Compositions and methods of treating cardiovascular disease

Abstract

Described herein are compositions (e.g., an inhibitory nucleic acid molecule) for reducing expression of small EDRK-rich factor 2 (SERF2) and methods thereof for (i) treating cardiovascular disease (e.g., PAD, CAD, heart failure, cardiomyopathy, or stroke) in a subject; (ii) treating or reducing the likelihood of CTLI in a subject; (iii) alleviating chronic ischemic rest pain in a subject having CTLI; and / or (iv) promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease). The inhibitory nucleic acid molecule may be a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), an anti-sense oligonucleotide (ASO), a microRNA (miRNA), a short hairpin RNA (shRNA), or a gapmeR described herein, or a composition (e.g., pharmaceutical composition) thereof.

Description

[0001] COMPOSITIONS AND METHODS OF TREATING CARDIOVASCULAR DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 626,188, filed on January 29, 2024, which is incorporated herein by reference in its entirety for any purpose.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 29, 2025 is named “51665-005WO2_Sequence_Lisiting_1_29_25” and is 41 ,937 bytes in size.

[0006] TECHNICAL FIELD

[0007] This disclosure relates to inhibitory nucleic acid molecules useful for targeting small EDRK-rich factor 2 (SERF2), and methods of using such inhibitory nucleic acid molecules for (i) treating cardiovascular disease (e.g., coronary artery disease (CAD), peripheral artery disease (PAD), heart failure, cardiomyopathy, or stroke) in a subject; (ii) treating or reducing the likelihood of chronic limbthreatening ischemia (CTLI) in a subject; (iii) alleviating chronic ischemic rest pain in a subject having CTLI; and / or (iv) promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease).

[0008] BACKGROUND

[0009] Cardiovascular disease remains the leading cause of morbidity and mortality worldwide. Moreover, for patients with diabetes, longevity and quality of life is negatively impacted due, in part, to impaired angiogenesis. Elevated blood glucose levels (e.g., hyperglycemia) can alter endothelial cell (EC) metabolism, induce oxidative stress in tissue, and promote apoptosis in ECs. These metabolic perturbations can negatively impact normal cell signaling pathways during angiogenesis. There remains an unmet need for therapeutics that can treat or alleviate symptoms of cardiovascular diseases by promoting angiogenesis. The role of microRNAs (miRNAs) in angiogenesis is relatively unexplored.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention provides an inhibitory nucleic acid molecule comprising sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and (ii) the target nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6, or a variant thereof (e.g., see Table 3).

[0012] In some embodiments, the target nucleic acid molecule comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, or a variant thereof (e.g., see Table 3).

[0013] In some embodiments, the target nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6, or a variant thereof (e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule is 15 to 2,543 nucleotides in length (e.g., 50 to 2543 nucleotides in length, 100 to 2,543 nucleotides in length, 250 to 2,543 nucleotides in length, 500 to 2,543 nucleotides in length, 750 to 2,543 nucleotides in length, 1 ,000 to 2,543 nucleotides in length, 1 ,500 to 2,543, or 2,000 to 2,543 nucleotides in length).

[0014] In some embodiments, the inhibitory nucleic acid molecule is 15 to 3,429 nucleotides in length (e.g., 50 to 3,429 nucleotides in length, 100 to 3,429 nucleotides in length, 250 to 3,429 nucleotides in length, 500 to 3429 nucleotides in length, 750 to 3,429 nucleotides in length, 1 ,000 to 3,429 nucleotides in length, 1 ,500 to 3,429 2,000 to 3,429, or 3,000 to 3,429 nucleotides in length).

[0015] In some embodiments, the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length (e.g., 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,

[0016] 43, 44, 45, 46, 47, 48, or 49 nucleotides in length), 50 to 99 nucleotides in length (e.g., 50, 51 , 52, 53, 54,

[0017] 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82,

[0018] 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 nucleotides in length), 100 to 2,543 nucleotides in length (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1 ,025, 1 ,050, 1 ,075, 1 ,100, 1 ,125, 1 ,150, 1 ,175, 1 ,200, 1 ,225, 1 ,250, 1 ,275, 1 ,300, 1 ,325, 1 ,350, 1 ,375, 1 ,400, 1 ,425, 1 ,450, 1 ,475, 1 ,500, 1 ,525, 1 ,550, 1 ,575, 1 ,600, 1 ,625, 1 ,650, 1 ,675, 1 ,700, 1 ,725, 1 ,750, 1 ,775, 1 ,800, 1 ,900, 2,000, or 2,543 nucleotides in length), or 100 to 3,429 nucleotides in length (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1 ,025, 1 ,050, 1 ,075, 1 ,100, 1 ,125, 1 ,150, 1 ,175, 1 ,200, 1 ,225, 1 ,250, 1 ,275, 1 ,300, 1 ,325, 1 ,350, 1 ,375, 1 ,400, 1 ,425, 1 ,450, 1 ,475, 1 ,500, 1 ,525, 1 ,550, 1 ,575, 1 ,600, 1 ,625, 1 ,650, 1 ,675, 1 ,700, 1 ,725, 1 ,750, 1 ,775, 1 ,800, 1 ,900, 2,000, 2,500, 3,000, or 3,429 nucleotides in length).

[0019] In some embodiments, the inhibitory nucleic acid molecule is 18 to 28 nucleotides in length (e.g., 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 nucleotides in length).

[0020] In some embodiments, the inhibitory nucleic acid molecule is 18 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 19 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 20 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 21 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 22 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 23 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 24 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 25 nucleotides in length.

[0021] In some embodiments, the inhibitory nucleic acid molecule is 19, 20, or 21 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 19 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 20 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 21 nucleotides in length.

[0022] In some embodiments, the inhibitory nucleic acid molecule comprises at least 84% (e.g., 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or variant thereof (e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule includes at least 89% (e.g., 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or variant thereof (e.g., see Table 3).

[0023] In some embodiments, the inhibitory nucleic acid molecule includes at least 94% (e.g., 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or variant thereof (e.g., see Table 3).

[0024] In some embodiments, the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule, or variant thereof (e.g., see Table 3).

[0025] In some embodiments, the inhibitory nucleic acid molecule further includes a modification.

[0026] In some embodiments, the modification includes: (a) a non-natural or modified nucleoside or nucleotide; and / or (b) a covalently or non-covalently conjugated moiety.

[0027] In some embodiments: (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LNA), a 2'-O-methyl (2’-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

[0028] In some embodiments, the targeting moiety is vascular cell adhesion protein 1 (VCAM1). In some embodiments, the targeting moiety is arginylglycylaspartic acid (RGD).

[0029] In some embodiments, the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

[0030] In some embodiments, the inhibitory nucleic acid molecule is a dsRNA.

[0031] In some embodiments, the inhibitory nucleic acid molecule is a miRNA.

[0032] In some embodiments, the inhibitory nucleic acid molecule is an shRNA.

[0033] In some embodiments, the inhibitory nucleic acid molecule is an ASO.

[0034] In some embodiments, the inhibitory nucleic acid molecule is a gapmeR.

[0035] In some embodiments, the inhibitory nucleic acid molecule is an siRNA.

[0036] In some embodiments, the siRNA comprises an antisense strand comprising at least 84% (e.g., 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0037] In some embodiments, the antisense strand comprises at least 89% (e.g., 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] In some embodiments, the antisense strand comprises at least 94% (e.g., 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0039] In some embodiments, the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0040] In some embodiments, the siRNA further comprises a sense strand comprising at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the siRNA includes: (a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 3; or (b) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 4.

[0041] In some embodiments, the siRNA contains 3’ overhangs selected from the group consisting of: (i) a single uracil overhang at one or more 3’ ends of the siRNA; (ii) a double uracil overhang at one or more 3’ ends of the siRNA; (iii) a single thymine overhang at one or more 3’ ends of the siRNA; (iv) a double thymine overhang at one or more 3’ ends of the siRNA; or (v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA.

[0042] In some embodiments, the siRNA targets the nucleotide sequence of SEQ ID NO: 5.

[0043] In some embodiments, the inhibitory nucleic acid molecule is a miRNA.

[0044] In some embodiments, the miRNA comprises a modification selected from: (a) a non-natural or modified nucleoside or nucleotide; and / or (b) a covalently or non-covalently conjugated moiety.

[0045] In some embodiments: (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LNA), a 2'-O-methyl (2’-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

[0046] In some embodiments, the targeting moiety is VCAM1 . In some embodiments, the targeting moiety is RGD.

[0047] In some embodiments, the miRNA comprises a nucleotide sequence comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15.

