Compositions and methods for inhibiting adenylate cyclase 9 (AC9)

Abstract

Methods of reducing serum low density lipoprotein (LDL) levels in a subject are disclosed. The methods comprise inhibiting the expression or function of adenylate cyclase in the subject, where the inhibiting comprises administering to the subject an inhibitory nucleic acid molecule, such as siRNA.

Description

Technical Field

[0001] The present disclosure relates to inhibitory nucleic acid molecules and compositions and methods thereof that lower low-density lipoprotein (LDL) and increase LDL receptor (LDLr) in a subject. Background Art

[0002] Adenylate cyclase (AC), also known as adenylate cyclase and adenylyl cyclase, is a regulatory enzyme that regulates signaling pathways and physiological responses in cells by converting adenosine triphosphate (ATP) to 3',5'-cyclic AMP (cAMP), a key second messenger. 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. [Physiol. Rev.] 102: 815-857) and autoinhibited by its C-terminal cytoplasmic domain (C2b) ( et al. (2018) Cell Signal. 51: 266-275) and is internalized upon stimulation by G-protein coupled receptors (GPCRs) (Lazar et al. (2020) eLife 9: e58039). Like other ACs, AC9 can form heterodimers with AC5 and AC6 (Baldwin et al. (2019) Mol. Pharmacol. 9: 349-360). Notably, expression of full-length AC9 blocks endogenous GPCR-related stimulation of AC and cAMP production, whereas C-terminally truncated AC9 does not ( et al. (2018) Cell Signal. 51: 266-275).

[0003] Throughout the body, low-density lipoprotein (LDL) transports fat molecules to cells via the bloodstream. LDL receptors (LDLr) on the surface of receptive cells can bind and internalize LDL, thereby internalizing LDL and lowering LDL levels in the blood. Excessive LDL in the blood is associated with an increase in cardiovascular complications, such as coronary heart disease, which causes a quarter of all deaths in industrialized countries (Goldstein et al. (2015) Cell 161:161-172). Therefore, regulating circulating LDL by modulating LDLr has therapeutic implications.

[0004] Several mechanisms are involved in the regulation of LDLr expression. Downregulation of LDLr protein can be mediated by inducible degraders of LDLr (IDOLs), which stimulate proteasomal degradation of LDLr (Zelcer et al. (2009) Science 325:100-104). Additionally, 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). Conversely, transcriptional upregulation of LDLr can be mediated by sterol regulatory element binding protein-2 (SREBP-2), which translocates to the nucleus of cells and stimulates LDLr gene expression when intracellular cholesterol is low (Goldstein et al. (2015) Cell 161:161-172). AC-cAMP and protein kinase A (PKA) can further influence the upregulation of LDLr. For example, phosphodiesterase (PDE) inhibitors can induce SREBP2 nuclear translocation through a PKA-dependent mechanism (Shimizu-Albergine et al. (2013) Proc Natl Acad Sci. [Proceedings of the National Academy of Sciences of the United States of America], 113: E5685-5693), thereby increasing LDLr transcription. In addition, the presence of a functional cAMP response element (CRE) in the LDLr promoter can stimulate LDLr transcription (Liu et al. (2000) J Biol Chem. [Journal of Biological Chemistry] 275: 5214-5221).

[0005] There remains a need for compositions and methods that can lower LDL and / or increase LDLr in a subject. Summary of the Invention

[0006] The present invention provides compositions and methods for reducing low-density lipoprotein (LDL) in the serum of a subject. In addition, the present invention provides compositions and methods for increasing LDL receptor (LDLr) in a subject.

[0007] In a first aspect, the present invention provides a method of reducing low-density lipoprotein (LDL) levels in the serum of a subject, the method comprising inhibiting the expression or function of adenylate cyclase in the subject, wherein the inhibition comprises administering an inhibitory nucleic acid molecule to the subject.

[0008] In a second aspect, the present invention provides a method of increasing LDL receptor expression in a subject, the method comprising inhibiting the expression or function of adenylate cyclase in the subject, wherein the inhibition comprises administering an inhibitory nucleic acid molecule to the subject.

[0009] In some aspects, the adenylate cyclase is adenylate cyclase type 9 (AC9).

[0010] In some aspects, the AC9 contains the mRNA sequence of SEQ D NO: 16; and / or the DNA sequence of SEQ ID NO: 17.

[0011] In some aspects, the inhibitory nucleic acid molecule is an antisense oligonucleotide (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA (dsRNA), or micro RNA (miRNA).

[0012] In some aspects, the inhibitory nucleic acid molecule is siRNA.

[0013] 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.

[0014] In some aspects, 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.

[0015] In some aspects, 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.

[0016] 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.

[0017] In some aspects, the siRNA molecule contains 3' overhangs, such as: a single uracil overhang at one or more 3' ends of the siRNA; a double uracil overhang at one or more 3' ends of the siRNA; a single thymine overhang at one or more 3' ends of the siRNA; a double thymine overhang at one or more 3' ends of the siRNA; or a single cytosine and single thymine overhang at one or more 3' ends of the siRNA.

[0018] In some aspects, the siRNA comprises the nucleotide sequence of any one or more of SEQ ID NOs: 1-10.

[0019] In some aspects, the siRNA comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, and the antisense strand comprising the sequence of SEQ ID NO: 2. In some aspects, the siRNA comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 3, and the antisense strand comprising the sequence of SEQ ID NO: 4. In some aspects, the siRNA comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 5, and the antisense strand comprising the sequence of SEQ ID NO: 6. In some aspects, the siRNA comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 7, and the antisense strand comprising the sequence of SEQ ID NO: 8. In some aspects, the siRNA comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 9, and the antisense strand comprising the sequence of SEQ ID NO: 10.

[0020] In some aspects, the siRNA includes non-natural or modified nucleosides or nucleotides.

[0021] In some aspects, the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate (PS) linkages between nucleosides, and 2'-fluoro (2'-F) modified nucleosides.

[0022] In some aspects, the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15.

[0023] In some aspects, the method of any of the preceding aspects further comprises administering a second therapeutic agent to the subject.

[0024] In some aspects, the second therapeutic agent is selected from the group consisting of a statin, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, an ATP citrate lyase (ACL) inhibitor, a lipoprotein (a) (Lp(a)) inhibitor, angiopoietin-like protein 3 (ANGPTL3) inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTP) inhibitor, an apolipoprotein B (ApoB) inhibitor, a bile acid binding resin, and colchicine.

[0025] In some aspects, the statin is atorvastatin.

[0026] In some aspects, the PCSK9 inhibitor is an siRNA molecule or a monoclonal antibody targeting PCSK9.

[0027] In some aspects, the ACL inhibitor is bepedic acid.

[0028] In some aspects, the Lp(a) inhibitor is a siRNA molecule that targets Lp(a).

[0029] In some aspects, the MTP inhibitor is lomitapide.

[0030] In some aspects, the ApoB inhibitor is mipomersen.

[0031] In a third aspect, the present invention provides an siRNA molecule comprising: a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 3, and the antisense strand comprising the sequence of SEQ ID NO: 4; a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 5, and the antisense strand comprising the sequence of SEQ ID NO: 6; a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 7, and the antisense strand comprising the sequence of SEQ ID NO: 8; or a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 9, and the antisense strand comprising the sequence of SEQ ID NO: 10.

[0032] In some aspects, the siRNA includes non-natural or modified nucleosides or nucleotides.

[0033] In some aspects, the modification is selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate (PS) linkages between nucleosides, and 2'-fluoro (2'-F) modified nucleosides.

[0034] In some aspects, the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are included to illustrate embodiments of the present disclosure and to provide a further understanding of how the same may be implemented.

[0036] Figure 1A Western blot showing AC9, LDLr, and ABCA1 protein levels in HepG2 cells following siRNA-mediated knockdown of AC9 relative to siScramble control. Actin is shown as a loading control.

[0037] Figure 1B Figure 2 is a graph showing the quantification of AC9, LDLr, and ABCA1 protein levels in HepG2 cells after siRNA-mediated knockdown of AC9 (left column, for AC9, LDLr, and ABCA1, respectively) relative to the siScramble control (right column, for AC9, LDLr, and ABCA1, respectively). Figure 1ADensitometry data for Western blots in and normalized to those for actin loading controls. Protein expression is expressed as a percentage relative to the control siScramble. Error bars represent mean ± standard deviation. n = 4-6. Paired t-test: * = p ≤ 0.05; ** = p ≤ 0.01; and *** = p ≤ 0.001, relative to siScramble.

