Methods for inhibiting adenylate cyclase type 9 (AC9) and uses thereof

Abstract

Described herein are compositions and methods useful for reducing the risk of a major adverse cardiovascular event, treating a cardiovascular condition, as well as decreasing glucose levels, reducing a risk of developing hyperglycemia, and treating hyperglycemia in a subject by decreasing the presence of an adenylate cyclase type 9 (AC9) and / or glucose-6-phosphatase catalytic subunit (G6PC) using inhibitory nucleic acid molecules.

Description

[0001] METHODS FOR INHIBITING ADENYLATE CYCLASE TYPE 9 (AC9) AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] This disclosure relates to compositions and methods useful for decreasing glucose levels and / or treating or reducing the risk of developing hyperglycemia in a subject.

[0004] BACKGROUND

[0005] Adenylate cyclases (ACs), also referred as adenyl cyclases and adenylyl cyclases, are regulatory enzymes that modulate signaling pathways and physiological responses in cells by converting adenosine triphosphate (ATP) to 3’,5’-cyclic AMP (cAMP), a key secondary 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. 102: 815-857), auto-inhibited by its C-terminal cytosolic domain (C2b) (Palvolgyi et al. (2018) Cell Signal. 51 :266-275), and endocytosed after stimulation by G-protein-coupled receptors (GPCRs) (Lazar et al. (2020) eLife 9: e58039).

[0006] Diabetes affects roughly 537 million adults worldwide. Diabetes results when the body does not produce enough insulin (Type I diabetes) or resists the effects of insulin (Type 2 diabetes); this may ultimately result in hyperglycemia in the subject. Because hyperglycemia can result in severe health consequences, diabetics (and non-diabetics frequently suffering from hyperglycemia) should monitor and maintain their blood glucose at baseline levels. However, blood glucose maintenance can be challenging, particularly in view of certain prescription drugs (e.g. , statins) known to naturally increase blood glucose levels, thereby increasing one’s risk of developing hyperglycemia. Therefore, there remains a need for compositions and methods of decreasing blood glucose in a subject having or at risk of developing hyperglycemia (e.g., diabetics).

[0007] Additionally, cardiovascular disease remains the leading cause of morbidity and mortality worldwide, with millions of individuals experiencing acute or chronic events that significantly impair quality of life and shorten survival. Among the most devastating conditions of cardiovascular disease are those collectively referred to as major adverse cardiovascular event (MACE). These events include acute and life-threatening clinical manifestations, such as myocardial infarction (Ml), stroke, cardiac arrest, arrhythmia (e.g., atrial fibrillation or ventricular tachycardia), and cardiovascular death of a subject. Patients who experience MACE frequently suffer long-term disability, recurrent hospitalizations, and increased risk of mortality. Therefore, there remains a need for compositions and methods of treating cardiovascular conditions and / or reducing the risk of MACE in a subject (e.g., a subject having or at risk of developing hyperglycemia (e.g., diabetics)).

[0008] SUMMARY OF THE INVENTION

[0009] The invention provides, inter alia, compositions (e.g., inhibitory nucleic acid molecules) and methods useful for reducing the risk of a major adverse cardiovascular event, treating a cardiovascular condition, as well as treating hyperglycemia or reducing the risk of hyperglycemia in a subject. In one aspect, the invention provides a method of reducing the risk of a major adverse cardiovascular event (MACE) in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0010] In some embodiments of the foregoing aspect, the MACE is selected from the group including myocardial infarction (Ml), a stroke, cardiac arrest, arrhythmia, and cardiovascular death.

[0011] In another aspect, the invention provides a method of treating a cardiovascular condition in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0012] In some embodiments of the foregoing aspect, the cardiovascular condition is selected from the group including: Ml, stroke, cardiac arrest, and arrhythmia.

[0013] In another aspect, the invention provides a method of decreasing glucose levels in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0014] In another aspect, the invention provides a method of decreasing adenylate cyclase type 9 (AC9, which is also referred to as ADCY9 herein) and / or glucose-6-phosphatase catalytic subunit (G6PC) in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0015] In some embodiments of any of the foregoing aspects, the subject is diagnosed with hyperglycemia.

[0016] In another aspect, the invention provides a method of reducing a risk of developing hyperglycemia in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0017] In another aspect, the invention provides a method of treating hyperglycemia in a subject, the method including inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting includes administration of an inhibitory nucleic acid molecule to the subject.

[0018] In some embodiments of any of the foregoing aspects, the method further includes administering in combination a second therapeutic agent to the subject.

[0019] In some embodiments, the second therapeutic agent is selected from the group consisting of a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, a biguanide, or a combination thereof (e.g. , a biguanide, such as metformin, in combination with a DPP-4 inhibitor, an insulin secretagogue, or a SGLT2 inhibitor).

[0020] In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®), saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or Ml NOD I AB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) ortosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0021] In some embodiments: (a) the the meglitinide is a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®); or (b) the sulfonylurea is selected from the group consisting of glipizide (e.g., GLUCOTROL® or MINODIAB®), glimepiride (e.g., AMARYL®), glyburide (e.g., GLYNASE®), or gliclazide (e.g., DIAMICRON® or DACADIS®).

[0022] In another aspect, the invention provides the use of an inhibitory nucleic acid moledule in the manufacture of a medicament for reducing the risk of MACE in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0023] In some embodiments of the foregoing aspect, the MACE is selected from the group including myocardial infarction (Ml), a stroke, cardiac arrest, arrhythmia, and cardiovascular death.

[0024] In another aspect, the invention provides the use of an inhibitory nucleic acid molecule in the manufacture of a medicament for treating a cardiovascular condition in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0025] In some embodiments of the foregoing aspect, the cardiovascular condition is selected from the group including: Ml, stroke, cardiac arrest, and arrhythmia.

[0026] In another aspect, the invention provides the use of an inhibitory nucleic acid molecule in the manufacture of a medicament for decreasing glucose levels in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0027] In another aspect, the invention provides the use of an inhibitory nucleic acid molecule in the manufacture of a medicament for decreasing AC9 or G6PC in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0028] In another aspect, the invention provides the use of an inhibitory nucleic acid molecule in the manufacture of a medicament for reducing a risk of developing hyperglycemia in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0029] In another aspect, the invention provides the use of an inhibitory nucleic acid molecule in the manufacture of a medicament for treating hyperglycemia in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

[0030] In some embodiments of any of the foregoing aspects, the subject has or is at risk of developing diabetes (e.g., type 1 diabetes or type 2 diabetes).

[0031] In some aspects, the subject is diagnosed with hyperglycemia.

[0032] In some embodiments of any of the foregoing aspects, the subject is to receive and / or has recently received (e.g., within the past 48 hours, e.g., within the past 1 , 2, 3, 4, 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, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48 hours) a statin, a steroid, a beta blocker, a thiazide diuretic, a quinolone antibiotic, an androgen deprivation therapy, an antipsychotic, a protease inhibitor, and / or a calcineurin inhibitor.

[0033] In some embodiments: (a) the statin is selected from the group including: simvastatin (e.g., ZOCOR®), atorvastatin (e.g., LIPITOR®), and rosuvastatin (e.g., CRESTOR®); (b) the steroid is prednisone (e.g., DELTASONE®); (c) the beta blocker is selected from the group including: atenolol (e.g., TENORMIN®), metoprolol (e.g., LOPRSSOR® or TORPOL®), and propranolol (e.g., INDERAL LA®); (d) the thiazide diuretic is hydrochlorothiazide (e.g., APO-HYDRO®, AQUAZIDE®, BPZIDE®, DICHLOTRIDE®, ESIDREX®, HYDROCHLOROT®, HYDRODIURIL®, HYDROSALURIC®, HYPOTHIAZID®, MICROZIDE®, or ORETIC®) or metolazone (e.g., ZYTANIX®, METOZ®, ZAROXOLYN®, or MYKROX®); (e) the quinolone antibiotic is gatifloxacin (e.g., GATIFLO®, TEQUIN®, or ZYMAR®) or levofloxacin (e.g., LEVAQUIN® or LEVOFLOXACIN SYSTEMIC®); (f) the androgen deprivation therapy includes leuprolide (e.g., CAMCEVI®, ELIGARD®, LUPRON®, LUPRON DEPOT®, LUPRON DEPOT-PED®, or VIADUR®), goserelin (e.g., ZOLADEX®), triptorelin (e.g., TRELSTAR®, TRELSTAR DEPOT®, TRELSTAR LA®, or TRIPTODUR®), or degarelix (e.g., FIRMAGON®); (g) the antipsychotic is olanzapine (e.g., ZYPREXA® or ZENTIVA®) or clozapine (e.g., CLOZARIL®, DENZAPINE®, or ZAPONEX®); (h) the protease inhibitor is selected from the group including: atazanavir (e.g., REYATAZ®), darunavir (e.g., PREZISTA®), and ritonavir (e.g., NORVIR®); or (i) the calcineurin inhibitor is selected from the group including: cyclosporine (e.g., GENGRAF®, NEORAL®, or SANDIMMUNE®), sirolimus (e.g., RAPAMUNE®), and tacrolimus (e.g., ASTAGRAF XL, ENVARSUS XR, PROGRAF, or PROTOPIC).

[0034] In some embodiments of any of the foregoing aspects, the adenylate cyclase is AC9.

[0035] In some embodiments, the AC9 includes: (i) an mRNA sequence of SEQ ID NO: 16; and / or (ii) a DNA sequence of SEQ ID NO: 17.

[0036] In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is an anti-sense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a double-stranded RNA (dsRNA), or a microRNA (miRNA).

[0037] In some embodiments, the inhibitory nucleic acid molecule is an siRNA molecule.

[0038] In some embodiments, the siRNA molecule includes a sequence complementary to at least 15 (e.g, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or 25) contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 16-27. In some embodiments, the siRNA molecule includes a sequence complementary to at least 19 (e.g, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, or 25) contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 16-27.

[0039] In some embodiments, the siRNA molecule includes a sequence complementary to at least 21 (e.g, at least 21 , at least 22, at least 23, at least 24, or 25) contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10, 16, and 17.

[0040] In some embodiments, the siRNA molecule includes a sequence complementary to at least 25 (e.g, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or 40) contiguous nucleotides set forth within any one of SEQ ID NOs: 16 and 17.

[0041] In some embodiments, the siRNA molecule contains 3’ overhangs selected from the group consisting of: (i) a single uracil (U) overhang at one or more 3’ ends of the siRNA; (ii) a double uracil (UU) overhang at one or more 3’ ends of the siRNA; (Hi) a single thymine (T) overhang at one or more 3’ ends of the siRNA; (iv) a double thymine (TT) overhang at one or more 3’ ends of the siRNA; or (v) a single cytosine and single thymine (CT) overhang at one or more 3’ ends of the siRNA.

[0042] In some embodiments, the siRNA molecule includes a nucleotide sequence of any one or more of SEQ ID NOs: 1-10 and and 18-27.

[0043] In some embodiments, the siRNA molecule includes: (i) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (ii) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (Hi) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (iv) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (v) a sense strand including the sequence of SEQ ID NO: 9 an antisense strand including the sequence of SEQ ID NO: 10; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21 ; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; and (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27.