[0048] In some embodiments, the miRNA comprises a nucleotide sequence comprising at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15.

[0049] In some embodiments, the miRNA comprises a nucleotide sequence comprising at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15.

[0050] In some embodiments, the miRNA comprises the nucleotide sequence of SEQ ID NO: 15.

[0051] In some embodiments, the miRNA is miR-1282.

[0052] In some embodiments, the inhibitory nucleic acid molecule is formulated in a delivery vehicle.

[0053] In some embodiments, the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

[0054] In some embodiments, the vector is a viral vector.

[0055] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0056] In some embodiments, the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , and AAV12.

[0057] In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition.

[0058] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient, diluent, and / or carrier. In a second aspect, the invention provides a method of treating cardiovascular disease (e.g., coronary artery disease (CAD), peripheral artery disease (PAD), heart failure, cardiomyopathy, or stroke) in a subject, the method comprising administering the inhibitory nucleic acid molecule of the first aspect.

[0059] In a third aspect, the invention provides a method of treating or reducing the likelihood of critical limb-threatening ischemia (CTLI) in a subject, the method comprising administering the inhibitory nucleic acid molecule of the first aspect.

[0060] In a fourth aspect, the invention provides a method of alleviating chronic ischemic rest pain in a subject having CTLI, the method comprising administering the inhibitory nucleic acid molecule of the first aspect.

[0061] In a fifth aspect, the invention provides a method of promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease), the method comprising administering the inhibitory nucleic acid molecule of the first aspect.

[0062] In some embodiments of any of the foregoing aspects, the subject has previously experienced a myocardial infarction.

[0063] In some embodiments of any of the foregoing aspects, the subject has an ischemic injury.

[0064] In some embodiments of any of the foregoing aspects, the subject has a cardiovascular disease.

[0065] In some embodiments, the cardiovascular disease is CAD, PAD, heart failure, cardiomyopathy, or stroke.

[0066] In some embodiments of any of the foregoing aspects, the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

[0067] In some embodiments, the metabolic disorder is diabetes.

[0068] In some embodiments, the subject at risk of developing diabetes is prediabetic and / or has one or more of the following: (a) hyperglycemia; (b) glucose resistance; (c) insulin resistance; (d) hyperlipidemia; and (e) has a family history of diabetes.

[0069] In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is administered to the subject intravenously, intraperitoneally, subcutaneously, intraarticularly, or intramuscularly.

[0070] In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is delivered to the subject’s limb skeletal muscle (e.g., gastrocnemius muscle) and / or cardiac muscle.

[0071] In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is delivered to an endothelial cell.

[0072] In some embodiments of the second or third aspect, the further comprising administering an additional therapeutic agent.

[0073] In some embodiments, the additional therapeutic agent is a statin (e.g., atorvastatin), a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor (e.g., an siRNA or monoclonal antibody targeting PCSK9), a Factor X inhibitor (e.g., rivaroxaban, e.g., XARELTO®), or ezetimibe (e.g., ZETIA™).

[0074] In yet another aspect, the invention provides any and aii compositions, articles of manufacture, methods, and uses disclosed and / or described in the specification.

[0075] In yet another aspect, the invention provides a method of treating tissue repair after ischemic injury, including but not limited to, peripheral artery disease, critical limb-threatening ischemia, ischemia or injury in the limb by administering to the subject a iocal or systemic injection of a therapeutically effective amount of a nucleic acid comprising microRNA mimics for miR-1282.

[0076] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

[0079] FIG. 1A-FIG. 11 contain data characterizing miR-1282, an endothelial cell (EC) enriched hypoxia- induced miRNA, and its cis-antisense target, small EDRK-rich factor 2 (SERF2). All values shown are mean + / - standard error (SE) (n > 3). Data were calculated using Student’s t-test or two-way analysis of variance (ANOVA). “NSm” refers to a non-specific (NS) control for miR-1282. “1282m” refers to the miR- 1282 mimic. FIG. 1A Plasma miRNA-sequencing identified miR-1282 as the highest upregulated miRNAs in Fontaine lll / IV patients. FIG. 1B is a schematic showing the identification of mouse miR-1282 that can pair with SERF2 transcript. The sequence labeled “SERF2” refers to nucleotides 193-218 of SEQ ID NO: 6. The sequence labeled “hsa-miR-1282” is SEQ ID NO: 15. FIG. 1C is a schematic showing the identification of a previously unannotated mouse ortholog of human miR-1282 that can pair with SERF2 transcript. The sequence labeled “SERF2” refers to nucleotides 193-218 of SEQ ID NO: 6. The sequence labeled “Gm22953-201 ” (which is the transcript name of miR-1282 in mice) is SEQ ID NO: 15. FIG. 1D is a graph quantifying the expression profile of miR-1282 in various cell lines. FIG. 1E is a graph quantifying the expression profile of miR-1282 in the gastrocnemius muscle in Db / + and Db / Db mice following femoral artery ligation (FAL). FIG. 1F is a graph showing the effect of hypoxia on miR-1282 expression in mouse diabetic skeletal muscle endothelial cells (DmECs). FIG. 1G is a graph showing the effect of hypoxia on SERF2 expression in mouse DmECs. FIG. 1H is a graph showing the effect of miR-1282 mimic (1282m) on SERF2 mRNA expression in DmECs. FIG. 11 is a graph showing the effect of miR- 1282 mimic (1282m) on SERF2 protein expression in DmECs. All values are mean + / - SE (n > 3).

[0080] FIG. 2A-FIG. 2P contain data demonstrating that miR-1282 overexpression or SERF2 knockdown promotes angiogenesis and the secretion of proangiogenic proteins in diabetic endothelial cells. All values are mean + / - SE. Data were calculated using Student’s t-test (n > 4). “NSm” refers to a nonspecific (NS) control for miR-1282. “1282m” refers to the miR-1282 mimic. “siNS” refers to a NS control for siSERF2. “siSERF” refers to siRNA-mediated knockdown of SERF2. FIG. 2A is a representative image of a network tube formation assay on miR-1282 mimic- (1282m) or SERF2 siRNA (siSERF2)- transfected DmECs under normoxia conditions. FIG. 2B is a set of graphs quantifying the number of tubes under normoxia conditions after 6 hours of cells seeding in 1282m- or siSERF-transfected cells. FIG. 2C is a graph quantifying tube length under normoxia conditions after 6 hours of cells seeding in 1282m-transfected cells. FIG. 2D is a graph quantifying tube length under normoxia conditions after 6 hours of cells seeding in siSERF-transfected cells. FIG. 2E is a graph quantifying the number of branching points under normoxia conditions after 6 hours of cells seeding in 1282m-transfected cells. FIG. 2F is a graph quantifying the number of branching points under normoxia conditions after 6 hours of cells seeding in siSERF-transfected cells. FIG. 2G is a representative image of a network tube formation assay on miR-1282 mimic- (1282m) or SERF2 siRNA (siSERF2)-transfected DmECs under hypoxic conditions. FIG. 2H is a set of graphs quantifying the number of tubes under hypoxic conditions after 6 hours of cells seeding in 1282m- or siSERF-transfected cells. FIG. 21 is a graph quantifying tube length under hypoxic conditions after 6 hours of cells seeding in 1282m-transfected cells. FIG. 2J is a graph quantifying tube length under hypoxic conditions after 6 hours of cells seeding in siSERF-transfected cells. FIG. 2K is a graph quantifying the number of branching points under hypoxic conditions after 6 hours of cells seeding in 1282m-transfected cells. FIG. 2L is a graph quantifying the number of branching points under hypoxic conditions after 6 hours of cells seeding in siSERF-transfected cells. FIG. 2M is a graph quantifying secreted vascular endothelial growth factor A (VEGF-A), an angiogenesis-related biomarker, in miR-1282 transfected DmECs under normoxia. FIG. 2N is a graph quantifying secreted stromal cell-derived factor 1 (SDF-1 ), an angiogenesis-related biomarker, in miR-1282 transfected DmECs under normoxia. FIG. 20 is a graph quantifying secreted placental growth factor 2 (PLGF-2), an angiogenesis-related biomarker, in miR-1282 transfected DmECs under normoxia. FIG. 2P is a graph quantifying secreted amphiregulin, an angiogenesis-related biomarker, in miR-1282 transfected DmECs under normoxia.