[0038] Figure 1C Figure 2 shows that after siRNA-mediated knockdown of AC9, the expression of 3 Quantification of H-CE-LDL association. 3 H-cholesterol oleate (CE)-labeled human LDL was incubated with HepG2 cells at 20 μg protein / ml for 4 h. Finally, the cells were lysed in 0.2 N NaOH, and radioactivity was estimated using a beta counter and normalized to cellular protein estimated by the Lowry assay. Error bars represent mean ± standard deviation. n = 5. Paired t-test: * = p ≤ 0.05, relative to siScramble.

[0039] Figure 1D It is shown that after siRNA-mediated knockdown of AC9, 3 Figure 1 shows the quantification of H-CE-LDL cholesterol efflux. Briefly, transfected HepG2 cells were loaded with 3 H-cholesterol was added for 24 hours and equilibrated for 18 hours. Cholesterol efflux to apo AI was then performed at 10 μg / ml (4 hours). This dose of apo AI was considered to be saturating for the ABCA1 transporter. Finally, the culture medium was harvested, the cells were dissolved in 0.2N NaOH, and radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the culture medium by the radioactivity measured in the cells and culture medium. Error bars represent mean ± standard deviation. n = 4. Paired t-test: * = p ≤ 0.05, relative to siScramble.

[0040] Figure 2A Graph showing quantification of AC9, LDLr, SREBP2, and ABCA1 protein levels in HepG2 cells following siRNA-mediated AC9 knockdown relative to the siScramble control by nontargeted relative proteomics. Error bars represent mean ± standard deviation. n = 4. Paired t-test: * = p ≤ 0.05; *** = p ≤ 0.001, relative to siScramble.

[0041] Figure 2BFigure 2 shows quantification of PRKAR1A, PRKAR1B, and AKAP12 protein levels in HepG2 cells following siRNA-mediated knockdown of AC9 relative to the siScramble control by nontargeted relative proteomics. Error bars represent mean ± standard deviation. n = 4. Paired t-test: * = p ≤ 0.05; *** = p ≤ 0.001, relative to siScramble.

[0042] Figure 3 Figure 2 shows the effect of AC9 siRNA (siAC9) on the expression of Ato in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 5 μM atorvastatin (Ato) 24 h before cholesterol association assay. 3 Quantification of H-CE-LDL association. 3 H-cholesterol oleate (CE)-labeled human LDL was incubated with HepG2 cells at 20 μg protein / ml for 4 h. Finally, the 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 control and Ato, the left column is siScramble and the right column is siAC9. These results illustrate the additive effects of AC9 siRNA and atorvastatin on LDL cholesterol uptake by LDLr. Error bars represent mean ± standard deviation. n = 5. Repeated measures ANOVA with uncorrected Fisher LSD: * = p ≤ 0.05, relative to siScramble; a = p ≤ 0.05, relative to control.

[0043] Figure 4 Figure 2 is a graph showing 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 of an actin loading control. Protein expression is expressed as a percentage relative to the control without Ato. For both without Ato and with Ato, the left column is the control and the right column is cAMP. These results indicate that the combined action of exogenous cAMP and atorvastatin increases LDLr protein. Error bars represent mean ± standard deviation. n = 5. Paired t-test: * = p ≤ 0.05, relative to control; a = p ≤ 0.05, relative to "without Ato".

[0044] Figure 5AFigure 2 is a graph showing the quantification of LDLr protein expression in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (PKA inhibitor) 24 h before cell protein extraction. Briefly, the densitometry data from the western blot were calculated and normalized to the densitometry data of the actin loading control. Protein expression is expressed as a percentage relative to the siScramble control. For siScramble and siAC9, the left column is the control and the right column is H89. These results show that the increase in LDLr caused by AC9 siRNA is mainly PKA-dependent. Error bars represent mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05, relative to siScramble-control; b = p ≤ 0.05, relative to siAC9-control.

[0045] Figure 5B Figure 2 is a graph showing the quantification of ABCA1 protein expression in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (PKA inhibitor) 24 hours before cell protein extraction. Briefly, densitometry data from Western blots were calculated and normalized to the densitometry data of an actin loading control. Protein expression is expressed as a percentage relative to the siScramble control. For siScramble and siAC9, the left column is the control and the right column is H89. These results show that the increase in ABCA1 caused by AC9 siRNA is primarily PKA-dependent. Error bars represent mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05, relative to the siScramble control.

[0046] Figure 5C Figure 2 shows the effect of AC9 siRNA (siAC9) on the expression of PKA in HepG2 cells transfected with AC9 siRNA (siAC9) and treated with or without 2 μM H89 (PKA inhibitor) 24 h before the cholesterol association assay. 3 Quantification of H-CE-LDL association. 3 H-cholesterol oleate (CE) labeled human LDL was incubated with HepG2 cells at 20 μg protein / ml for 4 h. 3H-CE-LDL is considered saturating for the LDLr transporter. Finally, cells were lysed in 0.2N NaOH, and radioactivity was estimated using a beta counter and normalized to cellular protein estimated by the Lowry assay. For 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 mean ± standard deviation. n = 3. Paired t-test: a = p ≤ 0.05, relative to siScramble control.

[0047] Figure 5D Figure 2 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 (PKA inhibitor) 24 h before the cholesterol efflux assay. 3 H-cholesterol, balanced, and used for cholesterol efflux to apo AI at 10 μg / ml (4 h). For siScramble and siAC9, the left column is the control and the right column is H89. This dose of apo AI is considered to be saturating for the ABCA1 transporter. Finally, the culture medium was harvested, the cells were dissolved in 0.2N NaOH, and the radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the culture medium by the radioactivity measured in the cells and culture medium. Error bars represent mean ± standard deviation. n = 4. Paired t-test: a = p ≤ 0.05, relative to siScramble control.

[0048] Figure 6 Graph showing quantification of AC9, SREBP2, PCSK9, and LDLr mRNA expression in HepG2 cells by quantitative PCR 48 hours after transfection with AC9 siRNA (siAC9), relative to a siScramble control. These results demonstrate that AC9 siRNA increases SREBP2 and LDLR mRNA expression, but not PCSK9 mRNA expression. For AC9, SREBP2, LDLr, and PCSK9, the left bar represents siScramble, and the right bar represents siAC9. Error bars represent mean ± standard deviation. n = 6. Paired t-test: * = p ≤ 0.05 and *** = p ≤ 0.001, relative to siScramble.

[0049] Figure 7Graph showing quantification of SREBP2 transcriptional activity in HepG2 cells 24 and 48 hours after treatment with AC9 siRNA (siAC9) relative to siScramble control. Briefly, SREBP-2 transcriptional activity was estimated using a kit in which SREBP-2 contained in nuclear extracts specifically binds to an immobilized SREBP-responsive element and is detected by adding a specific primary antibody against SREBP-2. At each time point, the left column is siScramble and the right column is siAC9. These results show that AC9 siRNA increases SREBP2 transcriptional activity. Error bars represent mean ± standard deviation. n = 5. Paired t-test: * = p ≤ 0.05 and ** = p ≤ 0.01, relative to siScramble.

[0050] Figure 8 Figure 2 is a graph showing 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 of the actin loading control. Protein expression is expressed as a percentage relative to the siScramble control. These results indicate that SREBP2 siRNA can block the increase in LDLr protein caused by AC9 siRNA. Error bars represent mean ± standard deviation. n = 6. Paired t-test: a = p ≤ 0.05, relative to siScramble; b = p ≤ 0.05, relative to siAC9.

[0051] Figure 9A Figure 2 is a graph showing the quantification of cholesterol efflux to apo AI in HepG2 cells treated with AC9 siRNA (siAC9), SREBP2 siRNA (siSREBP2), or both siAC9 and siSREBP2, with siScramble used as a control. 3H-cholesterol, equilibrated, and used at 10 μg / ml for cholesterol efflux to apoA-I (4 h). This dose of apoA-I is considered saturating for the ABCA1 transporter. Finally, the culture medium was harvested, the cells were lysed in 0.2N NaOH, and the radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the culture medium by the radioactivity measured in the cells and culture medium. These results indicate that SREBP2 is involved in the effect of AC9 siRNA on cholesterol efflux via ABCA1. Error bars represent mean ± standard deviation. n = 6. Repeated measures ANOVA with uncorrected Fisher LSD: a = p ≤ 0.05, relative to siScramble; b = p ≤ 0.05, relative to siAC9.

