. The method of claim 7, wherein the siRNA comprises a sequence complementary to at least 21 consecutive nucleotides set forth in any one of SEQ ID NOs: 1-10, 16, and 17.

10. 9. The method of claim 8, wherein the siRNA comprises a sequence complementary to at least 25 consecutive nucleotides set forth in any one of SEQ ID NOs: 16 and 17.

11. the siRNA molecule (i) a single uracil overhang at one or more 3' ends of the siRNA; (ii) a double uracil overhang at one or more 3' ends of the siRNA; (iii) a single thymine overhang at one or more 3' ends of the siRNA; (iv) a double thymine overhang at one or more 3' ends of the siRNA; or (v) a single cytosine and a single thymine overhang at one or more 3' ends of the siRNA. The method of any one of claims 5 to 9, comprising a 3' overhang selected from the group consisting of:

12. The method of any one of claims 5 to 11, wherein the siRNA comprises one or more nucleotide sequences of any one of SEQ ID NOs: 1 to 10.

13. The siRNA (i) a sense strand comprising the sequence of SEQ ID NO: 1 and an antisense strand comprising the sequence of SEQ ID NO: 2; (ii) a sense strand comprising the sequence of SEQ ID NO: 3 and an antisense strand comprising the sequence of SEQ ID NO: 4; (iii) a sense strand comprising the sequence of SEQ ID NO: 5 and an antisense strand comprising the sequence of SEQ ID NO: 6; (iv) a sense strand comprising the sequence of SEQ ID NO: 7 and an antisense strand comprising the sequence of SEQ ID NO: 8; or (v) a sense strand comprising the sequence of SEQ ID NO: 9 and an antisense strand comprising the sequence of SEQ ID NO: 10; 13. The method of claim 12, comprising:

14. The method of any one of claims 5 to 13, wherein the siRNA comprises non-natural or modified nucleosides or nucleotides.

15. 12. The method of claim 11, wherein the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate internucleoside (PS) linkages, and 2'-fluoro (2'-F) modified nucleosides.

16. The method of any one of claims 5 to 12, wherein the siRNA molecule targets any one of the sequences set forth in SEQ ID NOs: 11 to 15.

17. The method of any one of claims 1 to 16, further comprising administering to the subject a second therapeutic agent.

18. 18. The method of claim 17, wherein the second therapeutic agent is selected from the group consisting of statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ATP citrate lyase (ACL) inhibitors, lipoprotein(a) (Lp(a)) inhibitors, angiopoietin-like 3 (ANGPTL3) inhibitors, cholesteryl ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, apolipoprotein B (ApoB) inhibitors, bile acid-binding resins, and colchicine.

19. 19. The method of claim 18, wherein the statin is atorvastatin.

20. 19. The method of claim 18, wherein the PCSK9 inhibitor is an siRNA molecule or a monoclonal antibody that targets PCSK9.

21. 19. The method of claim 18, wherein the ACL inhibitor is bempedoic acid.

22. 19. The method of claim 18, wherein the Lp(a) inhibitor is an siRNA molecule that targets Lp(a).

23. 19. The method of claim 18, wherein the MTP inhibitor is lomitapide.

24. 19. The method of claim 18, wherein the ApoB inhibitor is mipomersen.

25. (i) a sense strand comprising the sequence of SEQ ID NO: 3 and an antisense strand comprising the sequence of SEQ ID NO: 4; (ii) a sense strand comprising the sequence of SEQ ID NO: 5 and an antisense strand comprising the sequence of SEQ ID NO: 6; (iii) a sense strand comprising the sequence of SEQ ID NO: 7 and an antisense strand comprising the sequence of SEQ ID NO: 8; or (iv) a sense strand comprising the sequence of SEQ ID NO: 9 and an antisense strand comprising the sequence of SEQ ID NO:

10.

1. An siRNA molecule comprising:

26. 21. The siRNA molecule of claim 20, wherein the siRNA comprises non-natural or modified nucleosides or nucleotides.

27. 22. The siRNA molecule of claim 21, wherein the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate internucleoside (PS) linkages, and 2'-fluoro (2'-F) modified nucleosides.

28. The siRNA molecule of claim 22, wherein the siRNA molecule targets any one of the sequences set forth in SEQ ID NOs: 11 to 15.

29. 10. Use of an inhibitory nucleic acid molecule for reducing serum low density lipoprotein (LDL) levels in a subject, wherein the expression or function of adenylate cyclase is inhibited in said subject by administration of the inhibitory nucleic acid molecule.

30. 10. Use of an inhibitory nucleic acid molecule for reducing LDL receptor expression in a subject, wherein the expression or function of adenylate cyclase is inhibited in said subject by administration of the inhibitory nucleic acid molecule.

31. 31. The use according to claim 29 or 30, wherein the adenylate cyclase is adenylate cyclase type 9 (AC9).

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