[0081] FIG. 3A-FIG. 3D contain data demonstrating that miR-1282 overexpression or SERF2 knockdown inhibits apoptosis and reduces the level of protein aggregation and aggresome-like structures. All values are mean + / - SE (n > 4). Data were calculated using Student’s t-test. “NSm” refers to a nonspecific (NS) control for miR-1282. “1282m” refers to the miR-1282 mimic. “siNS” refers to a NS control for siSERF2. “siSERF” refers to siRNA-mediated knockdown of SERF2. FIG. 3A is a graph assessing protein aggregation in 1282m-transfected DmECs. FIG. 3B is a graph assessing protein aggregation in siSERF2-transfected DmECs. FIG. 3C is a flow-cytometric quantification of aggresome-like structures in 1282m-transfected DmECs. FIG. 3B is a flow-cytometric quantification of aggresome-like structures in siSERF2-transfected DmECs.

[0082] FIG. 4A-FIG. 4J contain data demonstrating that the overexpression of miR-1282 improved blood flow recovery and decreased protein aggregation in Db / Db mice following FAL. All values shown are mean + / - SE (n > 4). Data was calculated using Student’s t-test. “NSm” refers to a non-specific (NS) control for miR-1282. “1282m” refers to the miR-1282 mimic. FIG. 4A is a schematic of FAL surgery at different days. FIG. 4B is a graph of ischemia scores at day 14 post-FAL. FIG. 4C is a representative set of blood flow recovery images in ischemic legs treated either with control (NSm) or miR-1282 mimic (1282m). See FIG. 4F for a quantification thereof. FIG. 4D is a quantification of cluster of differentiation (CD)-31+VEvessel counts using Imaged. FIG. 4E is a quantification of smooth muscle action (SMA)+VEvessel counts using Imaged. FIG. 4F is a quantification of percent blood flow recovery in ischemic legs treated either with control (NSm) or miR-1282 mimic (1282m). FIG. 4G contains representative confocal images (200X) of ischemic gastrocnemius muscle sections stained with CD31 , aSMA, and DAPI. FIG. 4H contains representative immunofluorescence images of ischemic gastrocnemius muscle sections stained with Proteostat (an aggresome detection reagent) and SERF2. FIG. 41 is a quantification of Proteostat from FIG. 4H using Image d. FIG. 4J is a quantification of SERF2 from FIG. 4H using Image d.

[0083] FIG. 5A-FIG. 5H contain data demonstrating that overexpression of miR-1282 or inhibition of SERF2 phenocopied each other by inhibiting apoptosis in diabetic ECs. All values are mean + / - SE (n > 4). Data were calculated using Student’s t-test. “NSm” refers to a non-specific (NS) control for miR-1282. “1282m” refers to the miR-1282 mimic. “siNS” refers to a NS control for siSERF2. “siSERF” refers to siRNA-mediated knockdown of SERF2. FIG. 5A is a flow cytometric quantification of apoptosis in 1282m- transfected DmECs under normoxia. FIG. 5B is a flow cytometric quantification of apoptosis in siSERF2- transfected DmECs under normoxia. FIG. 5C is a flow cytometric quantification of apoptosis in 1282m- transfected DmECs under hypoxia. FIG. 5D is a flow cytometric quantification of apoptosis in siSERF2- transfected DmECs under hypoxia. FIG. 5E is a chemiluminescence assessment of apoptosis using Caspase-Gio 3 / 7 assay in 1282m-transfeced DmECs under normoxia. FIG. 5F is a chemiluminescence assessment of apoptosis using Caspase-Gio 3 / 7 assay in SERF2-transfeced DmECs under normoxia. FIG. 5G is a chemiluminescence assessment of apoptosis using Caspase-Gio 3 / 7 assay in 1282m- transfeced DmECs under hypoxia. FIG. 5H is a chemiluminescence assessment of apoptosis using Caspase-Gio 3 / 7 assay in SERF2-transfected DmECs under hypoxia.

[0084] FIG. 6A-FIG. 6H contains data demonstrating that miR-1282 overexpression or SERF2 knockdown enhanced proliferation and reduced oxidative stress in diabetic endothelial cells. All values are mean + / - SE (n > 3). Data were calculated using Student’s t-test. “NSm” refers to a non-specific (NS) control for miR-1282. “1282m” refers to the miR-1282 mimic. “siNS” refers to a NS control for siSERF2. “siSERF” refers to siRNA-mediated knockdown of SERF2. FIG. 6A is a flow cytometric quantification of cell proliferation by 5-ethynyl-2’-deoxyuridine (EdU) assay in 1282m-transfected DmECs under normoxia. FIG. 6B is a flow cytometric quantification of cell proliferation by EdU assay in siSERF2-transfected DmECs under normoxia. FIG. 6C is a flow cytometric quantification of cell proliferation by EdU assay in 1282m-transfected DmECs under hypoxia. FIG. 6D is a flow cytometric quantification of cell proliferation by EdU assay in siSERF2-transfected DmECs under hypoxia. FIG. 6E is graph assessing reactive oxygen species (ROS) in 1282m-transfected DmECs under normoxia. FIG. 6F is graph assessing ROS in siSERF2-transfected DmECs under normoxia. FIG. 6G is graph assessing ROS in 1282m-transfected DmECs under hypoxia. FIG. 6H is graph assessing ROS in siSERF2-transfected DmECs under hypoxia.

[0085] FIG. 7A-FIG. 7H contain data demonstrating the identification of molecular targets and pathways influenced by miR-1282 and SERF2 in diabetic ECs. FIG. 7A is a Venn diagram of RNA-sequencing datasets from miR-1282 overexpression and SERF2 knockdown experiments at 48 hours posttransfection during normoxia in diabetic ECs. The numbers shown are the number of differentially expressed genes (Iog2fc > 0.58, p < 0.05, n > 3). FIG. 7B is a Venn diagram of RNA-sequencing datasets from miR-1282 overexpression and SERF2 knockdown experiments at 48 hours post-transfection during hypoxia in diabetic ECs. The numbers shown are the number of differentially expressed genes (Iog2fc > 0.58, p < 0.05, n > 3). FIG. 7C is a heatmap of the top 20 overlapping differentially expressed genes during normoxia in diabetic ECs. FIG. 7D is a heatmap of the top 20 overlapping differentially expressed genes during hypoxia in diabetic ECs. FIG. 7E is a bubble plot of the top 10 overlapping gene set enriched pathways upon miR-1282 overexpression during normoxia in diabetic ECs (normalized enrichment score > 1 , nominal p < 0.05). FIG. 7F is a bubble plot of the top 10 overlapping gene set enriched pathways upon miR-1282 overexpression during hypoxia in diabetic ECs (normalized enrichment score > 1 , nominal p < 0.05). FIG. 7G is a chord plot of selected EC-associated gene ontology processes and associated overlapping differentially expressed genes upon miR-1282 overexpression during normoxia in diabetic ECs. FIG. 7H is a chord plot of selected EC-associated gene ontology processes and associated overlapping differentially expressed genes upon miR-1282 overexpression during hypoxia in diabetic ECs. FIG. 8 is a schematic depicting the uncovered involvement of miR-1282 in both healthy and diabetic critical limb-threatening ischemia (CLTI) conditions. In contrast to healthy controls, diabetic CLTI results in the downregulation of endothelial miR-1282 and upregulation of its cis-antisense target SERF2, a pro-aggregatory protein. Reduced level of miR-1282 in diabetic CLTI thereby fosters protein aggregation, ROS generation, and apoptosis while inhibiting angiogenesis and cellular proliferation.

[0086] FIG. 9A is a flow cytometry of assessment of apoptosis in 1282m-transfected DmECs under normoxia.

[0087] FIG. 9B is a flow cytometry of assessment of apoptosis in 1282m-transfected DmECs under hypoxia.

[0088] FIG. 9C is a fluorescence micrograph (400X) of aggresomes-like structures (arrows) and nucleus in 1282m-transfected DmECs. Notably, 1282m-transfected DmECs showed reduced aggregation of aggresome-like structures.

[0089] FIG. 10A is a flow cytometry of assessment of an EdU cell proliferation assay in 1282m- transfected DmECs under normoxia. The micrographs show the percent of cell population under S-phase (e.g., AF647 / EdU positive).

[0090] FIG. 10B is a flow cytometry of assessment of an EdU cell proliferation assay in 1282m- transfected DmECs under hypoxia. The micrographs show the percent of cell population under S-phase (e.g., AF647 / EdU positive).

[0091] DEFINTIONS

[0092] 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.

[0093] 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).

[0094] 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.

[0095] As used herein, the term “administered in combination” or “combined administration” 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 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.

[0096] 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.

[0097] 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.

[0098] As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of a composition 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 small EDRK-rich factor 2 (SERF2), it is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. 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.