[0052] Figure 9B 24h before the cholesterol efflux assay, HepG2 cells were transfected with AC9 siRNA (siAC9) and treated with or without 5 μM GSK-2033 (LXR inhibitor) for quantification of cholesterol efflux to apo AI, with siScramble used as a control. Briefly, transfected HepG2 cells were loaded with 3H-cholesterol, equilibrated, and used for cholesterol efflux to apo AI at 10 gg / ml (4h). This dose of apo AI is considered saturating for the ABCA1 transporter. Finally, the culture medium was harvested, the cells were dissolved in 0.2NNaOH, and radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the culture medium by the radioactivity measured in the cells and culture medium. For the control and GSK-2033, the left column is siScramble and the right column is siAC9. These results indicate that LXR is involved in the effect of AC9 siRNA on cholesterol efflux via ABCA1. Error bars represent 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.

[0053] Figure 10AFigure 2 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 a siScramble control. Briefly, densitometry data from Western blots were calculated and normalized to those of an actin loading control. Protein expression is expressed as a percentage relative to siScramble. These results demonstrate that LDLr siRNA blocked the increase in ABCA1 protein caused by AC9 siRNA. Error bars represent mean ± standard deviation. n = 4. Repeated-measures ANOVA with uncorrected Fisher's LSD: a = p ≤ 0.05, relative to siScramble; b = p ≤ 0.05, relative to siAC9.

[0054] Figure 10B Figure 2 is a graph showing the quantification of cholesterol efflux to apo AI in HepG2 cells treated with AC9 siRNA (siAC9), LDLr siRNA (siLDLr), or both siAC9 and siLDLr, with siScramble used as a control. 3 H-cholesterol, balanced, and used for cholesterol efflux to apo AI at 10 μg / ml (4 h). This dose of apo AI is considered to be saturating for the ABCA1 transporter. Finally, the culture medium was harvested, the cells were dissolved in 0.2N NaOH, and the radioactivity was estimated using a beta counter. The percentage of cholesterol efflux was calculated by dividing the radioactivity in the culture medium by the radioactivity measured in the cells and culture medium. These results indicate that LDLr siRNA blocked the increase in cholesterol efflux caused by AC9 siRNA. Error bars represent mean ± standard deviation. n = 13. Repeated measures ANOVA with uncorrected Fisher LSD: a = p ≤ 0.05, relative to siScramble; b = p ≤ 0.05, relative to siAC9.

[0055] Figure 11The following is a schematic diagram illustrating the mechanism of action of AC9 knockdown on LDL-lysozyme (LDLr) and ABCA1 expression and function. Briefly, reduced AC9 protein expression leads to increased cellular cAMP levels due to the removal of AC9's known inhibitory properties on other members of the family. This, in turn, leads to PKA activation and target phosphorylation. One of these is SREBP-2, whose activity is enhanced through increased expression and PKA-mediated phosphorylation. SREBP-2 is a key transcription factor responsible for increased LDL mRNA expression. This results in higher LDL-lysozyme protein levels, which is associated with enhanced LDL particle uptake. When LDL particles are degraded in lysosomes, cellular cholesterol accumulates, leading to activation of LXR activity. Both SREBP-2 and LXR increase ABCA1 mRNA expression, and PKA activation is associated with ABCA1 phosphorylation. Together, this results in higher ACBA1 protein levels and cholesterol efflux to the receptor apoA-I, a homeostatic physiological response to elevated cellular cholesterol levels. Therefore, AC9 knockdown leads to increased LDL particle uptake, which can translate into lower LDL cholesterol levels in the blood.

[0056] definition

[0057] Unless otherwise defined herein, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "include and included" is not restrictive.

[0058] As used herein, the term "about" when applied to one or more values ​​of interest refers to a value that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value, unless otherwise specified or otherwise obvious from the context (except where such a number would exceed 100% of the possible value).

[0059] As used herein, "administering" refers to providing or giving a therapeutic agent to a subject by any effective route. Exemplary routes of administration are described below.

[0060] As used herein, the term "administered in combination" or "combined administration" means that two or more agents are administered to a subject simultaneously or within a certain interval so that the effects of each agent on the patient may overlap. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the intervals between the administration of these agents are close enough to achieve a combined (e.g., synergistic) effect.

[0061] As used herein, the term "adjunct moiety" refers to any moiety capable of being 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, the "adjunct moiety" is attached to the inhibitory nucleic acid molecules disclosed herein by forming one or more covalent or non-covalent bonds with one or more conjugated groups attached to any portion of a phosphate linkage, a phosphorothioate linkage, a 5' position of a nucleotide sugar, or a nucleobase. One skilled in the art will readily appreciate the appropriate point of attachment of a particular adjunct moiety to a nucleic acid molecule.

[0062] As used herein, "delivery vehicle" refers to any substance (eg, molecule, peptide, conjugate, and construct) that at least partially facilitates the delivery of a nucleic acid molecule to a target cell in vivo.

[0063] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition described herein refer to an amount sufficient to achieve a beneficial or desired result when administered to a subject; thus, an "effective amount" or its synonyms depend on the context in which it is used. For example, in the case of lowering low-density lipoprotein (LDL), it is the amount of the composition sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as the given agent, pharmaceutical composition, route of administration, type of disease or disorder, identity of the subject being treated (e.g., age, sex, weight), or the like, but can still be routinely determined by one skilled in the art.

[0064] As used herein, a "formulation" includes at least an inhibitory nucleic acid molecule and a delivery vehicle.

[0065] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than within an organism (eg, an animal, plant, or microorganism).

[0066] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0067] 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 to inhibit the expression of a protein encoded by the target nucleic acid molecule. Exemplary inhibitory nucleic acid molecules are antisense oligonucleotides (ASOs), small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA (dsRNA), and microRNA (miRNA). Inhibitory nucleic acid molecules 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.

[0068] As used herein, "modified" refers to an altered state or structure of a nucleic acid molecule described herein. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one embodiment, the inhibitory nucleic acid molecules of the present invention are modified by the introduction of non-natural nucleosides and / or nucleotides. In other embodiments, the inhibitory nucleic acid molecules of the present invention are modified by conjugating auxiliary moieties.

[0069] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject, such as a mammal, e.g., a human, to prevent, treat or control a particular disease or condition that affects or may affect the subject.

[0070] As used herein, the term "pharmaceutically acceptable" refers to those 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 subjects, such as mammals (e.g., humans), without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0071] " Percentage (%) of sequence identity " with respect to reference polynucleotide or peptide sequence is defined as the percentage of nucleic acid or amino acid identical with the nucleic acid in the reference polynucleotide or peptide sequence in the candidate sequence after alignment sequence and, if necessary, introducing gap to obtain maximum sequence identity percentage.Comparison for determining nucleic acid or amino acid sequence identity percentage can be realized in various 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 the appropriate parameters for aligning sequences, including any algorithm required for maximum alignment over the full length of the compared sequences. For example, sequence comparison computer program BLAST can be used to generate sequence identity percentage values. As an illustration, the sequence identity percentage of a given nucleic acid or amino acid sequence A with, with, or for a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A with, with, or for a given nucleic acid or amino acid sequence B having a certain sequence identity percentage) is calculated as follows:

[0072] 100 multiplied by (fraction X / Y)

[0073] where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0074] As used herein, the term "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0075] As used herein, "treatment" and "treating" with respect to a disease or condition refer to methods for obtaining a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviating or improving one or more symptoms or conditions; reducing the extent of the disease or condition; stabilizing the state of the disease, disorder, or condition (i.e., not worsening); preventing the spread of the disease or condition; delaying or slowing the progression of the disease or condition; improving or alleviating the disease or condition; and alleviating (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extended survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in which a condition or disorder is to be prevented.

[0076] As used herein, the term "vector" is considered a replicon, such as a plasmid, phage, viral construct, or cosmid, to which another nucleic acid (e.g., DNA or RNA) segment can be attached. Vectors are used to transduce and express nucleic acid segments in cells. DETAILED DESCRIPTION

[0077] Described herein are compositions (e.g., inhibitory nucleic acid molecules) and methods thereof for reducing low-density lipoprotein (LDL) in a subject's serum. Furthermore, the present invention provides compositions (e.g., inhibitory nucleic acid molecules) and methods thereof for increasing LDL receptor (LDLr) expression in a subject.