[0044] In another aspect, the invention provides an siRNA molecule including: (i) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (ii) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (Hi) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (iv) a sense strand including the sequence of SEQ ID NO: 9 and an antisense strand including the sequence of SEQ ID NO: 10; (v) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; or (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27.

[0045] In another aspect, the invention provides an siRNA molecule including: (i) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (ii) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (Hi) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (iv) a sense strand including the sequence of SEQ ID NO: 9 and an antisense strand including the sequence of SEQ ID NO: 10; (v) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; or (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27 for use in a method of decreasing glucose levels in a subject, the method including administering the siRNA molecule to the subject.

[0046] In another aspect, the invention provides an siRNA molecule including: (i) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (ii) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (Hi) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (iv) a sense strand including the sequence of SEQ ID NO: 9 and an antisense strand including the sequence of SEQ ID NO: 10; (v) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; or (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27 for use in a method of decreasing AC9 or G6PC in a subject, the method including administering the siRNA molecule to the subject.

[0047] In some embodiments of any of the foregoing aspects, the subject is diagnosed with hyperglycemia.

[0048] In another aspect, the invention provides an siRNA molecule including: (i) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (ii) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (iii) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (iv) a sense strand including the sequence of SEQ ID NO: 9 and an antisense strand including the sequence of SEQ ID NO: 10; (v) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; or (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27 for use in a method of reducing a risk of developing hyperglycemia in a subject, the method including administering the siRNA molecule to the subject.

[0049] In another aspect, the invention provides an siRNA molecule including: (i) a sense strand including the sequence of SEQ ID NO: 3 and an antisense strand including the sequence of SEQ ID NO: 4; (ii) a sense strand including the sequence of SEQ ID NO: 5 and an antisense strand including the sequence of SEQ ID NO: 6; (iii) a sense strand including the sequence of SEQ ID NO: 7 and an antisense strand including the sequence of SEQ ID NO: 8; (iv) a sense strand including the sequence of SEQ ID NO: 9 and an antisense strand including the sequence of SEQ ID NO: 10; (v) a sense strand including the sequence of SEQ ID NO: 1 and an antisense strand including the sequence of SEQ ID NO: 2; (vi) a sense strand including the sequence of SEQ ID NO: 18 and an antisense strand including the sequence of SEQ ID NO: 19; (vii) a sense strand including the sequence of SEQ ID NO: 20 and an antisense strand including the sequence of SEQ ID NO: 21; (viii) a sense strand including the sequence of SEQ ID NO: 22 and an antisense strand including the sequence of SEQ ID NO: 23; (ix) a sense strand including the sequence of SEQ ID NO: 24 and an antisense strand including the sequence of SEQ ID NO: 25; or (x) a sense strand including the sequence of SEQ ID NO: 26 and an antisense strand including the sequence of SEQ ID NO: 27 for use in a method of treating hyperglycemia in a subject, the method including administering the siRNA molecule to the subject.

[0050] In some embodiments of any one of the foregoing aspects, the siRNA molecule includes a modification, wherein the modification is a non-natural or modified nucleoside or nucleotide.

[0051] In some embodiments, the modification is chosen from a 2'-O-methyl (2'-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2'-F) modified nucleoside.

[0052] In some embodiments, the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15.

[0053] In some embodiments, the subject has or is at risk of developing diabetes (e.g., type 1 diabetes or type 2 diabetes).

[0054] In some embodiments, the subject is to receive and / or has recently received (e.g., within the past 48 hours, e.g., within the past 1 , 2, 3, 4, 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, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48 hours) a statin, a steroid, a beta blocker, a thiazide diuretic, a quinolone antibiotic, an androgen deprivation therapy, an antipsychotic, a protease inhibitor, and / or a calcineurin inhibitor.

[0055] In some embodiments: (a) the statin is selected from the group including: simvastatin (e.g., ZOCOR®), atorvastatin (e.g., LIPITOR®), and rosuvastatin (e.g., CRESTOR®); (b) the steroid is prednisone (e.g., DELTASONE®); (c) the beta blocker is selected from the group including: atenolol (e.g., TENORMIN®), metoprolol (e.g., LOPRSSOR® or TORPOL®), and propranolol (e.g., INDERAL LA®); (d) the thiazide diuretic is hydrochlorothiazide (e.g., APO-HYDRO®, AQUAZIDE®, BPZIDE®, DICHLOTRIDE®, ESIDREX®, HYDROCHLOROT®, HYDRODIURIL®, HYDROSALURIC®, HYPOTHIAZID®, MICROZIDE®, or ORETIC®) or metolazone (e.g., ZYTANIX®, METOZ®, ZAROXOLYN®, or MYKROX®); (e) the quinolone antibiotic is gatifloxacin (e.g., GATIFLO®, TEQUIN®, or ZYMAR®) or levofloxacin (e.g., LEVAQUIN® or LEVOFLOXACIN SYSTEMIC®); (f) the androgen deprivation therapy includes leuprolide (e.g., CAMCEVI®, ELIGARD®, LUPRON®, LUPRON DEPOT®, LUPRON DEPOT-PED®, or VIADUR®), goserelin (e.g., ZOLADEX®), triptorelin (e.g., TRELSTAR®, TRELSTAR DEPOT®, TRELSTAR LA®, or TRIPTODUR®), or degarelix (e.g., FIRMAGON®); (g) the antipsychotic is olanzapine (e.g., ZYPREXA® or ZENTIVA®) or clozapine (e.g., CLOZARIL®, DENZAPINE®, or ZAPONEX®); (h) the protease inhibitor is selected from the group including: atazanavir (e.g., REYATAZ®), darunavir (e.g., PREZISTA®), and ritonavir (e.g., NORVIR®); or (i) the calcineurin inhibitor is selected from the group including: cyclosporine (e.g., GENGRAF®, NEORAL®, or SANDIMMUNE®), sirolimus (e.g., RAPAMUNE®), and tacrolimus (e.g., ASTAGRAF XL, ENVARSUS XR, PROGRAF, or PROTOPIC).

[0056] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject.

[0057] In some embodiments, the second therapeutic agent is selected from the group including: a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, or a biguanide. In some aspects: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0058] In some embodiments: (a) the the meglitinide is a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®); or (b) the sulfonylurea is selected from the group consisting of glipizide (e.g., GLUCOTROL® or Ml NOD I AB®), glimepiride (e.g., AMARYL®), glyburide (e.g., GLYNASE®), or gliclazide (e.g., DIAMICRON® or DACADIS®).

[0059] In some embodiments of any one of the foregoing aspects, the inhibitory nucleic acid molecule (e.g., siRNA molecule) is packaged inside a delivery vehicle.

[0060] In some embodiments, the delivery vehicle is a micelle, a liposome, an exosome, or a lipid nano particle (LNP).

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

[0063] FIG. 1 is a graph showing the quantification of glucose-6-phosphatase catalytic subunit (G6PC) mRNA levels measured by RNA sequencing after siRNA-mediated knockdown of adenylate cyclase type 9 (AC9) in HepG2 cells, relative to siScramble control. Error bars represent the mean ± standard deviation, n = 5. Paired t-test: * = p < 0.05 relative to siScramble.

[0064] FIG. 2 is a graph showing the quantification of G6PC mRNA levels measured by qPCR after siRNA-mediated knockdown of AC9 in HepG2 cells, relative to siScramble control. Error bars represent the mean ± standard deviation, n = 3. Paired t-test: *“ = p < 0.001 relative to siScramble.

[0065] FIG. 3 is a graph showing the quantification of glucose concentration in media of HepG2 cells transfected with an siRNA-mediated knockdown of AC9 and incubated 72 hours later in a glucose production media, relative to siScramble control. Glucose level is represented as a percentage of the control siScramble. Error bars represent the mean ± standard deviation, n = 1.

[0066] FIG. 4 is a set of graphs showing the plasma glucose level in AC9 wild-type (WT) and AC9- inactivated (AC9 GT / GT) mice fed with an atherogenic diet for 6 (left panel) or 16 (right panel) weeks and submitted to an intraperitoneal glucose tolerance test. Error bars represent the mean ± standard deviation, n = 13 (AC9 GT / GT) or 14 (AC9 WT). t-test: * = p< 0.05, ** = p< 0.01, *** = p< 0.001 different from AC9 WT.

[0067] FIG. 5 is a set of graphs showing the area under the curve (AUG) of the plasma glucose level derived from the intraperitoneal glucose tolerance test in AC9 WT and AC9 GT / GT mice fed with an atherogenic diet for 6 (left panel) or 16 (right panel) weeks. Error bars represent the mean ± standard deviation, n = 13 (AC9 GT / GT) or 14 (AC9 WT). t-test: * = p< 0.05, *** = p< 0.001 different from AC9 WT.

[0068] FIG. 6 is a set of graphs showing the insulin plasma level in AC9 wild-type and AC9 GT / GT mice fed with an atherogenic diet for 6 (left panel) or 16 (right panel) weeks and submitted to an intraperitoneal glucose tolerance test. Error bars represent the mean ± standard deviation, n = 13 (AC9 GT / GT) or 14 (AC9 WT). t-test: *** = p< 0.001 different from AC9 WT.

[0069] FIG. 7 is a graph showing the quantification of AC9 protein expression levels 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells using the indicated siRNA, relative to siScramble. Error bars represent the mean ± standard deviation, n = 8. Repeated measures ANOVA with uncorrected Fisher’s LSD: *“ = p < 0.001 relative to siScramble. FIG. 8 is a screenshot of the Exome Phenome-Wide Association Study (ExPheWas) Browser of AC9 (i.e., ADCY9).

[0070] FIG. 9 is a plot showing the Mendelian Randomization (MR) estimate of monocyte count on glycated haemoglobin (HbA1c) based on genetic variants close to the gene ADCY9, with effects displayed for each principal component (PC) on the exposure and outcome. The black diagonal line shows the inverse variance weighted (IVW) estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g. , to comply with the relevance assumption of MR). IVW effect and 95% confidence interval (Cl): 0.29 (0.12, 0.45). MR p-value: 6.9E'4. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using body mass index (BMI) or basal metabolic rate (BMR) as exposure.

[0071] FIG. 10 is a plot showing the MR estimate of monocyte count on glucose based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 0.25 (0.07, 0.43). MR p-value: 0.007. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on diabetes.

[0072] FIG. 11 is a plot showing the MR estimate of monocyte count on diabetes based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 4.54 (1.98, 10.37). MR p-value: 3.4E'4. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on diabetes.

[0073] FIG. 12 is a plot showing the MR estimate of monocyte count on Type 2 diabetes based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 2.00 (1.03, 3.91 ). MR p-value: 0.042. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on Type 2 diabetes. FIG. 13 is a graph showing the quantification of G6PC mRNA expression levels in HepG2 cells 72 hours after siRNA-mediated knockdown of AC9 (siAC9), with or without 5 M of atorvastatin (“Atorva”), during the last 24 hours, relative to siScramble. These data show that atorvastatin-induced G6PC mRNA upregulation is inhibited by siRNA-mediated knockdown of AC9 in HepG2. Error bars represent the mean ± standard deviation, n = 8. Repeated measures ANOVA with uncorrected Fisher’s LSD: * = p < 0.05 relative to siScramble; *** = p < 0.001 relative to siScramble; # = p < 0.05; and ### = p < 0.001.