[0099] As used herein, a “formulation” includes at least an inhibitory nucleic acid molecule and a delivery vehicle.

[0100] 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).

[0101] 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).

[0102] 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 a product (e.g., a mRNA) 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 the target’s expression by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more). In one embodiment, the target nucleic acid molecule encodes SERF2.

[0103] As used herein the term “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.

[0104] 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.

[0105] 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.

[0106] “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:

[0107] 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.

[0108] As used herein, an inhibitory nucleic acid molecule (e.g., an siRNA, a dsRNA, a miRNA, a shRNA, an ASO, or a gapmeR) having “sufficient complementarity” to a target nucleic acid molecule (e.g., a target mRNA, e.g., SERF2) means that the inhibitory nucleic acid molecule includes a nucleotide sequence capable of hybridizing to, and triggering the destruction of, the target nucleic acid molecule (e.g., by RISC-mediated cleavage or Rnase H-mediated cleavage of the target nucleic acid molecule). The inhibitory nucleic acid molecule can be designed such that every nucleotide is complementary to a nucleotide in the target nucleic acid molecule. Alternatively, mismatched nucleotides may be introduced so long as there remains hybridization and destruction of the target nucleic acid molecule.

[0109] 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.

[0110] 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. 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.

[0111] 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.

[0112] DETAILED DESCRIPTION

[0113] Described herein are compositions (e.g., an inhibitory nucleic acid molecule) for reducing expression of a target nucleic acid molecule (e.g., an mRNA molecule encoding small EDRK-rich factor 2 (SERF2), e.g., any one of SEQ ID NOs: 6-14) and methods thereof for (i) treating cardiovascular disease (e.g., coronary artery disease (CAD), peripheral artery disease (PAD), heart failure, cardiomyopathy, or stroke) in a subject; (ii) treating or reducing the likelihood of chronic limb-threatening ischemia (CTLI) in a subject; (iii) alleviating chronic ischemic rest pain in a subject having CTLI; and / or promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease). The inhibitory nucleic acid molecule may be a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), an anti-sense oligonucleotide (ASO), a microRNA (miRNA), a short hairpin RNA (shRNA), or a gapmeR described herein, or a composition (e.g., pharmaceutical composition) thereof.

[0114] Inhibitory Nucleic Acid Molecules

[0115] Exemplary inhibitory nucleic acid molecules of the disclosure are siRNAs, dsRNAs, ASOs, miRNAs, gapmeRs, and shRNAs; however, any nucleic acid molecule capable of reducing SERF2 (e.g., any one of SEQ ID NOs: 6-14), or a variant thereof, is envisioned for use of the methods described herein. In some instances, the inhibitory nucleic acid molecules of the disclosure may be referred to as RNA inhibitory (RNAi) molecules.

[0116] For any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, gapmeR, or other inhibitory nucleic acid molecules capable of reducing expression of a target gene) the inhibitory nucleic acid molecule contains at least some sequence complementarity to the nucleotide sequence of SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a 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 a sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0117] For any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, gapmeR, or other inhibitory nucleic acid molecules capable of reducing expression of a target nucleic acid) the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 to 3429 contiguous nucleotides (e.g., 15 to 49, 19 to 28, 23 to 25, 50 to 99, 100 to 200, 150 to 300, 200 to 400, 300 to 700, 500 to 1000, 1000 to 5000, 100 to 3429, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 36 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotide) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0118] In some embodiments, the inhibitory nucleic acid is an siRNA targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. In some embodiments, the inhibitory nucleic acid is a dsRNA targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an ASO targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. In some embodiments, the inhibitory nucleic acid is a gapmeR targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. In some embodiments, the inhibitory nucleic acid is a miRNA targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an shRNA targeting SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. 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.

[0119] 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).

[0120] In some embodiments, siRNAs of the disclosure may include a nucleotide sequence of 10 to 30 nucleotides in length (e.g., 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 26 to 30, 27 to 30, 28 to 30, or 29 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).

[0121] In some embodiments, the siRNA includes a sequence complementary at least 15 to 30 contiguous nucleotides (e.g., 15 to 25, 19 to 28, 23 to 25, 25 to 28, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 36 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0122] 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.

[0123] 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), between 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), between 20 and 28 nucleotides (e.g., 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, or 28 nucleotides), between 23 and 25 nucleotides (e.g., 23, 24, or 25 nucleotides), or between 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 17 nucleotides. In some embodiments, the antisense strand is 18 nucleotides. In some embodiments, the antisense strand is 19 nucleotides. In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0124] 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), between 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), between 20 and 28 nucleotides (e.g., 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, or 28 nucleotides), between 23 and 25 nucleotides (e.g., 23, 24, or 25 nucleotides), or between 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the 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.

[0125] 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% base-paired due to mismatches. One or more mismatches may be present within the ds siRNA without impacting the siRNA’s ability to reduced expression of a target gene of interest.

[0126] The nucleotide sequence of an siRNA of the disclosure may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3) 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3), or a portion thereof. In some embodiments, the siRNA is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3), or a portion thereof.

[0127] 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the siRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. For example, the siRNA may hybridize to a target sequence of SEQ ID NO: 5 (e.g., see Table 2). The target gene of interest may be SERF2 (e.g., SEQ ID NO: 6, or a variant thereof, e.g., see Table 3).

[0128] 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.

[0129] Different siRNAs can be combined for decreasing mRNA expression of SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the siRNA sequence may contain at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA sequence may contain the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ).

[0130] In some embodiments, the siRNA contains at least 15 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 16 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 17 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 18 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 19 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 20 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains 21 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains 22 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains 23 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains 24 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ). In some embodiments, the siRNA contains 25 contiguous nucleotides set forth within SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., see Table 1 ).

[0131] In any of the foregoing embodiments, the siRNA further contains the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Table 1 below provides the antisense and sense strands of exemplary siRNA sequences of the invention.

[0132] TABLE 1. EXEMPLARY siRNA SEQUENCES

[0133] A = adenine; C = cytosine; G = guanine; U = uracil; T = thymine. In some embodiments, the siRNA of the disclosure may target a nucleotide sequence of SEQ ID

[0134] NO: 5 or SEQ ID NO: 6 (e.g., see Table 2), or a complementary sequence thereof, or variant thereof (e.g., see Table 3) with at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% thereto. TABLE 2. TARGET SEQUENCE

[0135] A = adenine; C = cytosine; G = guanine; U = uracil.

[0136] In some embodiments, the siRNA comprises a sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3). The nucleotide sequence of SEQ ID NO: 6 is set forth in Table 2 while variants (e.g., isoforms) are set forth in Table 3.

[0137] TABLE 3. SERF2 VARIANTS

[0138] Double-stranded RNA (ds RNA) 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.

[0139] 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 3429 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 150, about 155, about 160, about

[0140] 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about

[0141] 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about

[0142] 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 380, about

[0143] 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about

[0144] 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about

[0145] 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2200, about 2400, about 2543, about 2600, about 2800, about 3000, about 3250, or about 3429 nucleotides in length).

[0146] In some embodiments, dsRNAs of the disclosure may include a sense strand and an antisense strand, each containing a nucleotide sequence of 25 to 3429 nucleotides in length, or longer (e.g., 25 to 3429, 50 to 3429, 75 to 3429, 100 to 3429, 125 to 3429, 150 to 3429, 175 to 3429, 200 to 3429, 225 to 3429, 250 to 3429, 275 to 3429, 300 to 3429, 325 to 3429, 350 to 3429, 375 to 3429, 400 to 3429, 425 to 3429, 450 to 3429, 475 to 3429, 500 to 3429, 550 to 3429, 600 to 3429, 650 to 3429, 700 to 3429, 750 to 3429, 800 to 3429, 850 to 3429, 900 to 3429, 950 to 3429, 1000 to 3429, 1050 to 3429, 1100 to 3429, 1150 to 3429, 1200 to 3429, 1300 to 3429, 1400 to 3429, 1500 to 3429, 1600 to 3429, 1700 to 3429, 1800 to 3429, 1900 to 3429, 2000 to 3429, 2100 to 3429, 2200 to 3429, 2300 to 3429, 2400 to 3429, 2500 to 3429, 3000 to 3429, 3100 to 3429, 3200 to 3429, or 3300 to 3429 nucleotides in length, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,

[0147] 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,

[0148] 310, 320, 330, 340, 350, 360, 370, 380, 380, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,

[0149] 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600,

[0150] 1700, 1800, 1900, 2000, 2200, 2400, 2543, 2600, 2800, 3000, 3250, 3400, or 3429 nucleotides in length).