[0078] The inhibitory nucleic acid molecules described herein (e.g., small interfering RNA (siRNA), double-stranded RNA (dsRNA), antisense oligonucleotides (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 lowering LDL and / or increasing LDLr in a subject. In doing so, the methods can be used to lower the concentration of LDL in the blood (e.g., serum) of a subject.

[0079] Inhibitory nucleic acid molecules

[0080] Exemplary inhibitory nucleic acid molecules of the present disclosure are siRNA, dsRNA, ASO, miRNA, and shRNA; however, any nucleic acid molecule capable of reducing AC9 mRNA and / or protein expression is contemplated for use in the methods described herein. In some cases, the inhibitory nucleic acid molecules of the present disclosure can be referred to as RNA inhibition (RNAi) molecules.

[0081] For any inhibitory nucleic acid molecule described herein (e.g., siRNA, dsRNA, ASO, miRNA, shRNA, or other inhibitory nucleic acid molecule capable of reducing target gene expression), the inhibitory nucleic acid molecule contains 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 that is complementary to at least 15 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 16 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 17 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 18 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 19 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 20 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 21 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 22 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 23 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 24 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 25 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 26 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 27 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 28 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 29 consecutive nucleotides set forth in SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence that is complementary to at least 30 consecutive nucleotides set forth in SEQ ID NO: 16.

[0082] 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 a miRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an shRNA targeting AC9. Each of these forms will be further described below.

[0083] Small interfering RNA (siRNA)

[0084] The siRNA disclosed herein is a single-stranded (ss) or double-stranded (ds) nucleic acid molecule made of DNA, RNA, or both DNA and RNA (e.g., chimeras) that is complementary to the target gene and prevents the target's mRNA from being translated into protein. Once the siRNA molecule enters the cell, it is incorporated into the RNA-induced silencing complex (RISC). After the siRNA hybridizes with the target mRNA, the RISC complex will cut the target mRNA, thereby inactivating the target mRNA, resulting in reduced levels of the target's mRNA and protein.

[0085] In some embodiments, the siRNA of the present disclosure can 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).

[0086] In some embodiments, the siRNA of the disclosure can comprise a nucleotide sequence of 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).

[0087] Any length known in the art and previously unknown within the scope of this disclosure may be used in the present invention.

[0088] In some embodiments, the siRNA contains an antisense strand. In some embodiments, the antisense strand of the siRNA molecules of the present disclosure is between 10 and 30 nucleotides in length (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), between 15 and 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 between 18 and 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.

[0089] In some embodiments, the siRNA contains a sense strand. In some embodiments, the sense strand of the siRNA molecule of the present disclosure is between 10 and 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 between 14 and 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.

[0090] In some embodiments, the sense and antisense strands of the siRNA molecules of the present disclosure are fully complementary. In some embodiments, the sense and antisense strands of the siRNA molecules of the present disclosure are fully complementary to the extent that their lengths overlap with each other. Depending on the sequence of the first and second strands, complementarity need not be complete or perfect, meaning that the first and second strands are not 100% base paired due to mismatches. One or more mismatches can be present in a dssiRNA without affecting the ability of the siRNA to reduce expression of the target gene of interest.

[0091] The nucleotide sequence of the siRNA disclosed herein can contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9 mRNA) such that the siRNA can hybridize to 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 (e.g., AC9 mRNA) or a portion thereof. In some embodiments, the siRNA is complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof.

[0092] In some embodiments, the siRNA nucleotide sequence may contain sufficient complementarity to the exon sequence of the target gene of interest (e.g., the exon of AC9). In some embodiments, the siRNA nucleotide sequence may contain sufficient complementarity to the intron sequence of the target gene of interest (e.g., the intron of AC9). In some embodiments, the siRNA of the present disclosure may contain sufficient complementarity to the pre-mRNA transcript or mRNA transcript encoding AC9. The target sequence of interest may be any one of SEQ ID NOs: 11-15 (e.g., see Table 2). The target gene of interest (e.g., AC9) may be any one of SEQ ID NOs: 16-17 (e.g., see Table 3).

[0093] In certain embodiments, siRNA as described herein has 0-7 nucleotide 3 ' overhangs or 0-4 nucleotide 5 ' overhangs. In certain embodiments, the siRNA molecule has a single uracil (e.g., U) overhang at each 3 ' end of the siRNA. In certain embodiments, the siRNA molecule has a double uracil (e.g., UU) overhang at each 3 ' end of the siRNA. In certain embodiments, the siRNA molecule has a single thymine (e.g., T) overhang at each 3 ' end of the siRNA. In certain embodiments, the siRNA molecule has a double thymine (e.g., TT) overhang at each 3 ' end of the siRNA. In certain embodiments, the siRNA molecule has a cytosine and thymine (e.g., CT) overhang at each 3 ' end of the siRNA.

[0094] Different siRNAs can be combined to reduce protein expression of a target gene of interest (e.g., AC9). In the methods of the present invention, a combination of two siRNAs can be used, such as two different siRNAs, three different siRNAs, four different siRNAs, or five different siRNAs targeting the same target gene (e.g., AC9 or a variant thereof). In some embodiments, the siRNA sequence can contain 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 (e.g., see Table 1), or their complements. In some embodiments, the siRNA sequence can contain the sequence of any one or more of SEQ ID NOs: 1-10 (e.g., see Table 1), or their complements.

[0095] In some embodiments, the siRNA contains at least 15 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains at least 16 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains at least 17 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains at least 18 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains at least 19 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains at least 20 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains 21 consecutive nucleotides listed within any one of SEQ ID NOs: 1-10 (e.g., see Table 1).

[0096] Table 1. Exemplary siRNA sequences

[0097]

[0098]

[0099] A = adenine; C = cytosine; G = guanine; T = thymine; U = uracil. Note: When reading from 5' to 3', the RNA sequence of SEQ ID NO: 1 contains thymine nucleotides at positions 20-21; when reading from 5' to 3', the RNA sequence of SEQ ID NO: 2 contains thymine nucleotides at position 21.

[0100] In some embodiments, the siRNA of the present disclosure can target the nucleotide sequence of any one of SEQ D NOs: 11-15 (e.g., see Table 2), or a complementary sequence thereof, or a variant thereof that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0101] Table 2. Target sequences

[0102] SEQ ID NO mRNA sequence (5' to 3') SEQ ID NO: 11 CCUGAUGAAAGAUUACUUU SEQ ID NO: 12 CGAAAUGGAAGAUGGGAAA SEQ ID NO: 13 CGAAACAGGAAUAGAAGAA SEQ ID NO: 14 AGGAAGAGGUCAUAAAGAA SEQ ID NO: 15 GCACCAAGAUCCAGAGCAU

[0103] In some embodiments, the siRNA comprises a sequence that is complementary to at least 15 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 16 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 17 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 18 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 19 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 20 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 21 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 22 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 23 consecutive nucleotides listed within SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 24 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 25 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 26 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 27 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 28 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 29 consecutive nucleotides listed in SEQ ID NO: 16. In some embodiments, the siRNA comprises a sequence that is complementary to at least 30 consecutive nucleotides listed in SEQ ID NO: 16. The nucleotide sequence of SEQ ID NO: 16 is listed in Table 3.

[0104] In some embodiments, the siRNA comprises a sequence that is complementary to at least 15 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 17 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 17 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 18 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 19 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 20 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 21 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 22 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 23 consecutive nucleotides listed within SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 24 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 25 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 26 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 27 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 28 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 29 consecutive nucleotides listed in SEQ ID NO: 17. In some embodiments, the siRNA comprises a sequence that is complementary to at least 30 consecutive nucleotides listed in SEQ ID NO: 17. The nucleotide sequence of SEQ ID NO: 17 is listed in Table 3.

[0105] Table 3. Adenylate cyclase 9 sequence

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] Double-stranded RNA (dsRNA)

[0220] The dsRNA disclosed herein is a ds nucleic acid molecule made from DNA, RNA, or both DNA and RNA (e.g., a chimera) that is complementary to the target gene and prevents the target's mRNA from being translated into protein. Typically, dsRNA is longer than siRNA and is processed to form siRNA molecules within the cell. siRNA is then incorporated into an RNA-induced silencing complex (RISC). After siRNA hybridizes with the target mRNA, the RISC complex will cut the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target.