[0074] FIG. 14 is a graph showing the quantification of G6PC mRNA expression levels in HepG2 cells 72 hours after siRNA-mediated knockdown of AC9 (siAC9), with or without the indicated compound, during the last 24 hours, relative to siScramble. These data show that AC9 siRNA reshapes hepatic G6PC mRNA response to combinatorial forskolin (FSK) and 3-isobutyl-1 - methylxanthine (IMBX) (“FSK / IBMX”), and cyclic adenosine monophosphate (cAMP). HepG2 cells were treated with the combination of 10 M of FSK and 100 pM of IBMX, or 300 pM of cAMP. Error bars represent the mean ± standard deviation, n = 4 for FSK+IBMX and n = 3 for cAMP treatment. Repeated measures ANOVA with uncorrected Fisher’s LSD: * = p < 0.05 relative to siScramble; ** = p < 0.01 relative to siScramble; *** = p < 0.001 relative to siScramble; # = p < 0.05; and ## = p < 0.01.

[0075] FIG. 15 is a graph showing the quantification of G6PC mRNA expression levels in HepG2 cells 72 hours after siRNA-mediated knockdown of AC9 (siAC9), with or without 100 nM of insulin, during the last 24 hours, relative to siScramble. These data show that AC9 siRNA reshapes hepatic G6PC mRNA response to insulin. Error bars represent the mean ± standard deviation, n = 4. Paired t- test: ** = p < 0.01 ; and *“ = p < 0.001 .

[0076] FIG. 16 is a schematic showing the principal transcription factors involved in the regulation of G6PC expression in HepG2 cells. cAMP = cyclic adenosine monophosphate; PKA = protein kinase A; pCREB = phosphorylated cAMP response element-binding protein; G6PC = glucose-6-phosphatase catalytic subunit; PGC1a = peroxisome proliferator-activated receptor gamma coactivator 1a; HNF4a = hepatocyte nuclear factor 4a.

[0077] FIG. 17A is a graph showing the quantification of PGC1a and HNF4a mRNA expression 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that AC9 siRNA lowers G6PC-regulating transcription factors in HepG2 cells. Error bars represent the mean ± standard deviation, n = 5 for PGC1a and n = 3 for HNF4a. Paired t-test: ** = p < 0.01 ; and *** = p < 0.001.

[0078] FIG. 17B is a graph showing the quantification of HNF4a protein expression 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. Note, PGC1a was undetectable. Error bars represent the mean ± standard deviation. These data show that AC9 siRNA lowers G6PC-regulating transcription factors in HepG2 cells, n = 4. Paired t-test: ** = p < 0.01 ; and *** = p < 0.001.

[0079] FIG. 18 is a schematic illustrating the role of glucose-6-phosphate transporter (G6PT) and G6PC in the pathway of glucose production. Pi = inorganic phosphate.

[0080] FIG. 19A is a graph showing the quantification of G6PT mRNA expression levels 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that AC9 siRNA increases mRNA expression of G6PT. Error bars represent the mean ± standard deviation, n = 4.

[0081] FIG. 19B is a graph showing the quantification of glucose-6-phosphate levels 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells. These data show that AC9 siRNA increases glucose-6-phosphate levels. Error bars represent the mean ± standard deviation, n = 4. Paired t-test: “ = p < 0.01.

[0082] FIG. 20 is a schematic showing the role of phosphoenolpyruvate Carboxykinase 1 (PCK1 ) and glycogen phosphorylase (PYGL) enzymes in hepatic gluconeogenesis and glycogenolysis.

[0083] FIG. 21 A is a graph showing the quantification of PCK1 mRNA levels 72 hours after siRNA- mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that AC9 siRNA reduces mRNA expression PCK1. Error bars represent the mean ± standard deviation, n = 4. Paired t-test: **** = p < 0.0001.

[0084] FIG. 21 B is a graph showing the quantification of PYGL mRNA levels 72 hours after siRNA- mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that AC9 siRNA reduces mRNA expression PYGL. Error bars represent the mean ± standard deviation, n = 4. Paired t-test: * = p < 0.05.

[0085] FIG. 22A is a simplified schematic of glucose metabolism.

[0086] FIG. 22B is a graph showing the quantification of hexokinase mRNA levels after siRNA- mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that AC9 siRNA increases mRNA expression hexokinase. Error bars represent the mean ± standard deviation, n = 4. Paired t-test: “ = p < 0.01 .

[0087] FIG. 23 is a schematic exemplifying a method for measuring G6Pase activity.

[0088] FIG. 24A is a graph showing the quantification of G6Pase activity levels 24 hours after treatment with combinatorial FSK / IBMX (10 pM FSK and 100pM IBMX), or insulin (100 nM) in HepG2 cells relative to control. These data show that G6Pase activity is increased by FSK / IBMX and decreaed by insulin. Error bars represent the mean ± standard deviation, n = 8 for FSK / IBMX and n = 4 for insulin. Paired t-test: *“ = p < 0.001.

[0089] FIG. 24B is a graph showing the quantification of G6Pase activity levels 72 hours after siRNA-mediated knockdown of AC9 (siAC9) in HepG2 cells relative to siScramble. These data show that G6Pase activity is reduced by siAC9. Error bars represent the mean ± standard deviation, n = 8. Paired t-test: **** = p < 0.0001.

[0090] FIG. 25 is a schematic exemplifying a method for measuring the intracellular level of glycogen.

[0091] FIG. 26 is a graph showing the quantification of glycogen levels 24 hours after treatment with combinatorial FSK / IBMX (10 pM FSK and 100 pM IBMX) or insulin (100 nM) in HepG2 cells relative to control. These data show that FSK / IBMX decreases glycogen levels while insulin increases glycogen levels in HepG2 cells. Error bars represent the mean ± standard deviation, n = 5. Repeated measures ANOVA with uncorrected Fisher’s LSD: * = p < 0.01; **** = p < 0.0001.

[0092] FIG. 27A is a graph showing the quantification of glucose production 2 hours after treatment with combinatorial FSK / IBMX (10 pM FSK and 100pM IBMX), insulin (100 nM), or the PKA inhibitor, H89 (5 M), in HepG2 cells relative to control. These data validate glucose production by HepG2 cells. Error bars represent the mean ± standard deviation, n = 10. Repeated measures ANOVA with uncorrected Fisher’s LSD: * = p < 0.05; and **** = p < 0.0001 .

[0093] FIG. 27B is a graph showing the quantification of glucose production 72 hours after siRNA- mediated knockdown of PCK1 (siPCKI ) or AC9 (siAC9) in HepG2 cells relative to siScramble n HepG2 cells. These data validate glucose production by HepG2 cells. Error bars represent the mean ± standard deviation, n = 3. Repeated measures ANOVA with uncorrected Fisher’s LSD: * = p < 0.05; and ** = p < 0.01.

[0094] FIG. 28 is a plot showing the MR estimate of monocyte count on self-reported heart attack / myocardial infarction (Ml) based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 3.32 (1.03, 10.74). MR p-value: 0.045. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on heart attacks and / or Ml.

[0095] FIG. 29 is a plot showing the MR estimate of monocyte count on hospital-recorded (e.g., International Classification of Disease (ICD) codes) Ml based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 2.72 (1.19, 6.22). MR p-value: 0.018. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on Ml.

[0096] FIG. 30 is a plot showing the MR estimate of monocyte count on hospital-recorded (e.g., Phecode records) Ml based on genetic variants close to the gene ADCY9, with effects displayed for each PC on the exposure and outcome. The black diagonal line shows the IVW estimate for the causal effect. In the IVW estimator, the regression is weighted by the precision of the estimates, and so PCs with smaller error bars are given a larger weight. Small circles represent the effect of PCs that are excluded from the analysis because they are not associated with the exposure (e.g., to comply with the relevance assumption of MR). IVW effect and 95% Cl: 2.72 (1.19, 6.21 ). MR p-value: 0.017. The association p-value between the selected gene and exposure in ExPheWas is 1.8E'16. Notably, there was no effect when using BMI or BMR as exposure. These data support a causal effect of the ADCY9 drug target on Ml. DEFINITIONS

[0097] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

[0098] As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0099] As used herein, “administration” refers to providing or giving a subject a therapeutic agent by any effective route. Exemplary routes of administration are described herein below.

[0100] As used herein, the terms “administering in combination” or “administered in combination” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 120, 90, 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

[0101] As used herein, the term "auxiliary moiety" refers to any moiety, including, but not limited to, a small molecule, a peptide, a carbohydrate, a neutral organic polymer, a positively charged polymer, a therapeutic agent, a targeting moiety, an endosomal escape moiety, and any combination thereof, which can be conjugated to a nucleic acid molecule. In some embodiments, an "auxiliary moiety" is linked to an inhibitory nucleic acid molecule disclosed herein by forming one or more covalent or non- covalent bonds with one or more conjugating groups attached to a phosphate linkage, a phosphorothioate linkage, a 5' positions of a nucleotide sugar, or any portion of a nucleobase. One skilled in the art will readily understand appropriate points of attachment of a particular auxiliary moiety to a nucleic acid molecule.

[0102] As used herein, the term “cardiovascular condition” refers to an acute or chronic clinical manifestation of a cardiovascular disease, such as myocardial infarction (e.g., acute Ml), stroke (e.g., ischemic stroke or hemorrhagic stroke), cardiac arrest, and cardiac arrhythmia (e.g., atrial fibrillation or ventricular tachycardia).

[0103] As used herein, “delivery vehicle” refers to any substance (e.g., molecule, peptide, conjugate, and construct) that facilitates, at least in part, the in vivo delivery of a nucleic acid molecule to targeted cells.

[0104] As used herein, the term “effective amount” described herein refer to a quantity sufficient to, when administered to the subject, effect beneficial or desired results; as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of decreasing glucose levels, it is an amount of the composition sufficient to reduce glucose levels in the subject by about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) as compared to a subject that does not receive the composition. In another example, in the context of decreasing AC9 and / or G6PC, it is an amount of the composition sufficient to decrease AC9 and / or G6PC (e.g., mRNA or protein levels) in the subject by about 5% or more (e g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) as compared to a subject that does not receive the composition. In yet another example, in the context of reducing a risk of developing hyperglycemia, it is an amount of the composition sufficient to reduce a subject’s risk (or the rick of a population of subject’s) of developing hyperglycemia by about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) as compared to a subject (or a population of subject’s) that does not receive the composition. In yet another example, in the context of treating hyperglycemia, it is an amount of the composition sufficient to reduce the subject’s glucose levels by about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more), as compared to a subject (or a population of subject’s) that does not receive the composition. Alternatively, in the context of treating hyperglycemia, it is an amount of the composition sufficient to bring the subject’s glucose level back to a baseline level. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical compositions, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0105] As used herein, a “formulation” or a “package” includes at least an inhibitory nucleic acid molecule and a delivery vehicle.