[0151] In some embodiments, the dsRNA includes a sequence complementary at least 25 to 3429 contiguous nucleotides (e.g., 25 to 3429, 50 to 3429, 75 to 3429, 100 to 3429, 125 to 3429, 150 to 3429, 175 to 3429, 200 to 3429, 225 to 3429, 250 to 3429, 275 to 3429, 300 to 3429, 325 to 3429, 350 to 3429, 375 to 3429, 400 to 3429, 425 to 3429, 450 to 3429, 475 to 3429, 500 to 3429, 550 to 3429, 600 to 3429, 650 to 3429, 700 to 3429, 750 to 3429, 800 to 3429, 850 to 3429, 900 to 3429, 950 to 3429, 1000 to 3429, 1050 to 3429, 1100 to 3429, 1150 to 3429, 1200 to 3429, 1300 to 3429, 1400 to 3429, 1500 to 3429, 1600 to 3429, 1700 to 3429, 1800 to 3429, 1900 to 3429, 2000 to 3429, 2100 to 3429, 2200 to 3429, 2300 to 3429, 2400 to 3429, 2500 to 3429, or 3000 to 3429 contiguous nucleotides, e.g., 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2543, 3000, or 3429 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0152] 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.

[0153] In some embodiments, the sense and antisense strands of a dsRNA molecule of the disclosure are completely complementary. In some embodiments, the sense and antisense strands of a 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% base-paired 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.

[0154] The nucleotide sequence of a dsRNA of the disclosure may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof) 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof. In some embodiments, the dsRNA is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof.

[0155] 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3). 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3). In some embodiments, the dsRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3. The target sequence may be SEQ ID NO: 5 (e.g., see Table 2).

[0156] Different dsRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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., SERF2 (e.g., SEQ ID NO: 6), or a variant 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.

[0157] In some embodiments, miRNAs of the disclosure may include a nucleotide sequence of about 6 to about 30 nucleotides in length (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length).

[0158] In some embodiments, miRNAs of the disclosure may include a nucleotide sequence of 6 to 30 nucleotides in length (e.g., 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 26 to 30, 27 to 30, 28 to 30, or 29 to 30 nucleotides in length, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0159] In some embodiments, the miRNA comprises a sequence complementary at least 6 to 30 contiguous nucleotides (e.g., 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 26 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotides, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[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. The nucleotide sequence of the miRNA may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof) 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof. In some embodiments, the miRNA is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof.

[0161] 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the miRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. The target sequence may be SEQ ID NO: 5 (e.g., see Table 2).

[0162] In some embodiments, the nucleotide sequence of the miRNA includes 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. The miRNA may further include a modification described herein (e.g., a non-natural or modified nucleoside or nucleotide, and / or a covalently or non- covalently conjugated moiety). In some embodiments, the miRNA is miR-1282 (e.g., SEQ ID NO: 15).

[0163] Different miRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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 target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3). In some embodiments, at least one of the miRNAs is miR-1282 (e.g., SEQ ID NO: 15), or a variant thereof having 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% sequence identity thereto. 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.

[0164] In some embodiments, shRNAs of the disclosure may include a nucleotide sequence of about 50 to about 100 nucleotides in length (e.g., 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 nucleotides in length).

[0165] In some embodiments, shRNAs of the disclosure may include a nucleotide sequence of 50 to 100 nucleotides in length (e.g., 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 nucleotides in length, e.g., 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 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., 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50,

[0166] 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50,

[0167] 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50,

[0168] 46 to 50, 47 to 50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,

[0169] 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length). In some embodiments, the hairpin size is between 4 to 50 nucleotides in length (e.g., 4 to 50, 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50,

[0170] 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50,

[0171] 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50,

[0172] 46 to 50, 47 to 50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,

[0173] 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), 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.

[0174] In some embodiments, the shRNA includes a sequence (e.g., a stem sequence) complementary at least 10 to 50 contiguous nucleotides (e.g., 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 contiguous nucleotides, e.g., 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, 48, 49, or 50 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0175] 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.

[0176] The nucleotide sequence of the shRNA may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof) 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof. In some embodiments, the shRNA is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof.

[0177] 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the shRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. The target sequence may be SEQ ID NO: 5 (e.g., see Table 2).

[0178] Different shRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof).

[0179] Anti-Sense Oligonucleotide (ASO)

[0180] 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.

[0181] In some embodiments, ASOs of the disclosure may include a nucleotide sequence of about 12 to about 50 nucleotides in length (e.g., 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length).

[0182] In some embodiments, ASOs of the disclosure may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to

[0183] 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to

[0184] 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 12, 13,

[0185] 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 ,

[0186] 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0187] In some embodiments, the ASO includes a sequence complementary at least 12 to 50 contiguous nucleotides (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50,

[0188] 31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50,

[0189] 42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 contiguous nucleotides, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37,

[0190] 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0191] 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.

[0192] The nucleotide sequence of the ASO may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof) 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof. In some embodiments, the ASO is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof.

[0193] 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). 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 SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the ASO of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. The target sequence may be SEQ ID NO: 5 (e.g., see Table 2).

[0194] Different ASOs can be combined for decreasing the protein expression of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3). A combination of two ASOs may be used in a method of the invention, such as two different ASOs, three different ASOs, four different ASOs, or five different ASOs targeting the same gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof, e.g., see Table 3).

[0195] GapmeR

[0196] GapmeRs of the disclosure are single (ss) nucleic acid molecules made of DNA and RNA with the central 8-10 nucleotide of the gapmeR being DNA that is complementary to a target gene of interest, which 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.

[0197] In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of about 12 to about 50 nucleotides in length (e.g., 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length).

[0198] In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to

[0199] 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to

[0200] 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 12, 13,

[0201] 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 ,

[0202] 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0203] In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of 8 to 9, 8 to 10, or 9 to 10 (e.g., 8, 9, or 10) internal DNA nucleotides.

[0204] In some embodiments, the gapmeR includes a sequence complementary at least 12 to 50 contiguous nucleotides (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50,

[0205] 31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50,

[0206] 42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 contiguous nucleotides, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides) set forth within SEQ ID NO: 6 (or a variant thereof, e.g., see Table 3).

[0207] 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.

[0208] The nucleotide sequence of the gapmeR may contain sufficient complementarity to a portion of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof) such that the gapmeR can hybridize with the target gene of interest. In some embodiments, the gapmeR 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., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof. In some embodiments, the gapmeR is complementary to the target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof), or a portion thereof.

[0209] In some embodiments, the nucleotide sequence of the gapmeR may contain sufficient complementarity to an exon sequence of a target gene of interest (e.g., an exon of SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the nucleotide sequence of the gapmeR may contain sufficient complementarity to an intron sequence of a target gene of interest (e.g., an intron of SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). In some embodiments, the gapmeR of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding SERF2 (e.g., SEQ ID NO: 6), or a variant thereof. The target sequence may be SEQ ID NO: 5 (e.g., see Table 2).

[0210] Different gapmeRs can be combined for decreasing the protein expression of a target gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof). A combination of two gapmeRs may be used in a method of the invention, such as two different gapmeRs, three different gapmeRs, four different gapmeRs, or five different gapmeRs targeting the same gene of interest (e.g., SERF2 (e.g., SEQ ID NO: 6), or a variant thereof).

[0211] Modifications to the Inhibitory Nucleic Acid Molecules

[0212] 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.

[0213] Modifications may be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

[0214] 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).

[0215] 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. Nucleoside Modifications

[0216] 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- thioalkyl, 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.

[0217] Sugar Modifications

[0218] 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-alkyl-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.

[0219] Internucleoside Linkage Modifications

[0220] 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.

[0221] Conjugates

[0222] 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).

[0223] 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.

[0224] 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., polyethylene glycol) and polypropylene glycol) and copolymers thereof, e.g., di- and triblock copolymers.

[0225] 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-butydimethylsilyl, t-butyldiphenylsilyl, 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.

[0226] 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).

[0227] In some embodiments, the targeting moiety is vascular ceil adhesion protein 1 (VCAM1). in some embodiments, the targeting moiety is arginylglycylaspartic acid (RGD).

[0228] 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). 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)).

[0229] Preparation of Inhibitory Nucleic Acid Molecules

[0230] 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.

[0231] 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. Non-limiting 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.

[0232] Compositions

[0233] The inhibitory nucleic acid molecules described herein may be formulated 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 -4, 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).