[0221] In some embodiments, the dsRNA of the present disclosure may include a sense strand and an antisense strand, each strand comprising a length of about 25 to about 5000 nucleotides or longer (e.g., a length of 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 115, about 120, about 125, about 130, or more). , 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 140 0, about 1500, 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).

[0222] In some embodiments, the dsRNA of the present disclosure may comprise a sense strand and an antisense strand, each strand comprising 25 to 5000 nucleotides in length or longer (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, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, , 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, 150 0, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides).

[0223] Any length known in the art and previously unknown within the scope of this disclosure may be used in the present invention.

[0224] In some embodiments, the sense and antisense strands of the dsRNA molecules of the present disclosure are fully complementary. In some embodiments, the sense and antisense strands of the dsRNA molecules of the present disclosure are fully complementary to the extent that their lengths overlap. Depending on the sequence of the first and second strands, complementarity need not be complete or perfect, meaning that the first and second strands are not 100% base paired due to mismatches. One or more mismatches can be present in a dsRNA without affecting the ability of the dsRNA to reduce expression of the target gene of interest.

[0225] The nucleotide sequence of the dsRNA disclosed herein can contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9 mRNA) such that the dsRNA can hybridize to the target gene of interest. In some embodiments, the 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 (e.g., AC9 mRNA) or a portion thereof. In some embodiments, the dsRNA is complementary to the target gene of interest (e.g., AC9 mRNA) or a portion thereof.

[0226] In some embodiments, the nucleotide sequence of the dsRNA may contain 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 contain 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 contain 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 (e.g., see Table 3).

[0227] Different dsRNAs can be combined to reduce protein expression of a target gene of interest (e.g., AC9). In the methods of the invention, a combination of two dsRNAs can be used, such as two different dsRNAs, three different dsRNAs, four different dsRNAs, or five different dsRNAs targeting the same target gene (e.g., AC9 or a variant thereof).

[0228] Antisense Oligonucleotides (ASOs)

[0229] The disclosed ASOs are single-stranded (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., chimeras) that are complementary to the target gene of interest and prevent the target mRNA from being translated into protein. After hybridizing with the target mRNA, RNase H will degrade the mRNA by hydrolysis, resulting in reduced levels of the target mRNA and protein.

[0230] In some embodiments, the ASOs of the present disclosure may comprise a nucleotide sequence of 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).

[0231] In some embodiments, the ASOs of the present disclosure may comprise a nucleotide sequence of 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).

[0232] Any length known in the art and previously unknown within the scope of this disclosure may be used in the present invention.

[0233] The nucleotide sequence of the ASO can contain sufficient complementarity to a portion of the target gene of interest (e.g., AC9 mRNA) such 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.

[0234] In some embodiments, the nucleotide sequence of the ASO may contain sufficient complementarity to the exon sequence of the target gene of interest (e.g., the exon of AC9). In some embodiments, the nucleotide sequence of the ASO may contain sufficient complementarity to the intron sequence of the target gene of interest (e.g., the intron of AC9). In some embodiments, the ASO of the present disclosure may contain sufficient complementarity to the 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 (e.g., see Table 3).

[0235] Different ASOs can be combined to reduce protein expression of a target gene of interest (e.g., AC9). In the methods of the present invention, a combination of two ASOs, such as two different ASOs, three different ASOs, four different ASOs, or five different ASOs targeting the same target gene (e.g., AC9 or a variant thereof) can be used.

[0236] microRNA (miRNA)

[0237] The miRNA disclosed herein is a single-stranded (ss) nucleic acid molecule made of DNA, RNA, or both DNA and RNA (e.g., a chimera) that is complementary to the target gene and prevents the target mRNA from being translated into protein. Once the miRNA molecule enters the cell, it is incorporated into the RNA-induced silencing complex (RISC). After the miRNA hybridizes with the target mRNA, the RISC complex will cut the target mRNA, thereby inactivating the target mRNA, resulting in reduced levels of the target mRNA and protein.

[0238] In some embodiments, the miRNA of the present disclosure can comprise a nucleotide sequence of 6 to 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).

[0239] In some embodiments, the miRNA of the present disclosure can comprise a nucleotide sequence of 6 to 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).

[0240] The nucleotide sequence of the miRNA disclosed herein may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9 mRNA) such 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.

[0241] In some embodiments, the nucleotide sequence of the miRNA may contain sufficient complementarity to the exon sequence of the target gene of interest (e.g., the exon of AC9). In some embodiments, the nucleotide sequence of the miRNA may contain sufficient complementarity to the intron sequence of the target gene of interest (e.g., the intron of AC9). In some embodiments, the miRNA of the present disclosure may contain sufficient complementarity to the 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 (e.g., see Table 3).

[0242] Different miRNAs can be combined to reduce protein expression of a target gene of interest (e.g., AC9). In the methods of the present invention, a combination of two or more miRNAs can be used, such as two different miRNAs, three different miRNAs, four different miRNAs, or five different miRNAs targeting the same target gene (e.g., AC9 or a variant thereof).

[0243] Short hairpin RNA (shRNA)

[0244] The shRNA disclosed herein is an ss or ds nucleic acid molecule made from DNA, RNA, or both DNA and RNA (e.g., a chimera) that is complementary to the target gene and prevents the target mRNA from being translated into protein. Once the shRNA molecule enters the cell, it is incorporated into the RNA-induced silencing complex (RISC). After the shRNA hybridizes with the target mRNA, the RISC complex will cut the target mRNA, thereby inactivating the target mRNA, resulting in reduced levels of the target mRNA and protein.

[0245] In some embodiments, the shRNA of the disclosure may comprise a nucleotide sequence of 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).

[0246] In some embodiments, the shRNA of the disclosure can comprise a nucleotide sequence of 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).

[0247] The shRNA disclosed herein contains a variable hairpin loop structure and a stem sequence. In some embodiments, the length of the stem sequence can be 10 to 50 nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the length of the hairpin size is between 4 and 50 nucleotides, although the loop size may be larger, it will not significantly affect the silencing activity. The shRNA molecules disclosed herein may contain mismatches, such as s GU mismatches between the two strands of the hRNA stem do not reduce potency. In some embodiments, shRNAs are designed to include one or several GU pairs in the hairpin stem to stabilize the hairpin, for example, during bacterial propagation.

[0248] The nucleotide sequence of the shRNA can contain sufficient complementarity to a portion of the target gene of interest (e.g., AC9 mRNA) such that the shRNA can hybridize to 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.

[0249] In some embodiments, the nucleotide sequence of the shRNA may contain sufficient complementarity to the exon sequence of the target gene of interest (e.g., the exon of AC9). In some embodiments, the nucleotide sequence of the shRNA may contain sufficient complementarity to the intron sequence of the target gene of interest (e.g., the intron of AC9). In some embodiments, the shRNA of the present disclosure may contain sufficient complementarity to the 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 (e.g., see Table 3).

[0250] Different shRNAs can be combined to reduce protein expression of a target gene of interest (e.g., AC9). In the methods of the present invention, a combination of two or more shRNAs can be used, such as two different shRNAs, three different shRNAs, four different shRNAs, or five different shRNAs targeting the same target gene (e.g., AC9 or a variant thereof).

[0251] Modification of inhibitory nucleic acid molecules

[0252] It is contemplated that any inhibitory nucleic acid molecule disclosed herein can be used in the methods disclosed herein in unmodified or modified form. Unmodified inhibitory nucleic acid molecules contain nucleobases including 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.

[0253] Modification can be achieved by systematically adding or removing linked nucleosides to produce longer or shorter sequences.

[0254] Modifications can be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative internucleoside linkages, which are described further below. Typically, these types of modifications are introduced to optimize the efficacy or biophysical properties of the molecule (e.g., to increase serum stability or circulating half-life, increase thermal stability, enhance transmembrane delivery, reduce immunogenicity, and / or target a specific location or cell type).

[0255] Modification can be further accomplished by covalently or non-covalently conjugating a moiety (eg, a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer) to the 5' end and / or the 3' end of the inhibitory nucleic acid molecule, as described in more detail below.

[0256] Nucleoside modification

[0257] 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 other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo Uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. Inhibitory nucleic acid molecules can also include nucleobases in which purine or pyrimidine bases are replaced by other heterocycles, such as 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. Pat. 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.