[0106] As used herein, the term “hyperglycemia” refers to a biological event in which a subject’s blood sugar (e.g., glucose) is above a baseline level (e.g., above the subject’s personal baseline level or above a baseline level of a population of subjects). For example, for a subject that does not have diabetes, hyperglycemia may be a blood glucose level that is greater than 125 mg / dL (e.g., about 130 mg / dL, about 140 mg / dL, about 150 mg / dL, about 160 mg / dL, about 170 mg / dL, about 180 mg / dL, or greater) while fasting (e.g., not eating for at least eight hours). In another example, for a subject that has prediabetes, hyperglycemia may be a blood glucose level that is between about 100 mg / dL to about 180 mg / dL (e.g., about 110 mg / dL, about 120 mg / dL, about 130 mg / dL, about 140 mg / dL, about 150 mg / dL, about 160 mg / dL, about 170 mg / dL, or about 180 mg / dL). In another example, for a subject that has diabetes, hyperglycemia may be a blood glucose level that is greater than 180 mg / dL (e.g., about 185 mg / dL, about 190 mg / dL, about 200 mg / dL, about 210 mg / dL, about 220 mg / dL, about 230 mg / dL, about 240 mg / dL, 250 mg / dL, or greater).

[0107] As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0108] As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0109] 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 expression of protein encoded by the target nucleic acid molecule. Exemplary inhibitory nucleic acid molecules are anti-sense oligonucleotides (ASOs), small interfering RNA (siRNAs), short hairpin RNA (shRNAs), double stranded RNAs (dsRNAs), and microRNA (miRNAs). Inhibitory nucleic acid molecules may reduce target protein expression by 5% or more (e.g., 5% 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more). In one embodiment, the target nucleic acid molecule encodes AC9.

[0110] As used herein, the term “major adverse cardiovascular event” or “MACE” refers to a clinically significant cardiovascular outcome, including but not limited to one or more of the following: Ml, stroke (e.g., ischemic stroke or hemorrhagic stroke), cardiac arrest, arrhythmia (e.g., atrial fibrillation or ventricular tachycardia), and cardiovascular death of a subject.

[0111] As used herein “modified” refers to a changed state or structure of a nucleic acid molecule described herein. Molecules may 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 conjugation of an auxiliary moiety.

[0112] 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, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.

[0113] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0114] “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0115] 100 multiplied by (the fraction X / Y) 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 appreciated that where 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.

[0116] 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.

[0117] As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” during an acute event can also mean returning the subject back to a baseline condition. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0118] As used herein, the term “vector” is considered a replicon, such as plasmid, phage, viral construct or cosmid, to which another nucleic acid (e.g., DNA or RNA) segment may be attached. Vectors are used to transduce and express the nucleic acid segment in cells.

[0119] DETAILED DESCRIPTION

[0120] Provided herein are compositions (e.g., inhibitory nucleic acid molecules) and methods useful for decreasing decreasing an adenylate cyclase (AC; e.g., AC type 9 (AC9)) and / or glucose-6- phosphatase catalytic subunit (G6PC). Advantageoulsy, these compositions and methods are useful for also decreasing glucose levels in a subject, reducing a subject’s risk of developing hyperglycemia, and treating hyperglycemia in a subject.

[0121] Inhibitory Nucleic Acid Molecules

[0122] Exemplary inhibitory nucleic acid molecules of the disclosure are small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), anti-sense oligonucleotides (ASOs), microRNAs (miRNAs), and short hairpin RNAs (shRNAs); however, any nucleic acid molecule capable of reducing an adenylate cyclase (e.g., AC9) mRNA and / or protein expression is envisioned for use in the methods described herein. In some instances, the inhibitory nucleic acid molecules of the disclosure may be referred as RNA inhibitory (RNAi) molecules.

[0123] For any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, shRNA, or other inhibitory nucleic acid molecules capable of reducing expression of a target gene, e.g., AC9) 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 sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NOs: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 16.

[0124] In some embodiments, the inhibitory nucleic acid is an siRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an dsRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an ASO targeting AC9. In some embodiments, the inhibitory nucleic acid is an miRNA targeting AC9. In some embodiments, the inhibitory nucleic acid is an shRNA targeting AC9. Each of these modalities is described further below. small interfering RNA (siRNA) siRNAs of the disclosure are single-stranded (ss) or double-stranded (ds) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Once an siRNA molecule enters a cell, it is incorporated into an RNA-induced silencing complex (RISC). Upon siRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target.

[0125] In some embodiments, siRNAs of the disclosure may include 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).

[0126] In some embodiments, siRNAs of the disclosure may include 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).

[0127] It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention.

[0128] In some embodiments, the siRNA contains an antisense strand. In some embodiments, lengths for an antisense strand of the siRNA molecules 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), 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 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.

[0129] In some embodiments, the siRNA contains a sense strand. In some embodiments, the sense strand of the siRNA molecules 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 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.

[0130] In some embodiments, the sense and antisense strands of an siRNA molecule of the disclosure are completely complementary. In some embodiments, the sense and antisense strands of an siRNA molecule of the disclosure are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% basepaired due to mismatches. One or more mismatches may be present within the ds siRNA without impacting the siRNA’s ability to reduce expression of a target gene of interest (e.g., AC9).

[0131] The nucleotide sequence of an siRNA molecule of the disclosure may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9 mRNA) such that the siRNA can hybridize with the target gene of interest. In some embodiments, the siRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., AC9), or a portion thereof. In some embodiments, the siRNA is complementary to the target gene of interest (e.g., AC9), or a portion thereof.

[0132] In some embodiments, the nucleotide sequence of the siRNA 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 siRNA may contain sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the siRNA of the disclosure may contain sufficient complementarity to a pre-mRN A transcript or an 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).

[0133] In some embodiments, the siRNAs described herein have 0-7 nucleotide 3’ overhangs or 0-4 nucleotide 5’ overhangs. In some embodiments, the siRNA molecule has a single uracil (e.g., U) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a double uracil (e.g., UU) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a single thymine (e.g., T) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a double thymine (e.g., TT) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a cytosine and thymine (e.g., CT) overhang at each 3’ end of the siRNA. Different siRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., AC9). A combination of two siRNAs may be used in a method of the invention, such as two different siRNAs, three different siRNAs, four different siRNAs, or five different siRNAs targeting the same gene of interest (e.g., AC9, or variants thereof).

[0134] In some embodiments, the siRNA sequence may contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one or more of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1 ), or a complementary sequence thereof. In some embodiments, the siRNA sequence may contain the sequence of any one or more of SEQ ID NOs: 1- 10 and 18-27 (e.g., see Table 1 ), or a complementary sequence thereof.

[0135] In some embodiments, the siRNA contains at least 15 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1). In some embodiments, the siRNA contains at least 16 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 17 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 18 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 19 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 18-27 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 20 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 (e.g., see Table 1). In some embodiments, the siRNA contains 21 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 (e.g., see Table 1 ).

[0136] TABLE 1. EXEMPLARY siRNA SEQUENCES

[0137] A = adenine; C = cytosine; G = guanine; T = thymine; U = uracil, w / o = without; Note: 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 a thymine nucleotide at position 21 , when reading from 5’ to 3’.

[0138] In some embodiments, the siRNA of the disclosure may target a nucleotide sequence of any one of SEQ ID NOs: 11-15 (e.g., see Table 2), or a complementary sequence thereof, or variant thereof with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% thereto.

[0139] TABLE 2. TARGET SEQUENCES

[0140] A = adenine; C = cytosine; G = guanine; U = uracil

[0141] In some embodiments, the siRNA comprises sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 16. In some embodiments, the siRNA comprises sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 16. The nucleotide sequence of SEQ ID NO: 16 is set forth in Table 3. In some embodiments, the siRNA comprises sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 17. In some embodiments, the siRNA comprises sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 17. The nucleotide sequence of SEQ ID NO: 17 is set forth in Table 3.

[0142] TABLE 3. ADENYLATE CYCLASE 9 SEQUENCE

[0143] A = adenine; C = cytosine; G = guanine; T = thymine

[0144] Double-stranded RNA (dsRNA) dsRNAs of the disclosure are ds nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Typically, dsRNAs are longer than an siRNA and are processed within a cell to form an siRNA molecule. The siRNA is then incorporated into an RNA-induced silencing complex (RISC). Upon siRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target.

[0145] In some embodiments, dsRNAs of the disclosure may include a sense strand and an antisense strand, each containing a nucleotide sequence of about 25 to about 5000 nucleotides in length, or longer (e.g., 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about

[0146] 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about

[0147] 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

[0148] 370, about 380, about 380, about 400, about 425, about 450, about 475, about 500, about 525, about

[0149] 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about

[0150] 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about

[0151] 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about

[0152] 1800, about 1900, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about

[0153] 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, about 5000, about

[0154] 6000, about 7000, about 8000, about 9000, or about 10000 nucleotides in length).

[0155] In some embodiments, dsRNAs of the disclosure may include a sense strand and an antisense strand, each containing a nucleotide sequence of 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,

[0156] 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,

[0157] 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 380, 400, 425, 450, 475, 500, 525,

[0158] 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000,

[0159] 1100, 1200, 1300, 1400, 1500, 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 in length).

[0160] It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention.

[0161] In some embodiments, the sense and antisense strands of an dsRNA molecule of the disclosure are completely complementary. In some embodiments, the sense and antisense strands of an dsRNA molecule of the disclosure are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% basepaired due to mismatches. One or more mismatches may be present within the ds dsRNA without impacting the dsRNA’s ability to reduced expression of a target gene of interest.

[0162] The nucleotide sequence of an dsRNA of the disclosure may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9) such that the dsRNA can hybridize with 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), or a portion thereof. In some embodiments, the dsRNA is complementary to the target gene of interest (e.g., AC9), or a portion thereof.

[0163] 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 disclosure may contain sufficient complementarity to a pre-mRN A transcript or an 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). Different dsRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., AC9). A combination of two dsRNAs may be used in a method of the invention, such as two different dsRNAs, three different dsRNAs, four different dsRNAs, or five different dsRNAs targeting the same gene of interest (e.g., AC9, or variants thereof).

[0164] Anti-Sense Oligonucleotide (ASO)

[0165] ASOs of the disclosure are single (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Upon hybridization to a target mRNA, RNase H will degrade the mRNA by hydrolyzation, resulting in reduced mRNA and protein levels of the target.

[0166] In some embodiments, ASOs of the disclosure may include 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,

[0167] 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).

[0168] In some embodiments, ASOs of the disclosure may include 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 ,

[0169] 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0170] It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention.

[0171] The nucleotide sequence of the ASO may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9) such that the ASO can hybridize with 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), or a portion thereof. In some embodiments, the ASO is complementary to the target gene of interest (e.g., AC9), or a portion thereof.

[0172] In some embodiments, the nucleotide sequence of the ASO 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 ASO may contain sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the ASO of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an 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).

[0173] Different ASOs can be combined for decreasing the protein expression of a target gene of interest (e.g., AC9). A combination of two ASOs may be used in a method of the invention, such as two different ASOs, different three ASOs, four different ASOs, or five different ASOs targeting the same gene of interest (e.g., AC9, or variants thereof) micro RNA (miRNA) miRNAs of the disclosure are single stranded (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Once a miRNA molecule enters a cell, it is incorporated into a RNA-induced silencing complex (RISC). Upon miRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target.

[0174] In some embodiments, miRNAs of the disclosure may include 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,

[0175] 25, 26, 27, 28, 29, 30, or 31 nucleotides in length).

[0176] In some embodiments, miRNAs of the disclosure may include 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,

[0177] 26, 27, 28, 29, or 30 nucleotides in length).