[0234] 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. 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). For example, a second therapeutic agent may be a blood pressure medication, an antiinflammatory medication (e.g., a steroid or colchicine), or immunosuppressive agent. In some embodiments, the second therapeutic agent is a statin. Non-limiting examples of second therapeutic agents are a statin (e.g., atorvastatin), a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor (e.g., an siRNA or monoclonal antibody targeting PCSK9), a Factor X inhibitor (e.g., rivaroxaban, e.g., XARELTO®), or ezetimibe (e.g., ZETIA™).

[0235] In some embodiments, the second therapeutic agent (e.g., statin) is administered in combination with an inhibitory nucleic acid molecule of the disclosure. In some embodiments, the subject is orally administered a statin. In some embodiments, the subject is administered a statin daily.

[0236] Methods of Treatment

[0237] The disclosure provides methods of (i) treating cardiovascular disease (e.g., PAD, CAD, heart failure, cardiomyopathy, or stroke) in a subject; (ii) treating or reducing the likelihood of CTLI in a subject; (iii) alleviating chronic ischemic rest pain in a subject having CTLI; and / or promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease). In some embodiments, the method includes the step of administering to a subject an inhibitory nucleic acid molecule described herein, wherein the inhibitory nucleic acid molecule targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject an siRNA described herein (e.g., any one or more of SEQ ID NOs: 1 -4, or a variant thereof), wherein the siRNA targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject a dsRNA described herein, wherein the dsRNA targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject an ASO described herein, wherein the ASO targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject a gapmeR described herein, wherein the gapmeR targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject an miRNA described herein, wherein the miRNA targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3). In some embodiments, the method includes the step of administering to a subject an shRNA described herein, wherein the shRNA targets SERF2 (e.g., SEQ ID NO: 6), or a variant thereof (e.g., see Table 3).

[0238] Any of the methods can administer a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, or gapmeR).

[0239] In some embodiments, the methods of (i) treating cardiovascular disease (e.g., PAD, CAD, heart failure, cardiomyopathy, or stroke) in a subject; (ii) treating or reducing the likelihood of CTLI in a subject; (iii) alleviating chronic ischemic rest pain in a subject having CTLI; and / or promoting angiogenesis in a subject (e.g., a subject having or at risk of developing a cardiovascular disease) further includes administering a second therapeutic agent (e.g., a nucleic acid molecule to be expressed within a cell, a polypeptide, or a drug). For example, a second therapeutic agent may be a blood pressure medication, an anti-inflammatory medication (e.g., a steroid or colchicine), or immunosuppressive agent. In some embodiments, the second therapeutic agent is a statin. Non-limiting examples of second therapeutic agents are a statin (e.g., atorvastatin), a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor (e.g., an siRNA or monoclonal antibody targeting PCSK9), a Factor X inhibitor (e.g., rivaroxaban, e.g., XARELTO®), or ezetimibe (e.g., ZETIA™). In some embodiments, the additional therapeutic agent can be administered prior to, subsequent to, or concurrently with an inhibitory nucleic acid described herein.

[0240] Delivery Vehicle

[0241] 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)).

[0242] 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 (AAV), poxviruses, herpes viral vectors, and Sindbis viral vectors. For example, the vector may be an AAV vAAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , or AAV12 vector.

[0243] 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.

[0244] 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.

[0245] 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, miRNA, shRNA, ASO, or gapmeR) may be formulated in a lipid nanoparticle such as those described in International Publication No. WO2012170930, 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-[(w-methoxy polyethylene 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-dil inoleyloxy- 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).

[0246] 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 ).

[0247] Subject

[0248] The subject to be treated may have a cardiovascular disease, including, but not limited to, CAD, PAD, heart failure, cardiomyopathy, or CTLI. The subject may also have, or be at risk of developing, a stroke. Furthermore, the subject may have previously experienced a myocardial infarction and / or has an ischemic injury. Additionally, the subject to be treated may have a metabolic disorder, or is at risk of developing a metabolic disorder, such as diabetes. Subjects at risk of developing diabetes may be prediabetic and / or have experienced one or more of the following risk factors: hyperglycemia, glucose resistance, insulin resistance, hyperlipidemia, or has a family history of diabetes.

[0249] The inhibitory nucleic acid molecules of the disclosure, and compositions thereof, may be delivered to the subject’s limb skeletal muscle (e.g., gastrocnemius muscle) or cardiac muscle. The inhibitory nucleic acid molecules of the disclosure, and compositions thereof, may be delivered to an endothelial cell in the subject (e.g., an endothelial cell in the subject’s limb skeletal muscle (e.g., gastrocnemius muscle) or cardiac muscle). Such delivery can promote angiogenesis in the subject, e.g., by stimulating endothelial cell proliferation.

[0250] Dosage

[0251] 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.

[0252] Routes of Administration

[0253] 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.

[0254] 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. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intraarticularly. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intramuscularly.

[0255] EXAMPLES

[0256] The following examples are 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 are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0257] Example 1. MicroRNA-1282 as a Cis-Antisense Regulator of Angiogenesis in Diabetic Critical limb Ischemia

[0258] This example describes the discovery of miR-1282’s role in regulating angiogenesis is subject’s suffering from peripheral artery disease (PAD). Additionally, this example describes the discovery of small EDRK-rich factor 2 (SERF2) as a therapeutic target for promoting angiogenesis, which can be used to treat or reduce the likelihood of critical limb-threatening ischemia (CTLI) (FIG. 8).

[0259] PAD is an atherosclerotic occlusive disease that leads to reduced blood flow to the lower extremities and endothelial dysfunction. Some patients develop CLTI, a severe manifestation of PAD defined as chronic ischemic rest pain with higher risk of amputation. A major risk factor for both PAD and CTLI is type 2 diabetes.

[0260] The pathophysiological contribution of microRNAs (miRNAs) in diabetic CTLI progression remains poorly elucidated. By overlapping plasma miRNA sequencing in PAD patients that develop CTLI and a CTLI mouse model, miRNAs were identified in the progression of diabetic limb ischemia (FIG. 1 A). A previously uninvestigated miRNA, miR-1282, was the highest upregulated miRNA in high-risk diabetic CTLI human subjects. Further, miR-1282 was found to be expressed in endothelial cells (ECs) (FIG. 1D). A mouse ortholog of human miR-1282, a cis-antisense miRNA, was found to strongly inhibit the expression of small EDRK-rich factor 2 (SERF2), a protein-coding gene located on the opposite DNA strand in both human and mouse endothelial cells (FIG. 1B and FIG. 1C). SERF2 is predicted to affect the formation of aggresomes, the inclusion bodies of misfolded proteins; however, its molecular function is poorly understood. MiR-1282 expression was decreased by diabetic stimuli such as hypoxia and glucose; and its expression inversely correlated with SERF2 expression in endothelial cells.

[0261] In vitro experiments on diabetic skeletal muscle endothelial cells identified that miR-1282 mimic overexpression or SERF2 siRNA-mediated inhibition suppressed protein aggregation (FIG. 3 and FIG. 9C), reduced reactive oxygen species (ROS) generation (FIG. 6E-6H and FIG. 10A-10B), and reduced apoptosis (FIG. 5 and FIG. 9A-9B) while fostering angiogenesis and cellular proliferation (FIG. 2) under normal and hypoxic conditions (FIG. 6A-6D). Transcriptomic studies by RNA-sequencing identified that miR-1282 regulated protein quality control (e.g., via SERF2) and regulated cell death (e.g., via lysosomal- associated membrane protein 1 (LAMP1 ) and caspase 3 (CASP3)), among others. In vivo experiments showed that intramuscular administration of miR-1282 significantly improved blood flow recovery, as assessed by laser doppler imaging, and decreased protein aggregation in the hindlimbs of diabetic db / db mice (FIG. 4), as compared to control injected mice.

[0262] Taken together, the cis-antisense regulatory network of miR-1282 revealed therapeutic interventions (e.g., SERF2) for regulating angiogenesis in CTLI.

[0263] Example 2. MicroRNA-1282 Rescues Diabetic Limb Ischemia Via a SERF2-Protein Aggregation Pathway

[0264] This example describes the discovery of miR-1282’s role in promoting endothelial angiogenesis and proteostasis during diabetic chronic limb-threatening ischemia (CLTI). Additionally, this example describes the discovery of small EDRK-rich factor 2 (SERF2) as a therapeutic target for promoting angiogenesis, which can be used to treat or reduce the likelihood of CTLI (FIG. 8).