[0258] Sugar modification

[0259] Modifications of the inhibitory nucleic acid molecules described herein can also include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2'-modifications that can modify the inhibitory nucleic acid molecules described herein include all possible orientations of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Other potential sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluorine (F). The 2'-sugar substituent can be in the arabinose (upper) position or the ribose (lower) position. In some embodiments, the 2'-arabinose 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 a 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0260] Internucleoside bond modification

[0261] 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, including 3′-alkylenephosphonates and 5′-alkylenephosphonates; phosphinates; phosphoramidates, including 3′-alkylenephosphonates; ′ -aminophosphoramidates and aminoalkylphosphoramidates; thiophosphoramidates; thioalkylphosphonates; thioalkylphosphotriesters: selenophosphoroates; and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these esters, and those with reversed polarity where one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages.

[0262] Conjugate

[0263] Any inhibitory nucleic acid molecule described herein can be modified by adding an auxiliary moiety (e.g., a cell penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety). The auxiliary moiety can be present as a 5' terminal modification (e.g., covalently bonded to the 5' terminal nucleoside), a 3' terminal modification (e.g., covalently bonded to the 3' terminal nucleoside), or an internucleoside linkage (e.g., covalently bonded to a phosphate or phosphorothioate in an internucleoside linkage).

[0264] CPPs are known in the art (eg, 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.

[0265] The inhibitory nucleic acid molecules of the present disclosure can include a covalently attached auxiliary portion based on a neutral polymer. Neutral polymers include poly (C1-6 alkylene oxides), such as poly (ethylene glycol) and poly (propylene glycol) and copolymers thereof, such as diblock copolymers and triblock copolymers.

[0266] Inhibitory nucleic acid molecules containing a hydrophobic moiety can exhibit superior cellular uptake compared to inhibitory nucleic acid molecules lacking the hydrophobic moiety. The hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyllithocholic acid, or oleoylcholine acid), a glycolipid, a phospholipid, a sphingolipid, an isoprenoid, a vitamin, a saturated fatty acid, an unsaturated fatty acid, a fatty acid ester, a triglyceride, a pyrene, a porphyrin, a texaporphyrin, an adamantane, an acridine, a biotin, a coumarin, a fluorescein, a rhodamine, a Texas Red, a digoxigenin, a dimethoxytrityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a cyanine dye (e.g., Cy3 or Cy5), a Hoechst 33258 dye, a psoralen, or an ibuprofen) covalently linked to the nucleic acid backbone (e.g., the 5'-end) of the inhibitory nucleic acid molecule.

[0267] The targeting moiety is selected based on its ability to target the oligonucleotide of the invention to a desired or selected cell population that expresses the corresponding binding partner (e.g., corresponding receptor or ligand) of the selected targeting moiety. For example, the oligonucleotide of the invention can be targeted to hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) by selecting a targeting moiety containing N-acetylgalactosamine (GalNAc).

[0268] The targeting moiety can include one or more ligands (e.g., 1 to 9 ligands, 1 to 6 ligands, 1 to 3 ligands, 3 ligands, or 1 ligand). The ligand can target cells expressing asialoglycoprotein receptor (ASGP-R), IgA receptor, HDL receptor, LDL receptor, or transferrin receptor. Non-limiting examples of ligands include N-acetylgalactosamine (e.g., triantennary N-acetylgalactosamine), glycyrrhetinic acid, glycyrrhizin, lactobionic acid, lactoferrin, IgA, or bile acids (e.g., lithocholoyltaurine or taurocholic acid).

[0269] 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 (e.g., Fcab or fusion protein (e.g., scFv)).

[0270] Preparation of inhibitory nucleic acid molecules

[0271] 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, a synthesis cycle based on phosphoramidite can be used to prepare the inhibitory nucleic acid molecules of the present disclosure. The synthesis cycle comprises the following steps: (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 connected by a phosphite, (3) repeating steps (1) and (2) one or more times as needed, (4) capping the 5'-end, and (5) oxidizing or sulfiding the internucleoside phosphite. Reagents and reaction conditions that can be used for oligonucleotide synthesis are known in the art.

[0272] As a result of solid phase synthesis, the inhibitory nucleic acid molecules disclosed herein can be connected to a solid support. Useable cleavable solid supports are known in the art. Non-limiting examples of solid supports include, for example, controlled pore glass or macroporous polystyrene bonded to a chain by a cleavable linker (e.g., a linker based on succinate) known in the art (e.g., UnyLinker™). By cutting the linker connecting the nucleic acid and the solid support, the nucleic acid connected to the solid support can be removed from the solid support.

[0273] Composition

[0274] Inhibitory nucleic acid molecules as described herein can be formulated into various compositions (e.g., pharmaceutical compositions) for administration to subjects in a biocompatible form suitable for in vivo administration. For example, inhibitory nucleic acid molecules as described herein (e.g., siRNA molecules of SEQ ID NO: 1-10 or variants thereof) can be administered in a suitable diluent, carrier, or excipient, and can further contain a preservative, such as to prevent the growth of microorganisms. Conventional procedures and ingredients for selecting and preparing suitable compositions are described in, for example, Remington, JP The Science and Practice of Pharmacy, Easton, Pennsylvania Mark Publishing Company, 2012, 22nd edition, and The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0275] Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, such as non-human animals, such as non-human mammals. It is fully understood that pharmaceutical compositions suitable for administration to humans are modified to make these compositions suitable for administration to various animals, and ordinary veterinary pharmacologists can design and / or perform such modifications only by ordinary (if any) experiments. It is contemplated that the subject to whom the pharmaceutical composition is administered includes, but is not limited to, humans and / or other primates and mammals.

[0276] The compositions containing the inhibitory nucleic acids described herein can further include a second therapeutic agent (e.g., a nucleic acid molecule, polypeptide, or drug to be expressed in a cell). For example, the second therapeutic agent can be a blood pressure medication, an anti-inflammatory drug (e.g., a steroid or colchicine), or an immunosuppressant. In some embodiments, 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., bepedic acid), lipoprotein (a) (Lp(a)) inhibitors (e.g., siRNA targeting Lp(a)), angiopoietin-like protein 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).

[0277] In some embodiments, the second therapeutic agent (e.g., statin) is administered in combination with an inhibitory nucleic acid molecule of the present disclosure. In some embodiments, the statin is administered orally to the subject. In some embodiments, the statin is administered daily to the subject.

[0278] Treatment

[0279] The present disclosure provides methods for reducing LDL in the serum of a subject. In some embodiments, the method comprises the step of administering to the subject an inhibitory nucleic acid molecule described herein, wherein the inhibitory nucleic acid molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject an siRNA molecule described herein (e.g., SEQ ID NO: 1-10 or variants thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject a dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject an ASO molecule described herein, wherein the ASO molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of 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 comprises the step of administering to the subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0280] The present disclosure provides methods for increasing LDLr in a subject. In some embodiments, the method comprises the step of administering to the subject an inhibitory nucleic acid molecule described herein, wherein the inhibitory nucleic acid molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject an siRNA molecule described herein (e.g., SEQ ID NO: 1-10 or variants thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject a dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of administering to the subject an ASO molecule described herein, wherein the ASO molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method comprises the step of 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 comprises the step of administering to the subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0281] Any of these methods can administer a composition (e.g., a pharmaceutical composition) or a 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).

[0282] Delivery vehicle

[0283] Any suitable delivery vehicle can be used to deliver the inhibitory nucleic acid molecules of the present disclosure to a subject (e.g., a human). For example, the delivery vehicle of any inhibitory nucleic acid molecule described herein can be a vector, a plasmid, or a nanoparticle (e.g., a micelle, a liposome, an exosome, or a lipid nanoparticle (LNP)).

[0284] The inhibitory nucleic acid molecules and compositions thereof disclosed herein can be delivered to a subject via a vector (e.g., a viral vector). Any suitable viral vector system can be used, including, for example, adenovirus (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdovirus (e.g., vesicular stomatitis virus), retrovirus, adeno-associated vector, poxvirus, herpes virus vector, and Sindbis virus vector.