[0178] The nucleotide sequence of the miRNA may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9) such that the miRNA can hybridize with 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), or a portion thereof. In some embodiments, the miRNA is complementary to the target gene of interest (e.g., AC9), or a portion thereof.

[0179] In some embodiments, the nucleotide sequence of the miRNA 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 miRNA may contain sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the miRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an 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).

[0180] Different miRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., AC9). A combination of two or more miRNAs may be used in a method of the invention, such as two different miRNAs, three different miRNAs, four different miRNAs, or five different miRNAs targeting the same gene of interest (e.g., AC9, or variants thereof) short hairpin RNA (shRNA) shRNAs of the disclosure are ss or ds nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Once a shRNA molecule enters a cell, it is incorporated into a RNA-induced silencing complex (RISC). Upon shRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target.

[0181] In some embodiments, shRNAs of the disclosure may include 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).

[0182] In some embodiments, shRNAs of the disclosure may include 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). shRNAs of the disclosure contain a variable hairpin loop structure and a stem sequence. In some embodiments the stem sequence may be 10 to 50 nucleotides in length (e.g., 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length). In some embodiments, the hairpin size is between 4 to 50 nucleotides in length, although the loop size may be larger without significantly affecting silencing activity. shRNA molecules of the disclosure may contain mismatches, for example G-U mismatches between two strands of the shRNA stem without decreasing potency. In some embodiments, shRNAs are designed to include one or several G-U pairings in the hairpin stem to stabilize hairpins during propagation in bacteria, for example.

[0183] The nucleotide sequence of the shRNA may contain sufficient complementarity to a portion of a target gene of interest (e.g., AC9) such that the shRNA can hybridize with the target gene of interest. In some embodiments, the shRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., AC9), or a portion thereof. In some embodiments, the shRNA is complementary to the target gene of interest (e.g., AC9), or a portion thereof.

[0184] In some embodiments, the nucleotide sequence of the shRNA 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 shRNA may contain sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of AC9). In some embodiments, the shRNA of the disclosure may contain sufficient complementarity to a pre-mRN A transcript or an 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).

[0185] Different shRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., AC9). A combination of two or more shRNAs may be used in a method of the invention, such as two different shRNAs, three different shRNAs, four different shRNAs, or five different shRNAs targeting the same gene of interest (e.g., AC9, or variants thereof).

[0186] Modifications to the Inhibitory Nucleic Acid Molecules

[0187] It is contemplated that any of the inhibitory nucleic acid molecules disclosed herein may be used in the methods disclosed herein in an unmodified or in a modified form. Unmodified inhibitory nucleic acid molecules contain nucleobases that include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below.

[0188] Modifications may be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

[0189] Modifications may 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 molecule’s efficacy or biophysical properties (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, reduce immunogenicity, and / or targeting to a particular location or cell type).

[0190] Modification may further be achieved by covalently or non-covalently conjugating a moiety (e.g., a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer) to the 5’ end and / or 3’ end of the inhibitory nucleic acid molecule, as described in more detail below.

[0191] Nucleoside Modifications

[0192] Modification of the inhibitory nucleic acid molecules described herein include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalky I, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. The inhibitory nucleic acid molecules may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine, and / or 2-pyridone. Further modification of the inhibitory nucleic acid molecules described herein may include nucleobases disclosed in US 3,687,808; Kroschwitz, J. I., 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, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J. ed., 1993, pp. 289-302.

[0193] Sugar Modifications

[0194] Modifications of the inhibitory nucleic acid molecules described herein may also include one or more of the following 2’ sugar modifications: 2’-O-methyl (2’-O-Me), 2'-methoxyethoxy (2'-O- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., a 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 OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alky l-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2 -5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0195] Internucleoside Linkage Modifications

[0196] Modifications of the inhibitory nucleic acid molecules described herein may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.

[0197] Conjugates

[0198] Any of the inhibitory nucleic acid molecules described herein may be modified via the addition of an auxiliary moiety, e.g., a cell penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety. The auxiliary moiety may be present as a 5’ terminal modification (e.g., covalently bonded to a 5’-terminal nucleoside), a 3’ terminal modification (e.g., covalently bonded to a 3’-terminal nucleoside), or an internucleoside linkage (e.g., covalently bonded to phosphate or phosphorothioate in an internucleoside linkage).

[0199] CPPs are known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2:43-51). Specific examples of CPPs are provided in WO2011157713, which is incorporated herein by reference in its entirety.

[0200] Inhibitory nucleic acid molecules of the disclosure may include covalently attached neutral polymer-based auxiliary moieties. Neutral polymers include poly(C1-6 alkylene oxide), e.g., poly( ethylene glycol) and polypropylene glycol) and copolymers thereof, e.g., di- and triblock copolymers.

[0201] An inhibitory nucleic acid molecule containing a hydrophobic moiety may exhibit superior cellular uptake, as compared to an inhibitory nucleic acid molecule lacking the hydrophobic moiety. A hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsily I, t-butyldiphenylsily I, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently linked to the nucleic acid backbone (e.g., 5’- terminus) of the inhibitory nucleic acid molecule.

[0202] A targeting moiety is selected based on its ability to target oligonucleotides of the invention to a desired or selected cell population that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) for the selected targeting moiety. For example, an oligonucleotide of the invention could be targeted to hepatocytes expressing asialoglycoprotein receptor (ASGP-R) by selecting a targeting moiety containing N-acetylgalactosamine (GalNAc).

[0203] A targeting moiety may 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 may target a cell expressing asialoglycoprotein receptor (ASGP-R), IgA receptor, HDL receptor, LDL receptor, or transferrin receptor. Non-limiting examples of the ligands include N-acetylgalactosamine (e.g., a triantennary N-acetylgalactosamine), glycyrrhetinic acid, glycyrrhizin, lactobionic acid, lactoferrin, IgA, or a bile acid (e.g., lithocholyltaurine or taurocholic acid).

[0204] The ligand may be a small molecule, e.g., a small molecule targeting a cell expressing asialoglycoprotein receptor (ASGP-R). A non-limiting example of a small molecule targeting an asialoglycoprotein receptor is N-acetylgalactosamine. Alternatively, the ligand can be an antibody or an antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)).

[0205] Preparation of Inhibitory Nucleic Acid Molecules

[0206] Inhibitory nucleic acid molecules of the disclosure may be prepared using techniques and methods known in the art for the oligonucleotide synthesis. For example, inhibitory nucleic acid molecules of the disclosure may be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of (1 ) de-blocking a 5’-protected nucleotide to produce a 5’- deblocked nucleotide, (2) coupling the 5’-deblocked nucleotide with a 5’-protected nucleoside phosphoramidite to produce nucleosides linked through a phosphite, (3) repeating steps (1) and (2) one or more times as needed, (4) capping the 5’-terminus, and (5) oxidation or sulfurization of internucleoside phosphites. The reagents and reaction conditions useful for the oligonucleotide synthesis are known in the art.

[0207] The inhibitory nucleic acid molecules disclosed herein may be linked to solid support as a result of solid-phase synthesis. Cleavable solid supports that may be used are known in the art. Nonlimiting examples of the solid support include, e.g., controlled pore glass or macroporous polystyrene bonded to a strand through a cleavable linker (e.g., succinate-based linker) known in the art (e.g., UnyLinkerTM). A nucleic acid linked to solid support may be removed from the solid support by cleaving the linker connecting a nucleic acid and solid support.

[0208] Compositions

[0209] The inhibitory nucleic acid molecules described herein may be formulated or packaged into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo. For example, the inhibitory nucleic acid molecules described herein (e.g., the siRNA molecules of SEQ ID NOs: 1-10 and 18-27, or variants thereof) may be administered in a suitable diluent, carrier, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nded. And in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0210] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates and mammals.

[0211] Compositions containing the inhibitory nucleic acids described herein may further include a second therapeutic agent (e.g., a nucleic acid molecule to be expressed within a cell, a polypeptide, or a drug).

[0212] For example, a second therapeutic agent may be a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, a biguanide, or a combination thereof (e.g., a biguanide, such as metformin, in combination with a DPP- 4 inhibitor, an insulin secretagogue, or a SGLT2 inhibitor). Exemplary DPP-4 inhibitors include alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®), saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®). Exemplary GLP-1 receptor agonists include exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®). Exemplary SGLT2 inhibitors include canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®). Exemplary AGIs include acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®). Exemplary insulin secretagogues include meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)). Exemplary thiazolidinediones include pioglitazone (e.g., ACTOS®) or% rosiglitazone (e.g., AVANDIA®). An exemplary biguanide includes metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0213] In some embodiments, the second therapeutic agent (e.g., the DPP-4 inhibitor, the GLP-1 receptor agonist, the SGLT2 inhibitor, the AGI, the insulin secretagogue, the thiazolidinedione, the biguanide, or some combination thereof) is administered in combination with an inhibitory nucleic acid molecule of the disclosure.

[0214] Methods

[0215] The disclosure provides methods of (i) reducing the risk of a major adverse cardiovascular event (MACE) in a subject, (ii) treating a cardiovascular condition in a subject, (iii) decreasing glucose levels in a subject, (iv) decreasing AC9 and / or G6PC in a subject, (v) reducing a risk of developing hyperglycemia in a subject, and (vi) treating hyperglycemia in a subject. Advantageoulsy, these methods are not only useful for subjects that frequently suffer from hyperglycemia (e.g. , prediabetics and diabetics), these methods are further useful for those subjects that are prescribed medication that naturally increases one’s blood glucose. When these medications are administered in combination with the compositions described above, a clinical benefit may result in the subject, such as a reduction in blood glucose and / or a reduction in the probability of a future hyperglycemic event. Over time, a reduction in the number and / or severity of a subject’s hyperglycemic events can reduce any unwanted complications of hyperglycemia, such as blurred vision, nausea and vomiting, fatigue, nerve damage, frequent urination and manifestation of a major adverse cardiovascular event.

[0216] Methods of Reducing the Risk of a MACE in a Subject

[0217] Provided herein are methods of reducing the risk of a major adverse cardiovascular event (MACE) in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). MACE refers to a clinical endpoint resulting from the onset of one or more cardiovascular events in a subject, such as a diagnosable onset of Ml, stroke (ischemic or hemorrhagic), cardiac arrest, or arrhythmia (e.g., atrial fibrillation or ventricular tachycardia). It can also include cardiovascular death ( I. e. , death of a subject due to a cardiovascular condition or disease). A subject may be considered to have experienced MACE upon the occurrence of one or more (e.g., 1 , 2, 3, 4, or more) clinically diagnosable outcomes related to a cardiovascular condition (e.g., Ml (e.g., acute Ml), stroke (e.g., ischemic or hemorrhagic stroke), cardiac arrest, and cardiac arrhythmias (e.g., atrial fibrillation or ventricular tachycardia)). Patients at risk of MACE often share common cardiometabolic risk factors, such as diabetes (e.g., diabetes mellitus), hyperglycemia (e.g., chronie hyperglycemia), and obesity (e.g., a waist circumferance of about >80 cm for human males or about >78 cm for human females). Alterations in glucose metabolism play a particularly important role in the risk of MACE; for example, chronic hyperglycemia and insulin resistance accelerate atherosclerosis, impair endothelial function, and promote a pro-inflammatory and pro-thrombotic state, thereby increasing the risk of MACE. Additional risk factors include advanced age, smoking, chronic kidney disease, and hypertension, each of which further compounds the burden of cardiovascular morbidity.