[0265] Patients with diabetes are at higher risk of CLTI, a severe form of peripheral artery disease (PAD) causing restricted blood flow to the lower limbs due, in part, to impaired angiogenesis. As described in Example 1 , by integrating plasma miRNA sequencing data from PAD patients having diabetes and a diabetic CLTI mouse model (FIG. 1A), a conserved miRNA, miRNA miR-1282, was identified. Notably, miR-1282 is in cis-antisense orientation to small EDRK-rich factor 2 (SERF2), a gene associated with amyloid aggregation (FIG. 1B and FIG. 1C).

[0266] Using miR-1282 overexpression or SERF2 knockdown experiments, the role of miR-1282 and SERF2 in angiogenesis, apoptosis, protein aggregation, and oxidative stress was uncovered in diabetic mouse skeletal muscle endothelial cells (ECs). In vivo, miR-1282 mimics were delivered intramuscularly to assess their impact on blood flow recovery, angiogenesis, and protein aggregation (FIG. 3 and FIG. 4). Mechanistic insights in ECs were gained via RNA-seq, predictive algorithms, and proteomic analyses. miR-1282 inhibited the expression of its cis-antisense target SERF2 by 98% (FIG. 1H and FIG. 11). miR-1282 is a hypoxia-induced endothelial-enriched miRNA (FIG. 1F), and its expression was inversely correlated with SERF2 expression (FIG. 1G). miR-1282 levels were markedly reduced after femoral artery ligation (FAL) in diabetic db / db mice (FIG. 1E). Overexpression of miR-1282 or SERF2 knockdown enhanced angiogenesis (FIG. 2) and reduced protein aggregation (FIG. 3), apoptosis (FIG. 5 and FIG. 9A-9B), and oxidative stress (FIG. 6E-6H) in both normal and hypoxic conditions in vitro (FIG. 6A-6D). Delivery of miR-1282 mimics in db / db mice improved blood flow recovery by 108% and angiogenesis by 98%, and reduced protein aggregation by 48% and tissue necrosis (FIG. 8A-8C and FIG. 9C). Coupling RNA-seq profiling and prediction algorithms of ECs upon miR-1282 overexpression or SERF2 knockdown revealed epiregulin (EREG), BAG cochaperone 5 (BAG5), caspase 3 (CASP3), arginase 1 (ARG1 ), and heat shock protein 90 alpha family class A member 1 (HSP90AA1 ) as potential downstream regulators (FIG. 7A-7F). Pathway enrichment analysis implicated inhibition of endothelial apoptosis, ER stress, and protein stability among the most dysregulated processes (FIG. 7C-7D and FIG. 7G-7H).

[0267] Taken together, this example describes the discovery of miR-1282 as a promoter of EC function (e.g., angiogenesis) that is also capable of diminishing protein aggregation in diabetic CLTI mice via suppression of SERF2. These findings uncover SERF2 as a therapeutic target in treating diabetic CLTI.

[0268] Numbered Embodiments

[0269] 1 . An inhibitory nucleic acid molecule comprising sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and (ii) the target nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

[0270] 2. The inhibitory nucleic acid molecule of embodiment 1 , wherein the target nucleic acid molecule comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

[0271] 3. The inhibitory nucleic acid molecule of embodiment 1 or 2, wherein the target nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

[0272] 4. The inhibitory nucleic acid molecule of any one of embodiments 1 -3, wherein the inhibitory nucleic acid molecule is 15 to 2543 nucleotides in length.

[0273] 5. The inhibitory nucleic acid molecule of embodiment 4, wherein the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length, 50 to 99 nucleotides in length, or 100 to 2543 nucleotides in length.

[0274] 6. The inhibitory nucleic acid molecule of embodiment 5, wherein the inhibitory nucleic acid molecule is 19 to 28 nucleotides in length.

[0275] 7. The inhibitory nucleic acid molecule of embodiment 6, wherein the inhibitory nucleic acid molecule is 19, 20, or 21 nucleotides in length.

[0276] 8. The inhibitory nucleic acid molecule of any one of embodiments 1 -7, wherein the inhibitory nucleic acid molecule comprises at least 85% complementarity to the target nucleic acid molecule.

[0277] 9. The inhibitory nucleic acid molecule of embodiment 8, wherein the inhibitory nucleic acid molecule comprises at least 90% complementarity to the target nucleic acid molecule.

[0278] 10. The inhibitory nucleic acid molecule of embodiment 9, wherein the inhibitory nucleic acid molecule comprises at least 95% complementarity to the target nucleic acid molecule.

[0279] 11 . The inhibitory nucleic acid molecule of embodiment 10, wherein the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule. 12. The inhibitory nucleic acid molecule of any one of embodiments 1 -1 1 , further comprising a modification.

[0280] 13. The inhibitory nucleic acid molecule of embodiment 12, wherein the modification comprises:

[0281] (a) a non-natural or modified nucleoside or nucleotide; and / or

[0282] (b) a covalently or non-covalently conjugated moiety.

[0283] 14. The inhibitory nucleic acid molecule of embodiment 13, wherein:

[0284] (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LN A), a 2'-O-methyl (2’-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or

[0285] (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

[0286] 15. The inhibitory nucleic acid molecule of any one of embodiments 1 -14, wherein the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a doublestranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

[0287] 16. The inhibitory nucleic acid molecule of embodiment 15, wherein the inhibitory nucleic acid molecule is an siRNA.

[0288] 17. The inhibitory nucleic acid molecule of embodiment 16, wherein the siRNA comprises an antisense strand comprising at least 88% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0289] 18. The inhibitory nucleic acid molecule of embodiment 17, wherein the antisense strand comprises at least 92% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0290] 19. The inhibitory nucleic acid molecule of embodiment 18, wherein the antisense strand comprises at least 96% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0291] 20. The inhibitory nucleic acid molecule of embodiment 19, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0292] 21 . The inhibitory nucleic acid molecule of any one of embodiments 17-20, wherein the siRNA further comprises a sense strand comprising at least 88%, at least 92%, or at least 96% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0293] 22. The inhibitory nucleic acid molecule of embodiment 21 , wherein the siRNA comprises

[0294] (a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 3; or

[0295] (b) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 4.

[0296] 23. The inhibitory nucleic acid molecule of any one of embodiments 15-22, wherein the siRNA contains 3’ overhangs selected from the group consisting of:

[0297] (i) a single uracil overhang at one or more 3’ ends of the siRNA;

[0298] (ii) a double uracil overhang at one or more 3’ ends of the siRNA;

[0299] (iii) a single thymine overhang at one or more 3’ ends of the siRNA;

[0300] (iv) a double thymine overhang at one or more 3’ ends of the siRNA; or

[0301] (v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA.

[0302] 24. The inhibitory nucleic acid molecule of any one of embodiments 15-23, wherein the siRNA targets the nucleotide sequence of SEQ ID NO: 5. 25. The inhibitory nucleic acid molecule of embodiment 15, wherein the inhibitory nucleic acid molecule is a miRNA.

[0303] 26. The inhibitory nucleic acid molecule of embodiment 25, wherein the miRNA comprises a modification selected from:

[0304] (a) a non-natural or modified nucleoside or nucleotide; and / or

[0305] (b) a covalently or non-covalently conjugated moiety.

[0306] 27. The inhibitory nucleic acid molecule of embodiment 26, wherein:

[0307] (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LN A), a 2'-O-methyl (2'-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or

[0308] (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

[0309] 28. The inhibitory nucleic acid molecule of embodiment 26 or 27, wherein the miRNA comprises a nucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 15.

[0310] 29. The inhibitory nucleic acid molecule of embodiment 28, wherein the miRNA comprises a nucleotide sequence comprising at least 90% sequence identity to SEQ ID NO: 15.

[0311] 30. The inhibitory nucleic acid molecule of embodiment 29, wherein the miRNA comprises a nucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 15.

[0312] 31 . The inhibitory nucleic acid molecule of embodiment 30, wherein the miRNA comprises the nucleotide sequence of SEQ ID NO: 15.

[0313] 32. The inhibitory nucleic acid molecule of embodiment 31 , wherein the miRNA is miR-1282.

[0314] 33. The inhibitory nucleic acid molecule of any one of embodiments 1 -32, wherein the inhibitory nucleic acid molecule is formulated in a delivery vehicle.

[0315] 34. The inhibitory nucleic acid molecule of embodiment 33, wherein the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

[0316] 35. The inhibitory nucleic acid molecule of embodiment 34, wherein the vector is a viral vector.

[0317] 36. The inhibitory nucleic acid molecule of any one of embodiment 1 -35, wherein the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition.

[0318] 37. The inhibitory nucleic acid molecule of embodiment 36, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent, and / or carrier.