[0285] The inhibitory nucleic acid molecules and compositions thereof disclosed herein can be delivered to a subject via liposomes. Liposomes are artificially prepared vesicles that can be mainly composed of lipid bilayers and can be used as delivery vehicles for administering inhibitory nucleic acids and compositions thereof as described herein. Liposomes can have different sizes, such as but not limited to diameters of hundreds of nanometers and can contain a series of concentric bilayer multilamellar vesicles (MLVs) separated by narrow aqueous compartments, small unilamellar vesicles (SUVs) with a diameter of less than 50 nm, and large unilamellar vesicles (LUVs) with a diameter of between 50 nm and 500 nm. Liposome design can include but is not limited to opsonins or ligands to improve the attachment or activation events of liposomes to unhealthy tissues, such as but not limited to endocytosis. Liposomes can contain low or high pH to improve the delivery of pharmaceutical compositions.

[0286] The inhibitory nucleic acid molecules and compositions thereof disclosed herein can be delivered to a subject via exosomes. Exosomes produced by cells can be collected from cell culture media 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 (<20,000 g) centrifugation to precipitate larger particles, followed by high-speed (>100,000 g) centrifugation to precipitate exosomes, size filtration with an appropriate filter (e.g., a 0.22 micron filter), gradient ultracentrifugation (e.g., with a sucrose gradient), or a combination of these methods.

[0287] The inhibitory nucleic acid molecules of present disclosure and compositions thereof can be delivered to experimenter via LNP.For example, inhibitory nucleic acid molecules (for example, siRNA, dsRNA, ASO, miRNA or shRNA) can be formulated in lipid nanoparticles, as those described in International Publication No. WO 2012170930, which are incorporated herein by reference in their entirety. As non-limiting examples, LNP formulations can contain cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(ω-methoxy poly (ethylene glycol) 2000) carbamoyl)]-1,2-dimyristyloxy-propyl-3-amine (PEG-c-DOMG), distearoyl-rac-glycerol (DSG) and / or dimethylaminobutyrate (DMA). As non-limiting examples, compared with cationic lipids, DSPC and cholesterol, the lipid mol ratio of PEG-c-DOMG is 1%-5%. In another embodiment, the PEG-c-DOMG can be replaced by 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). Cationic lipids can be selected from any lipid known in the art, such as, but not limited to, 4-(dimethylamino)butanoic acid (6Z, 9Z, 28Z, 31Z)-heptatriacontane-6,9,28,31-tetraene-19-yl ester (DLin-MC3-DMA), 1,2-dilinoleyloxy-n, n-dimethyl-3-aminopropane (DLin-DMA), C 12-200 and N, N-dimethyl-2,2-bis-(9Z, 12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine (DLin-KC2-DMA).

[0288] Exemplary commercial reagents that can be used for lipid-based delivery of inhibitory nucleic acid molecules include, but are not limited to, TransIT-TKO TM (Mirus, catalog number MIR 2150), TransmessengerTM (Qiagen, catalog number 301525), Oligofectamine TM and Lipofectamine TM (Invitrogen, Cat. No. MIR12252-011 and Cat. No. 13778-075), siPORT TM (Ambion, Cat. No. 1631) and DharmaFECT TM (Fisher Scientific, catalog number T-2001-01).

[0289] dose

[0290] The actual dosage of the composition of the disclosure applied to the subject can be determined by physical and physiological factors, such as body weight, the severity of the disease, previous or ongoing therapeutic intervention, the patient's spontaneous onset and route of administration.Depending on dosage (for example, mg / kg) and route of administration, the number of administrations of preferred dosage and / or effective dose can change according to the response of the subject.In any case, the practitioner responsible for administration will determine the concentration of one or more active components in the composition and one or more appropriate dosages of individual subjects.Administer any suitable number of times every day, and continue the necessary time as required.The subject can be an adult or a child, suffering from or not suffering from comorbidity.

[0291] Route of administration

[0292] The compositions used in the methods described herein can be administered to a subject by any suitable route of administration. For example, compositions containing the inhibitory nucleic acids of the present disclosure can be administered intramuscularly, intravenously, intradermally, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation, by injection, by infusion, by continuous infusion, by direct local perfusion bathing target cells, by catheter, by lavage, in a cream, or in a lipid composition.

[0293] In some embodiments, the compositions used in the methods described herein can be administered intravenously to a subject. In some embodiments, the compositions used in the methods described herein can be administered subcutaneously to a subject.

[0294] Examples

[0295] Example 1. In vitro effects of siRNA targeting human AC9

[0296] The following examples describe the materials and methods used to obtain the results described herein.

[0297] Materials and methods

[0298] Cell culture

[0299] HepG2 (hepatocellular carcinoma) cells were grown in Eagle's Minimum Essential Medium (EMEM). The medium was supplemented with 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin. The cells were cultured at 37°C in 5% CO2 and harvested weekly using trypsin-EDTA. For the experiments, the cells were trypsinized, seeded, and cultured for at least 3 days prior to the assay.

[0300] siRNA transfection

[0301] Unless otherwise indicated, HepG2 cells were transfected with ADCY9, SREBP2, LDLr, or scramble siRNA in the presence of Lipofectamine RNAiMAX in Opti-MEM for 72 h.

[0302] Cellular cholesterol efflux

[0303] The cells were cultured at 37°C in a 2 μCi / ml [1,2- 3 [H] cholesterol was labeled for 24 hours in EMEM supplemented with 1% FBS. Cells were then equilibrated in EMEM supplemented with 0.2% BSA for 18 hours at 37°C, in the presence or absence of H89 or GSK-2033. Efflux assays were performed in the absence or presence of 10 μg / ml apo AI. At the end of the incubation period, the culture medium was harvested and the cells were lysed. Radioactivity was counted in the culture medium and cells in a beta counter. Percent efflux was calculated by subtracting the radioactivity count in the culture medium without the cholesterol acceptor from the radioactivity count in the culture medium with the acceptor present, then dividing by the sum of the radioactivity count in the culture medium plus the cell fraction.

[0304] Isolation and radiolabeling of lipoproteins

[0305] Lipoproteins were isolated from human plasma obtained from BioIVT (Westbury, NY). Prior to isolation, 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). 1,2-[ 3[H] cholesterol oleate was used to label LDL. Labeled LDL was then isolated by ultracentrifugation. The specific activity of labeled LDL in CE ranged from 7,000 to 12,000 cpm / μg protein.

[0306] Lipoprotein cell association assay

[0307] The cells were measured in 12-well plates at 37°C. 3 [H] CE-lipoprotein (20 μg protein / ml) cell association for 4 h. The cells were washed twice with 1 ml phosphate-buffered saline (PBS) and incubated in a total volume of 250 μl containing 125 μl culture medium (2×), 4% bovine serum albumin, pH 7.4 (total binding). Nonspecific association was assessed by adding 1.5 mg protein / ml unlabeled lipoprotein. At the end of the incubation, the cells were washed twice with 1 ml PBS containing 0.2% BSA (PBS-BSA) and then twice with 1 ml PBS. The cells were then dissolved in 1.5 ml 0.2N NaOH. Radioactivity counts in the homogenate were obtained using a beta counter. To compare CE ( 3 H) association of labeled lipoproteins, the association data were estimated as micrograms of protein per milligram of cellular protein (apparent uptake). To achieve this, [ 3 The specific activity of [H]CE-lipoprotein was expressed as counts per μg of lipoprotein protein. Specific association was calculated by subtracting nonspecific association from total association.

[0308] Immunoblotting

[0309] 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 a protease inhibitor cocktail). The lysate was microcentrifuged at 4°C for 20 min, and the supernatant was evaluated for cellular protein. Proteins (30-50 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently Western blotted onto a PVDF membrane.

[0310] Quantification of mRNA expression by reverse transcription-quantitative PCR

[0311] According to the manufacturer's protocol, total RNA from HepG2 cells was extracted using the RNeasy isolation kit. cDNA was synthesized using MultiScribe reverse transcriptase using components from a high-capacity cDNA reverse transcription kit according to the manufacturer's procedure. RNA was quantified by the Quant-it RiboGreen RNA assay kit according to the manufacturer's procedure, 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 PPIA and TBP were selected for normalization using Bio-Rad CFX Maestro software (which uses the GeNorm method). qPCR was performed using the SYBR-Green reaction mixture. qPCR conditions consisted of an initial denaturation at 95°C for 5 minutes, followed by 40 amplification cycles, each cycle consisting of 15 seconds at 95°C and 60 seconds at 60°C. The results were analyzed using the Δ-ΔCt method using Bio-Rad CFX Maestro software.

[0312] SREBP-2 transcriptional activity

[0313] SREBP-2 transcriptional activity was estimated using a kit, in which SREBP-2 contained in nuclear extracts obtained from transfected HepG2 cells specifically bound to immobilized SREBP-response element oligonucleotides according to the kit supplier's protocol and was detected by adding a specific primary antibody against SREBP-2.