[0218] In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0219] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s glucose levels may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) upon administration. In some embodiments, the subject’s glucose levels return to baseline.

[0220] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0221] Methods of Treating a Cardiovascular Condition in a Subject

[0222] Provided herein are methods of treating a cardiovascular condition (e.g., Ml (e.g., acute Ml), stroke (e.g., ischemic or hemorrhagic stroke), cardiac arrest, and cardiac arrhythmias (e.g., atrial fibrillation or ventricular tachycardia)) in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). Cardiovascular conditions are the underlying clinical manifestations (e.g., of a cardiovascular disease) that can result in a clinically significant cardiovascular event (e.g., MACE). Cardiovascular conditions include, but are not limited to, acute Ml, ischemic or hemorrhagic stroke, cardiac arrest, and cardiac arrhythmias (e.g., atrial fibrillation or ventricular tachycardia). These conditions represent a pathological state of a subject rather than clinical endpoints. They often arise from or are exacerbated by chronic cardiovascular diseases.

[0223] In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0224] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s glucose levels may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) upon administration. In some embodiments, the subject’s glucose levels return to baseline.

[0225] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0226] Methods of Decreasing Glucose Levels in a Subject

[0227] Provided herein are methods of decreasing glucose levels (e.g., blood glucose levels) in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0228] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s glucose levels may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) upon administration. In some embodiments, the subject’s glucose levels return to baseline.

[0229] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0230] Methods of Decreasing AC9 and / or G6PC in a Subject

[0231] Provided herein are methods of decreasing AC9 and / or G6PC in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0232] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s AC9 and / or G6PC levels (e.g., mRNA or protein levels) may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) upon administration. Notably, a reduction in AC9 may likely result in a reduction in G6PC (e.g., see Example 1 ).

[0233] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2 inhibitor, an AGI, an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0234] Methods of Reducing a Risk of Developing Hyperglycemia in a Subject

[0235] Provided herein are methods of reducing a risk of developing hyperglycemia in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0236] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s risk of hyperglycemia may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) upon administration, as compared to a subject that does not receive the inhibitory nucleic acid molecule.

[0237] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2 inhibitor, an AGI, an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0238] Methods of Treating Hyperglycemia in a Subject

[0239] Provided herein are methods of treating hyperglycemia in a subject (e.g., a subject diagnosed with prediabetes, diabetes, and / or hyperglycemia). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1-10 and 18-27, or a variant thereof), wherein the siRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17). In some embodiments, the method contains the step of administering to a 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 contains the step of administering to a 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 contains the step of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets AC9 (e.g., SEQ ID NO: 16 or 17).

[0240] The step of administering may include administering a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA). In some embodiments, the subject’s hyperglycemia may be reduced by about about 5% or more (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) upon administration. In some embodiments, the subject’s glucose levels return to baseline.

[0241] In some embodiments, the method further includes administering in combination a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group including: a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2 inhibitor, an AGI, an insulin secretagogue, a thiazolidinedione, or a biguanide. In some embodiments: (i) the DPP-4 inhibitor is selected from the group consisting of alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®, saxagliptin (e.g., ONGLYZA®), and sitagliptin (e.g., JANUVIA® or ZITUVIO®); (ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide (e.g., BYETTA® or BYDUREON®), lixisenatide (e.g., LYXUMIA® or ADLYXIN®), dulaglutide (e.g., TRULICITY®), and liraglutide (e.g., SAXENDA® or VICTOZA®); (iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin (e.g., INVOKANA®), dapagliflozin (e.g., FORXIGA®), and empagliflozin (e.g., JARDIANCE®); (iv) the AGI is acarbose (e.g., PRECOSE®) or miglitol (e.g., GLYSETR®); (v) the insulin secretagogue is a meglitinide (e.g, a repaglinide (e.g., PRANDIN®) or a nateglinide (e.g., STARLIX®)) or a sulfonylurea (e.g., a glipizide (e.g., GLUCOTROL® or MINODIAB®), a glimepiride (e.g., AMARYL®), a glyburide (e.g., GLYNASE®), or a gliclazide (e.g., DIAMICRON® or DACADIS®)); (vi) the thiazolidinedione is pioglitazone (e.g., ACTOS®) or rosiglitazone (e.g., AVANDIA®); or (vii) the biguanide is metformin (e.g., GLUCOPHAGE®, GLUMETZA®, FORTAMET®, or RIOMET®).

[0242] Subjects

[0243] In any of the methods described herein, the subject may be one that has diabetes (e.g., type 1 diabetes or type 2 diabetes), is prediabetic, or is at risk of developing diabetes. Subjects at risk include those with a family history of diabetes and / or are obese (e.g., have a waist circumferance of about >80 cm for males or about >78 cm for females). In any of the methods described herein, the subject may be diagnosed as having hyperglycemia.

[0244] In any of the methods described herein, the subject may be about to receive and / or has recently received (e.g. , within the past 48 hours, e.g., within the past 1 , 2, 3, 4, 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, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48 hours) an agent that, upon its administration, is known to increase blood glucose levels in a subject, such as a statin, a steroid, a beta blocker, a thiazide diuretic, a quinolone antibiotic, an androgen deprivation therapy, an antipsychotic, a protease inhibitor, and / or a calcineurin inhibitor. Exemplary statins that may increase blood glucose levels in a subject include simvastatin (e.g., ZOCOR®), atorvastatin (e.g., LIPITOR®), and rosuvastatin (e.g., CRESTOR®). An exemplary steroid that may increase blood glucose levels in a subject includes prednisone (e.g., DELTASONE®). Exemplary beta blockers that may increase blood glucose levels in a subject include atenolol (e.g., TENORMIN®), metoprolol (e.g., LOPRSSOR® or TORPOL®), and propranolol (e.g., INDERAL LA®). Exemplary thiazide diuretics that may increase blood glucose levels in a subject include hydrochlorothiazide (e.g., APO-HYDRO®, AQUAZIDE®, BPZIDE®, DICHLOTRIDE®, ESIDREX®, HYDROCHLOROT®, HYDRODIURIL®, HYDROSALURIC®, HYPOTHIAZID®, MICROZIDE®, or ORETIC®) and metolazone (e.g., ZYTANIX®, METOZ®, ZAROXOLYN®, or MYKROX®). Exemplary quinolone antibiotics that may increase blood glucose levels in a subject include gatifloxacin (e.g., GATIFLO®, TEQUIN®, or ZYMAR®) and levofloxacin (e.g., LEVAQUIN® or LEVOFLOXACIN SYSTEMIC®). Exemplary androgen deprivation therapies that may increase blood glucose levels in a subject include leuprolide (e.g., CAMCEVI®, ELIGARD®, LUPRON®, LUPRON DEPOT®, LUPRON DEPOT-PED®, or VIADUR®), goserelin (e.g., ZOLADEX®), triptorelin (e.g., TRELSTAR®, TRELSTAR DEPOT®, TRELSTAR LA®, or TRIPTODUR®), and degarelix (e.g., FIRMAGON®). Exemplary antipsychotics that may increase blood glucose levels in a subject include olanzapine (e.g., ZYPREXA® or ZENTIVA®) or clozapine (e.g., CLOZARIL®, DENZAPINE®, or ZAPONEX®). Exemplary protease inhibitors that may increase blood glucose levels in a subject include atazanavir (e.g., REYATAZ®), darunavir (e.g., PREZISTA®), and ritonavir (e.g., NORVIR®). Exemplary calcineurin inhibitors that may increase blood glucose levels in a subject include cyclosporine (e.g., GENGRAF®, NEORAL®, or SANDIMMUNE®), sirolimus (e.g., RAPAMUNE®), and tacrolimus (e.g., ASTAGRAF XL, ENVARSUS XR, PROGRAF, or PROTOPIC).

[0245] Delivery Vehicles

[0246] The inhibitory nucleic acid molecule of the disclosure may be delivered to a subject (e.g., a human) using any suitable delivery vehicle. For example, a delivery vehicle for any of the inhibitory nucleic acid molecules described herein may be a vector, plasmid, or nano particle, (e.g., a micelle, a liposome, an exosome, or a lipid nano particle (LNP)).

[0247] The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via a vector (e.g., a viral vector). Any suitable viral vector system can be used including, e.g., adenoviruses (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdoviruses (e.g., vesicular stomatitis virus), retroviruses, adeno-associated vectors, poxviruses, herpes viral vectors, and Sindbis viral vectors.

[0248] The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of the inhibitory nucleic acids described herein, and compositions thereof. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical composition.

[0249] The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via exosomes. Exosomes produced from cells can be collected from cell culture medium by any suitable method. Typically, a preparation of exosomes can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. For example, using standard methods, exosomes can be prepared by differential centrifugation, that is low speed (<20000 g) centrifugation to pellet larger particles followed by high speed (>100000 g) centrifugation to pellet exosomes, size filtration with appropriate filters (for example, 0.22 micrometer filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.

[0250] The inhibitory nucleic acid molecules of the disclosure, and compositions thereof, may be delivered to a subject via LNPs. For example, the inhibitory nucleic acid molecules (e.g., siRNA, dsRNA, ASO, miRNA, or shRNA) may be formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930, herein incorporated by reference in its entirety. As a non-limiting example, LNP formulations may contain cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(co-methoxy poly(ethylene glycol)2000)carbamoyl)]-1 ,2-dimyristyloxl-propyl-3-amine (PEG-c-DOMG), distearoyl-rac-glycerol (DSG) and / or dimethylaminobutanoate (DMA). As a non-limiting example, 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1 ,2- Distearoyl-sn-glycerol, methoxypoly ethylene glycol) or PEG-DPG ( 1 ,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 1 ,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane (DLin- DMA), C 12-200, and N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1 ,3-dioxolane-4- ethanamine (DLin-KC2-DMA).

[0251] Exemplary commercial reagents useful for lipid-based delivery of inhibitory nucleic acid molecules including, but not limited to, TRANSIT-TKO™ (Mirus, Catalog No. MIR 2150), TRANSMESSENGER™ (Qiagen, Catalog No. 301525), OLIGOFECTAMINE™ and LIPOFECTAMINE™ (Invitrogen, Catalog No. MIR 12252-011 and Catalog No. 13778-075), SIPORT™ (Ambion, Catalog No. 1631 ), and DHARMAFECT™ (Fisher Scientific, Catalog No. T-2001- 01).

[0252] Dosage

[0253] The actual dosage amount of a composition of the present disclosure administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage (e.g. , mg / kg) and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary. Subjects may be adult or pediatric humans, with or without comorbid diseases.

[0254] Routes of Administration

[0255] The compositions utilized in the methods described herein can be administered to a subject by any suitable route of administration. For example, a composition containing an inhibitory nucleic acid of the disclosure may be administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions.

[0256] In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intravenously. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject subcutaneously.

[0257] EXAMPLE

[0258] The following example is put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and is intended to be purely exemplary and is not intended to limit the scope of the disclosure.

[0259] Example 1. Effects of siRNA Targeting of Human AC9 in vitro

[0260] The following Example describes the materials and methods that were utilized for obtaining the results described herein.