[0319] 38. A method of treating peripheral artery disease (PAD) in a subject, the method comprising administering the inhibitory nucleic acid molecule of any one of embodiments 1 -37.

[0320] 39. A method of treating or reducing the likelihood of critical limb-threatening ischemia (CTLI) in a subject, the method comprising administering the inhibitory nucleic acid molecule of any one of embodiments 1 -37.

[0321] 40. A method of alleviating chronic ischemic rest pain in a subject having CTLI, the method comprising administering the inhibitory nucleic acid molecule of any one of embodiments 1 -37.

[0322] 41 . A method of promoting angiogenesis in a subject, the method comprising administering the inhibitory nucleic acid molecule of any one of embodiments 1 -37. 42. The method of any one of embodiments 39-41 , wherein the subject has a cardiovascular disease.

[0323] 43. The method of embodiment 44, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, heart failure, cardiomyopathy, or stroke.

[0324] 44. The method of any one of embodiments 38-43, wherein the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

[0325] 45. The method of embodiment 44, wherein the metabolic disorder is diabetes.

[0326] 46. The method of embodiment 45, wherein the subject at risk of developing diabetes is prediabetic and / or has one or more of the following:

[0327] (a) hyperglycemia;

[0328] (b) glucose resistance;

[0329] (c) insulin resistance;

[0330] (d) hyperlipidemia; and

[0331] (e) has a family history of diabetes.

[0332] 47. The method of any one of embodiments 38-46, wherein the inhibitory nucleic acid molecule is administered to the subject intravenously, intraperitoneally, subcutaneously, intraarticularly, or intramuscularly.

[0333] 48. The method of any one of embodiments 38-47, wherein the inhibitory nucleic acid molecule is delivered to the subject’s limb skeletal muscle and / or cardiac muscle.

[0334] 49. The method of any one of embodiments 38-49, wherein the inhibitory nucleic acid molecule is delivered to an endothelial cell.

[0335] 50. The method of any one of embodiments 38-49, further comprising administering an additional therapeutic agent.

[0336] 51 . Any and ail compositions, articles of manufacture, methods, and uses disclosed and / or described in the specification.

[0337] 52. A method of treating tissue repair after ischemic injury, including but not limited to, peripheral artery disease, critical limb-threatening ischemia, ischemia or injury in the limb by administering to the subject a local or systemic injection of a therapeutically effective amount of a nucleic acid comprising microRNA mimics for miR-1282.

[0338] Other Embodiments

[0339] 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.

[0340] 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.

[0341] Other embodiments are within the claims.

Claims

CLAIMS1 . An inhibitory nucleic acid molecule comprising sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and (ii) the target nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

2. The inhibitory nucleic acid molecule of claim 1 , wherein the target nucleic acid molecule comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

3. The inhibitory nucleic acid molecule of claim 1 , wherein the target nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

4. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is 15 to 2543 nucleotides in length.

5. The inhibitory nucleic acid molecule of claim 4, wherein the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length, 50 to 99 nucleotides in length, or 100 to 2543 nucleotides in length.

6. The inhibitory nucleic acid molecule of claim 5, wherein the inhibitory nucleic acid molecule is 19 to 28 nucleotides in length.

7. The inhibitory nucleic acid molecule of claim 6, wherein the inhibitory nucleic acid molecule is 19, 20, or 21 nucleotides in length.

8. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule comprises at least 84% complementarity to the target nucleic acid molecule.

9. The inhibitory nucleic acid molecule of claim 8, wherein the inhibitory nucleic acid molecule comprises at least 89% complementarity to the target nucleic acid molecule.

10. The inhibitory nucleic acid molecule of claim 9, wherein the inhibitory nucleic acid molecule comprises at least 94% complementarity to the target nucleic acid molecule.11 . The inhibitory nucleic acid molecule of claim 10, wherein the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule.

12. The inhibitory nucleic acid molecule of claim 1 , further comprising a modification.

13. The inhibitory nucleic acid molecule of claim 12, wherein the modification comprises:(a) a non-natural or modified nucleoside or nucleotide; and / or(b) a covalently or non-covalently conjugated moiety.

14. The inhibitory nucleic acid molecule of claim 13, wherein:(a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LN A), a 2’-O-methyl (2’-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or(b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

15. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

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

17. The inhibitory nucleic acid molecule of claim 16, wherein the siRNA comprises an antisense strand comprising at least 84% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

18. The inhibitory nucleic acid molecule of claim 17, wherein the antisense strand comprises at least 89% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

19. The inhibitory nucleic acid molecule of claim 18, wherein the antisense strand comprises at least 94% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

20. The inhibitory nucleic acid molecule of claim 19, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.21 . The inhibitory nucleic acid molecule of claim 17, wherein the siRNA further comprises a sense strand comprising at least 84%, at least 89%, or at least 94% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

22. The inhibitory nucleic acid molecule of claim 21 , wherein the siRNA comprises(a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 3; or(f) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 4.

23. The inhibitory nucleic acid molecule of claim 15, wherein the siRNA contains 3’ overhangs selected from the group consisting of:(i) a single uracil overhang at one or more 3’ ends of the siRNA;(ii) a double uracil overhang at one or more 3’ ends of the siRNA;(iii) a single thymine overhang at one or more 3’ ends of the siRNA;(iv) a double thymine overhang at one or more 3’ ends of the siRNA; or(v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA.

24. The inhibitory nucleic acid molecule of claim 15, wherein the siRNA targets the nucleotide sequence of any one of SEQ ID NOs: 1 -4.

25. The inhibitory nucleic acid molecule of claim 15, wherein the inhibitory nucleic acid molecule is a miRNA.

26. The inhibitory nucleic acid molecule of claim 25, wherein the miRNA comprises a modification selected from:(a) a non-natural or modified nucleoside or nucleotide; and / or(b) a covalently or non-covalently conjugated moiety.

27. The inhibitory nucleic acid molecule of claim 26, wherein:(a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LNA), a 2'-O-methyl (2'-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and / or(b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

28. The inhibitory nucleic acid molecule of claim 26, wherein the miRNA comprises a nucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 15.

29. The inhibitory nucleic acid molecule of claim 28, wherein the miRNA comprises a nucleotide sequence comprising at least 90% sequence identity to SEQ ID NO: 15.

30. The inhibitory nucleic acid molecule of claim 29, wherein the miRNA comprises a nucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 15.31 . The inhibitory nucleic acid molecule of claim 30, wherein the miRNA comprises the nucleotide sequence of SEQ ID NO: 15.

32. The inhibitory nucleic acid molecule of claim 31 , wherein the miRNA is miR-1282.

33. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is formulated in a delivery vehicle.

34. The inhibitory nucleic acid molecule of claim 33, wherein the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

35. The inhibitory nucleic acid molecule of claim 34, wherein the vector is a viral vector.

36. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition.

37. The inhibitory nucleic acid molecule of claim 36, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent, and / or carrier.

38. A method of treating peripheral artery disease (PAD) in a subject, the method comprising administering the inhibitory nucleic acid molecule of claim 1 .

39. A method of treating or reducing the likelihood of critical limb-threatening ischemia (CTLI) in a subject, the method comprising administering the inhibitory nucleic acid molecule of claim 1 .

40. A method of alleviating chronic ischemic rest pain in a subject having CTLI, the method comprising administering the inhibitory nucleic acid molecule of claim 1 .41 . A method of promoting angiogenesis in a subject, the method comprising administering the inhibitory nucleic acid molecule of claim 1 .

42. The method of any one of claims 39-41 , wherein the subject has a cardiovascular disease.

43. The method of claim 42, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, heart failure, cardiomyopathy, or stroke.

44. The method of any one of claims 38-41 , wherein the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

45. The method of claim 44, wherein the metabolic disorder is diabetes.

46. The method of claim 45, wherein the subject at risk of developing diabetes is prediabetic and / or has one or more of the following:(a) hyperglycemia;(b) glucose resistance;(c) insulin resistance;(d) hyperlipidemia; and(e) has a family history of diabetes.

47. The method of any one of claims 38-41 , wherein the inhibitory nucleic acid molecule is administered to the subject intravenously, intraperitoneally, subcutaneously, intraarticularly, or intramuscularly.

48. The method of any one of claims 38-41 wherein the inhibitory nucleic acid molecule is delivered to the subject’s limb skeletal muscle and / or cardiac muscle.

49. The method of any one of claims 38-41 , wherein the inhibitory nucleic acid molecule is delivered to an endothelial cell.

50. The method of any one of claims 38-41 further comprising administering an additional therapeutic agent.