[0314] Proteomic analysis using tandem mass tags (TMT)

[0315] HepG2 cells were transfected and harvested 3 days later for non-targeted proteomic analysis. Briefly, eight tandem mass spectrometry tags (TMTs) were used to label two different conditions (siScramble and siAC9) for four different assays. Once labeled, all samples were pooled and analyzed in a single liquid chromatography-mass spectrometry (LC-MS) experiment.

[0316] Other methods

[0317] The protein content was determined by the Lowry method using BSA as a standard.

[0318] result

[0319] The protein expressions of adenylate cyclase type 9 (AC9), low-density lipoprotein receptor (LDLr), and ATP-binding cassette subfamily A member 1 (ABCA1) in HepG2 cells were analyzed after siRNA-mediated AC9 knockdown (KD). Figure 1A The protein expression of AC9 was significantly decreased, while the protein expression of LDLr and ABCA1 was significantly increased ( Figure 1B ). After siRNA-mediated KD of AC9, 3 H-CE-LDL association ( Figure 1C )and 3 H-CE-LDL cholesterol efflux ( Figure 1D ) showed a significant increase.

[0320] After siRNA-mediated KD of AC9, the protein expression of AC9, LDLr, sterol regulatory element binding protein-2 (SREBP-2), and ABCA1 in HepG2 cells was analyzed by non-targeted relative proteomics. The peptide level of AC9 was significantly decreased, while the peptide levels of LDLr and SREBP2 were significantly increased ( Figure 2A ).

[0321] After siRNA-mediated KD of AC9, the protein expression of protein kinase cAMP-dependent type I regulatory subunit alpha (PRKKAR1A), protein kinase cAMP-dependent type I regulatory subunit beta (PRKAR1B), and A-kinase anchoring protein 12 (AKAP12) in HepG2 cells was analyzed by non-targeted relative proteomics. The levels of PRKAR1A, PRKAR1B, and AKAP12 peptides were significantly increased ( Figure 2B ).

[0322] After siRNA-mediated KD of AC9 (siAC9), the expression of 5 μM atorvastatin (Ato) in HepG2 cells was analyzed with and without co-treatment. 3 H-CE-LDL association. Co-treatment with siAC9 and Ato produced an additive effect on LDL cholesterol uptake by LDLr ( Figure 3 ).

[0323] LDLr expression in HepG2 cells was analyzed after treatment with 300 μM exogenous cAMP (to replicate the AC9 knockdown effect), 5 μM Ato, or both cAMP and Ato. The combined effect of exogenous cAMP and atorvastatin increased LDLr protein expression ( Figure 4 ).

[0324] After siRNA-mediated KD of AC9, LDLr and ABCA1 expression in HepG2 cells was analyzed with and without co-treatment with 2 μM H89 (PKA inhibitor). siRNA-mediated KD of AC9 resulted in increased LDLr and ABCA1 expression; however, this increased expression was reversed after co-treatment with the PKA inhibitor H89 ( Figures 5A-5B). In addition, the expression of siAC9 and H89 in HepG2 cells was observed. 3 H-CE-LDL association was reduced without reducing cholesterol efflux ( Figures 5C-5D Together, these results indicate that the increases in LDLr and ABCA1 observed after KD of AC9 are primarily PKA-dependent.

[0325] After siRNA-mediated KD of AC9, the gene expressions of AC9, SREBP2, proprotein convertase subtilisin / kexin type 9 (PCSK9), and LDLr in HepG2 cells were analyzed ( Figure 6 These results indicate that AC9 siRNA increases the mRNA expression of SREBP2 and LDLR, but does not increase the mRNA expression of PCSK9.

[0326] The SREBP2 transcriptional activity in HepG2 cells was analyzed 24 and 48 hours after siRNA-mediated KD of AC9. These results showed that AC9 siRNA increased SREBP2 transcriptional activity ( Figure 7 ).

[0327] After siRNA-mediated KD of AC9, SREBP2, or both AC9 and SREBP2, LDLr protein expression in HepG2 cells was analyzed ( Figure 8 AC9 KD resulted in increased LDLr protein expression. These results suggest that SREBP2 siRNA can block siAC9-mediated increase in LDLr protein.

[0328] Analysis of cholesterol efflux to apoA-I in HepG2 cells after siRNA-mediated KD of AC9, SREBP2, or both AC9 and SREBP2 revealed a significant increase in cholesterol efflux only upon KD of AC9 ( Figure 9A Next, cholesterol efflux in HepG2 cells was studied with and without co-treatment with 5 μM GSK-2033 (LXR inhibitor) after siRNA-mediated KD of AC9. The LXR inhibitor was able to significantly reduce the siAC9-mediated increase in ABCA1-mediated cholesterol efflux ( Figure 9B These results suggest that LXR is involved in the effect of AC9 siRNA on cholesterol efflux through ABCA1.

[0329] After siRNA-mediated KD of AC9, LDLr, or both AC9 and LDLr, ABCA1 protein expression in HepG2 cells was analyzed ( Figure 10A Only KD of AC9 alone increased ABCA1 expression. Cholesterol efflux also increased after KD of AC9 ( Figure 10B). Cholesterol efflux was also increased after co-treatment with AC9 and LDLrsiRNA, although significantly less than that of AC9 siRNA alone. These results indicate that LDLrsiRNA partially blocks siAC9-mediated increase in ABCA1 protein.

[0330] The above results indicate that AC9 KD affects the expression and function of LDLr and ABCA1 (see, for example, Figure 11 ).

[0331] AC9 and LDLr protein expression were analyzed in HepG2 cells transfected with different siRNA sequences targeting ADCY9 mRNA at different exons. AC9 protein expression was significantly reduced, while LDLr protein expression was significantly increased by all siRNA sequences used (Table 4).

[0332] Table 4: Effects of various siRNA sequences targeting ADCY9 mRNA on AC9 and LDLr protein expression in HepG2 cells

[0333]

[0334] Table 4 shows AC9 and LDLr protein levels in HepG2 cells transfected with various siRNA sequences targeting ADCY9 mRNA at different exons, relative to a siScramble control. Results were obtained by Western blotting, with actin serving as a loading control. Protein expression is expressed as a percentage relative to the siScramble control. n = 8. Paired t-test: * = p ≤ 0.05; ** = p ≤ 0.01; and *** = p ≤ 0.001, relative to siScramble.

[0335] Other embodiments

[0336] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0337] While the invention has been described with reference to particular embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any changes, uses, or adaptations which follow generally in accordance with the principles and which include departures from the invention which come within the known or customary practice in the art to which this invention pertains and which may be adapted to the essential characteristics as hereinbefore set forth and appearing within the scope of the claims.

[0338] Other embodiments are within the claims.

Claims

1. A method of 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 inhibition comprises administering an inhibitory nucleic acid molecule to the subject.

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

3. The method of claim 1 or 2, wherein the adenylate cyclase is adenylate cyclase type 9 (AC9).

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

17.

5. The method of any one of claims 1-4, 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). The method of claim 4 , wherein the inhibitory nucleic acid molecule is siRNA.

7. 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. 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. 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. 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 method of any one of claims 5-9, wherein the siRNA molecule contains a 3' overhang selected from the group consisting of: (i) a single uracil overhang at one or more 3' ends of the siRNA; (ii) a biuracil 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) single cytosine and single thymine overhangs at one or more 3' ends of the siRNA.

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

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

10.

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

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

16. The method of any one of claims 5-12, wherein the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15.

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

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 protein 3 (ANGPTL3) inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, apolipoprotein B (ApoB) inhibitors, bile acid-binding resins, and colchicine.

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

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

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

22. The method of claim 18, wherein the Lp(a) inhibitor is a siRNA molecule targeting Lp(a).

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

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

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

10.

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

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

28. The siRNA molecule of claim 22, wherein the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15.

29. Use of an inhibitory nucleic acid molecule to reduce serum low-density lipoprotein (LDL) levels in a subject, wherein the expression or function of adenylate cyclase in the subject is inhibited by administering the inhibitory nucleic acid molecule.

30. Use of an inhibitory nucleic acid molecule to reduce LDL receptor expression in a subject, wherein the expression or function of adenylate cyclase in the subject is inhibited by administering the inhibitory nucleic acid molecule.

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

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