[0261] Material and Methods

[0262] Cell culture 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 g / ml streptomycin. Cells were incubated in 5% CO2 at 37°C and were harvested once a week with trypsin- EDTA. siRNA transfection:

[0263] HepG2 cells were transfected with an adenylate cyclase type 9 (AC9) or a scramble siRNA in the presence of Lipofectamine RNAiMAX in Opti-MEM for 72 h unless otherwise stated.

[0264] Quantification of mRNA Expression by

[0265] Reverse Transcription-Quantitative PCR

[0266] HepG2 cells total RNA was extracted using QIAGEN RNeasy Plus isolation kits according to the manufacturer’s protocol. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kits and MultiScribe Reverse Transcriptase according to Agilent’s protocol. Primers were designed using the Beacon designer software v.8 and obtained from IDT. The reference genes for normalization, HBS1 like translational GTPase (HBS1 L) and phosphoglycerate kinase 1 (PGK1 ), were selected by using the Bio-Rad CFX Maestro software based on the GeNorm method. The qPCR was performed using SYBR-Green reaction mix (Bio-Rad). The qPCR conditions consisted of an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of amplification, with each cycle consisting of 95°C for 15 seconds, and 60°C for 60 seconds. Results were analysed with the delta-delta Ct method using Bio-Rad CFX Maestro software.

[0267] RNA sequencing

[0268] HepG2 cell culture, silencing of adenylate cyclase 9 (ADCY9; otherwise referred herein as AC9) and RNA were performed as above. Then RNA was used for Illumina TruSeq stranded cDNA library preparation following manufacturer instructions. The library was sequenced on an Illumina HiSeq 2500 using paired-ends 100 bp mode. The RNA-Seq data were analyzed using the standard RNA-Seq workflow by McGill University and Genome Quebec Innovation Centre.

[0269] Untargeted proteomic

[0270] Before harvesting the HepG2 proteome, HepG2 silencing of AC9 was performed as described above. Then, Tandem Mass Tags (TMT) technology was used to label peptides from different samples, allowing for multiplexed analysis and comparison of protein abundance of multiple conditions simultaneously with liquid chromatography-mass spectrometry (LC-MS / MS).

[0271] G6Pase activity

[0272] G6Pase activity measurements were conducted on isolated microsomes. Briefly, cells plated in 6-well plates were homogenized in 500 L per well of homogenization buffer (e.g. , 300 mM sucrose, 20 mM HEPES, pH 7.2) with a 2-mL Dounce homogenizer by 20 strokes using the tight pestle. Samples were then sonicated on ice (e.g., 3 cycles of 5 seconds with 5 seconds rest) and centrifuged at 12,000 x g for 20 minutes at 4°C. Supernatant were subsequently centrifuged at 100,000 x g for 60 minutes at 4°C in a TLA 100.3 Ti rotor. Pellets were finally resuspended in 50 pL of homogenization buffer and protein quantification was done using the Lowry method. For measuring activity, 30 pg of microsomes were diluted in 30 pL of 100 mM phosphate buffer pH 7.2. Then, 5 pL of a 10 mM Nicotinamide Adenine Dinucleotide (NAD) solution, 5 pL of a 20 U / mL glucose dehydrogenase solution, and 5 pL of a 20 mM Glucose-6-phosphate (G6P) solution were added. Each protein sample had a control well (without G6P) to determine specific activity of G6Pase. The plate was incubated at 37°C for 90 minutes. Optical density was read at 340 nm. Glucose concentration was determined with a glucose standard curve.

[0273] Immunoblotting

[0274] For Western blotting, cells were washed with cold phosphate buffered saline (PBS) and then scraped and lysed in ice-cold lysis buffer (e.g., 50 mM Tris, pH 7.5, 150 mM NaCI, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, and a protease inhibitor cocktail). Lysate was microcentrifugated for 20 min at 4°C and supernatant was assessed for cell proteins. Proteins (e.g., 30-50 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) followed by Western blotting to a polyvinylidene fluoride (PVDF) membrane.

[0275] Glucose production assay

[0276] HepG2 cells were seeded in 6-well plates and after 72h, cells were washed with PBS to remove the remaining glucose. The medium was replaced with a glucose production buffer (e.g., glucose-free DMEM, without phenol red supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate). After 2 h at 37° C, the supernatants were collected to measure glucose concentration with the Amplex Red Glucose Assay Kit (ThermoFisher Scientific) following the manufacturer’s instructions.

[0277] Glucose-6-Phosphate quantification

[0278] HepG2 cells were transfected for 72h, washed with PBS and harvested to be used for G6P quantification with Glucose-6-Phosphate Assay Kit (Sigma-Aldrich) following the manufacturer’s instructions.

[0279] Glycogen quantification

[0280] Cells were washed once with PBS and then lysed in 300 pL KOH 30% with agitation at room temperature for 15 minutes. Lysates were transferred in eppendorfs and boiled at 100°C for 30 minutes. After cooling down, 20 pL of saturated Na2SO4 were added as coprecipitant followed by 600 pL (e.g., 2 vol) of absolute ethanol. Samples were centrifuged at 15,000 x g for 15 minutes at 4°C and pellets were suspended in 300 pL of H2O. Glycogen was precipitate by adding 2 volumes of absolute ethanol and centrifuge again. Pellets were suspended in 250 pL of 50 mM acetate buffer pH 4.5 containing 1 mg / mL of amyloglucosidase (Sigma). Samples and glycogen standards were incubated overnight at 37°C. Fifty microliters of each reaction were transferred to a 96-well plate. To each well the following was added; 30 L of 100 mM phosphate buffer pH 7.2, 10 L of 10 mM NAD and 10 pL of glucose dehydrogenase solution (20 U / mL) (Sigma). The plate was incubated for 2 hours at 37°C before reading OD at 340 nm. Glycogen concentrations were calculated with the glycogen standard curve.

[0281] Glucose tolerance test in atherosclerotic mice

[0282] Wild-type (WT) and AC9 KO (via gene trapping with GT / GT) mice fed with a cholesterol- enriched atherogenic diet for 6 and 16 weeks were food-deprived during 5 hours with ad libitum access to water. A bolus of glucose was administered via an intraperitoneal injection, and glycemia was measured from blood sampled at the tail vein using a glucometer at TO (before injection), 15, 30, 60, and 90 minutes. Tail vein blood samples were collected via a capillary for insulin assays.

[0283] Results

[0284] AC9 protein expression was analyzed by Western blot in HepG2 cells after siRNA-mediated knockdown (KD) of AC9 (siAC9). Several different siRNAs were tested (e.g. , see Table 1 ), all of which significantly reduced AC9 in HepG2 cells (FIG. 7 and Table 4).

[0285] Table 4. AC9 PROTEIN EXPRESSION FOLLOWING KNOCKDOWN n = 8; Repeated measures ANOVA with uncorrected Fisher’s LSD: *** = p < 0.001 different from siScramble

[0286] Glucose-6-phosphatase catalytic subunit (G6PC) mRNA expression was analyzed by RNA sequencing in HepG2 cells after siRNA-mediated KD of AC9. The mRNA expression of G6PC was significantly reduced in siAC9, relative to an siScramble control (FIG. 1 ). Similar results were observed by qPCR analysis (FIG. 2). Notably, HepG2 cells transfected with siAC9 and incubated 72 hours later in a glucose production media resulted in a 42% reduction in glucose concentrations relative to the siScramble control (FIG. 3). These results illustrate the impact of AC9 siRNA on G6PC gene expression and hepatic gluconeogenesis and glycogenolysis.

[0287] We next investigated these impacts in vivo. AC9 WT and AC9 KO (AC9 GT / GT) mice were fed an atherogenic diet for 6 or 16 weeks and submitted to an intraperitoneal glucose tolerance test (IPGTT). The results of this test show that AC KO mice had significantly reduced glucose levels compared to WT across the vast majority of time points (FIG. 4). Measuring the area under the curve (AUG) of FIG. 4 supports this observation, showing that blood glucose levels in AC9 KO mice were significantly reduced during the intraperitoneal glucose tolerance test (FIG. 5). Plasma insulin levels in these AC9 KO mice were not significantly modulated compared to AC9 WT mice, with the exception of a significant increase at 15 minutes of the IPGTT assay in AC9 KO mice fed with an atherogenic diet for 6 weeks (FIG. 6). Taken together, these in vivo experiments support the observed impact of AC9 siRNA on G6PC gene expression and G6Pase activity and the resulting effect on hepatic gluconeogenesis and glycogenolysis.

[0288] Next, the Exome Phenome-Wide Association Study (ExPheWas) Browser, an interactive web-based tool for exploring associations between genetic variants and phenotypes, was utilized for exploring an association between adenylate cyclase 9 ADCY9 (i.e., AC9) and various outcomes (e.g., see FIG. 8). Using genetic variants near the ADCY9 gene, Mendelian Randomization (MR) provided an estimate on the effect of monocyte count on glycated hemoglobin (HbA1c) (FIG. 9), glucose (FIG. 10), diabetes (FIG. 11 ) and Type 2 diabetes (FIG. 12). Taken together, these data show that genetic variants influencing ADCY9 expression or function are associated with subjects having diabetes (e.g., Type 2 diabetes). Therefore, targeting ADCY9 with an inhibitory nucleic acid molecule described herein may reduce the risk of a subject from developing hyperglycemia (e.g., diabetes, e.g., Type 2 diabetes). Additionally, MR provided an estimate on the effect of monocyte count on self-reported heart attack / myocardial infarction (Ml) events (FIG. 28) and hospital-recorded heart attack / MI events (FIG. 29 and FIG. 30). Taken together, these data show that genetic variants influencing ADCY9 expression or function are associated with Ml risk in a way that supports a causal relationship. Therefore, targeting ADCY9 with an inhibitory nucleic acid molecule described herein may reduce the risk of Ml in a subject (e.g., a subject having hyperglycemia, e.g., diabetes, e.g., Type 2 diabetes).

[0289] Next, the effects of various agents (e.g., atorvastatin, forskolin (FSK), 3-isobutyl-1 - methylxanthine (IMBX), cyclic adenosine monophosphate (cAMP), and insulin) in combination with siAC9 were investigated for their effect on G6PC expresison. Atorvastatin (e.g., LIPITOR®) is a medication used to lower cholesterol levels and reduce the risk of cardiovascular disease. It is known to increase levels of G6PC. Notably, atorvastatin (5 M) induced significant G6PC mRNA upregulation, which was then significantly reduced upon siRNA-mediated knockdown of AC9 (FIG.

[0290] 13), suggesting that targeting AC9 may be a strategy to mitigate the diabetogenic side effects of statin therapy. FSK is known to activate AC9 and increase cAMP inside of cells and IBMX, a nonselective phosphodiesterases (PDE) inhibitor, also allows for cAMP accumulation inside of cells. While the expected stimulatory effect of these treatments (e.g., combinatorial FSK and IBMX (“FSK / IMBX”), or cAMP) on G6PC mRNA expression was preserved in AC9 knockdown cells, the overall expression levels remained significantly lower than in siScramble-transfected cells, even after stimulation (FIG.

[0291] 14). Similarly, the G6PC mRNA response to insulin (100 nM) is preserved after siRNA-mediated knockdown of AC9 (FIG. 15). These findings suggest that AC9 is a viable target for controlling glucose level without compromising responsiveness to other key regulatory signals.

[0292] Next, the effect of siAC9 on transcriptional regulators of glucose production were examined. FIG. 16 provides a schematic of gluconeogenesis-related transcription factors that regulate G6PC expression. siAC9 was found to lower two G6PC-regulating transcription factors in HepG2 cells: peroxisome proliferator-activated receptor gamma coactivator 1a (PGC1a) (FIG. 17A) and hepatocyte nuclear factor 4a (HNF4a) (FIG. 17B). These results suggest that the reduction of G6PC mRNA expression and activity induced by AC9 siRNA is caused by the decrease expression of these transcriptional factors. Other factors of gluconeogenesis (FIG. 18) were also investigated, such as glucose-6- phosphate transporter (G6PT) and glucose-6-phosphate. Notably, siAC9 was found to increase mRNA expression of both G6PT (FIG. 19A) and glucose-6-phosphate (FIG. 19B). FIG. 20 provides a schematic of the role of phosphoenolpyruvate Carboxykinase 1 (PCK1 ) and glycogen phosphorylase (PYGL) enzymes in hepatic gluconeogenesis and glycogenolysis. Notably, siAC9 reduced mRNA expression of both PCK1 (FIG. 21 A) and PYGL (FIG. 21 B), suggesting that the mechanism by which AC9 knockdown reduces glucose production is not limited to its effect on G6PC. FIG. 22A is a simplified schematic of glucose metabolism, with hexokinase being the catalyst of glucose-6- phosphate production. Notably, siAC9 increased mRNA expression hexokinase (FIG. 22B).

[0293] FIG. 23 is a schematic of an exemplary method for measuring G6Pase activity. Notably, the HepG2 cell model responded appropriately to regulators of glucose metabolism, with G6Pase activity increasing in response to combinatorial FSK (10 M) and IBMX (1 OO M) and decreasing in response to insulin (100 nM) (FIG. 24A). Importantly, G6Pase activity is decreased upon siRNA-mediated knockdown of AC9 (FIG. 24B), indicating that reduction of G6PC gene expression translates into a functional reduction of G6Pase activity. FIG. 25 is a schematic exemplifying a method for measuring the intracellular level of glycogen. Notably, combinatorial FSK (10 M) and IBMX (100pM) decreased glycogen levels while insulin (100 nM) increased glycogen levels in HepG2 cells (FIG. 26), further validating the HepG2 cell model.

[0294] Lastly, the effects of various agents (e.g., FSK / IMBX, insulin, and the PKA inhibitor, H89) were investigated for their effect on glucose production and to validate the glucose production protocol and cellular model. As expected, combinatorial FSK (10 pM) and IMBX (100 pM) significantly increased glucose production, while insulin (100 nM) and the protein kinase A (PKA) inhibitor, H89 (5 pM), significantly decreased glucose production (FIG. 27A). Notably, siRNA-mediated knockdown of PCK1 (siPCKI ) or AC9 (siAC9) in HepG2 cells significantly decreased glucose production (FIG. 27B), further validating the above observations.

[0295] Taken together, these show that AC9 knockdown effectively reduces glucose production by decreasing the expression and activity of G6PC. This effect was observed in both human HepG2 cells and in AC9 knockout mice. Additionally, the findings indicate that AC9 inhibition could mitigate the diabetogenic side effects of statin therapy. Moreover, these data show that genetic variants influencing ADCY9 expression or function are associated with risk of diabetes and Ml. These findings suggest that targeting AC9 represents a therapeutic strategy for treating hyperglycemia and diabetes, as well as reducing the risk of major adverse cardiovascular events.

[0296] Other Embodiments

[0297] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0298] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0299] Other embodiments are within the claims.

Claims

1. CLAIMSWhat is claimed is:

1. A method of reducing the risk of a major adverse cardiovascular event (MACE) in a subject, the method comprising inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject2. The method of claim 1 , wherein the MACE is selected from the group consisting of myocardial infarction (Ml), a stroke, cardiac arrest, arrhythmia, and cardiovascular death.

3. A method of treating a cardiovascular condition in a subject, the method comprising inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject.

4. The method of claim 3, wherein the cardiovascular condition is selected from the group consisting of: Ml, stroke, cardiac arrest, and arrhythmia.

5. A method of decreasing blood glucose level in a subject, the method comprising inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject.

6. A method of decreasing glucose-6-phosphatase catalytic subunit (G6PC) in a subject, the method comprising inhibiting the expression or function of adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject.

7. The method of any one of claims 1-6, wherein the subject is diagnosed with hyperglycemia.

8. A method of reducing a risk of developing hyperglycemia in a subject, the method comprising inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject.

9. A method of treating hyperglycemia in a subject, the method comprising inhibiting the expression or function of an adenylate cyclase in the subject, wherein the inhibiting comprises administration of an inhibitory nucleic acid molecule to the subject.

10. The method of any one of claims 1-9, wherein the subject has or is at risk of developing diabetes.

11. The method of any one of claims 1-10, wherein the subject is to receive and / or has recently received a statin, a steroid, a beta blocker, a thiazide diuretic, a quinolone antibiotic, an androgen deprivation therapy, an antipsychotic, a protease inhibitor, or a calcineurin inhibitor.

12. The method of claim 11 , wherein:(a) the statin is selected from the group consisting of simvastatin, atorvastatin, and rosuvastatin;(b) the steroid is prednisone;(c) the beta blocker is selected from the group consisting of: atenolol, metoprolol, and propranolol;(d) the thiazide diuretic is hydrochlorothiazide or metolazone;(e) the quinolone antibiotic is gatifloxacin or levofloxacin;(f) the androgen deprivation therapy comprises leuprolide, goserelin, triptorelin, or degarelix;(g) the antipsychotic is olanzapine or clozapine;(h) the protease inhibitor is selected from the group consisting of atazanavir, darunavir, and ritonavir; or(i) the calcineurin inhibitor is selected from the group consisting of cyclosporine, sirolimus, and tacrolimus.

13. The method of any one of claims 1-12, wherein the adenylate cyclase is adenylate cyclase type 9 (AC9).

14. The method of claim 13, wherein the AC9 comprises:(i) an mRNA sequence of SEQ ID NO: 16; and / or(ii) a DNA sequence of SEQ ID NO: 17.

15. The method of any one of claims 1-14, 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).

16. The method of claim 15, wherein the inhibitory nucleic acid molecule is an siRNA molecule.

17. The method of claim 16 or 16, wherein the siRNA molecule comprises a sequence complementary to at least 15 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 16-27.

18. The method of claim 17, wherein the siRNA molecule comprises a sequence complementary to at least 19 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10 and 16-27.

19. The method of claim 18, wherein the siRNA molecule comprises a sequence complementary to at least 21 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-10, 16, and 17.

20. The method of claim 19, wherein the siRNA molecule comprises a sequence complementary to at least 25 contiguous nucleotides set forth within any one of SEQ ID NOs: 16 and 17.

21. The method of any one of claims 15-20, wherein the siRNA molecule contains 3’ overhangs selected from the group consisting of:(i) a single uracil overhang at one or more 3’ ends of the siRNA molecule;(ii) a double uracil overhang at one or more 3’ ends of the siRNA molecule;(Hi) a single thymine overhang at one or more 3’ ends of the siRNA molecule;(iv) a double thymine overhang at one or more 3’ ends of the siRNA molecule; or(v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA molecule.

22. The method of any one of claims 15-21 , wherein the siRNA molecule comprises a nucleotide sequence of any one or more of SEQ ID NOs: 1-10 and 18-27.

23. The method of claim 22, wherein the siRNA molecule comprises:(i) a sense strand comprising the sequence of SEQ ID NO: 1 and an antisense strand comprising the sequence of SEQ ID NO: 2;(ii) a sense strand comprising the sequence of SEQ ID NO: 3 and an antisense strand comprising the sequence of SEQ ID NO: 4;(Hi) a sense strand comprising the sequence of SEQ ID NO: 5 and an antisense strand comprising the sequence of SEQ ID NO: 6;(iv) a sense strand comprising the sequence of SEQ ID NO: 7 and an antisense strand comprising the sequence of SEQ ID NO: 8;(v) a sense strand comprising the sequence of SEQ ID NO: 9 and an antisense strand comprising the sequence of SEQ ID NO: 10;(vi) a sense strand comprising the sequence of SEQ ID NO: 18 and an antisense strand comprising the sequence of SEQ ID NO: 19;(vii) a sense strand comprising the sequence of SEQ ID NO: 20 and an antisense strand comprising the sequence of SEQ ID NO: 21 ;(viii) a sense strand comprising the sequence of SEQ ID NO: 22 and an antisense strand comprising the sequence of SEQ ID NO: 23;(ix) a sense strand comprising the sequence of SEQ ID NO: 24 and an antisense strand comprising the sequence of SEQ ID NO: 25; or(x) a sense strand comprising the sequence of SEQ ID NO: 26 and an antisense strand comprising the sequence of SEQ ID NO: 27.

24. The method of any one of claims 15-23, wherein the siRNA molecule comprises a modification, wherein the modification is a non-natural or modified nucleoside or nucleotide.

25. The method of claim 24, wherein the wherein the modification is chosen from a 2'-O-methyl (2 -0- Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2-F) modified nucleoside.

26. The method of any one of claims 15-25, wherein the siRNA molecule targets the sequence of any one of SEQ ID NOs: 11-15.

27. The method of any one of claims 1-26, wherein the method further comprises administering in combination a second therapeutic agent to the subject.

28. The method of claim 27, wherein the second therapeutic agent is selected from the group consisting of a dipeptidyl-peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1 ) receptor agonist, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an alpha-glucosidase inhibitor (AGI), an insulin secretagogue, a thiazolidinedione, a biguanide, or a combination thereof.

29. The method of claim 28, wherein:(i) the DPP-4 inhibitor is selected from the group consisting of alogliptin, linagliptin, saxagliptin, and sitagliptin;(ii) the GLP-1 receptor agonist is selected from the group consisting of exenatide, lixisenatide, dulaglutide, and liraglutide;(iii) the SGLT2 inhibitor is selected from the group consisting of canagliflozin, dapagliflozin, and empagliflozin;(iv) the AGI is acarbose or miglitol;(v) the insulin secretagogue is a meglitinide or a sulfonylurea;(vi) the thiazolidinedione is pioglitazone or^osiglitazone; or(vii) the biguanide is metformin.

30. The method of claim 29, wherein:(a) the the meglitinide is a repaglinide or a nateglinide; or(b) the sulfonylurea is selected from the group consisting of glipizide, glimepiride, glyburide, or gliclazide.31 . Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for decreasing glucose levels in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

32. Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for decreasing G6PC in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

33. Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for reducing a risk of developing hyperglycemia in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

34. Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for treating hyperglycemia in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

35. Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for reducing the risk of MACE in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

36. Use of an inhibitory nucleic acid molecule in the manufacture of a medicament for treating a cardiovascular condition in a subject, wherein the expression or function of an adenylate cyclase in the subject is inhibited by an administration of the inhibitory nucleic acid molecule.

37. The use of any one of claims 31-36, wherein the adenylate cyclase is adenylate cyclase type 9 (AC9).

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