Treatment of aortic aneurysm

Abstract

The present disclosure provides methods for treating abdominal aortic aneurysm. More specifically, the present disclosure provides methods for treating abdominal aortic aneurysms with one or more inhibitors of APOC3, ANGPTL3, ANGPTL8, or a combination thereof, APOA5, an active fragment or variant thereof, or a nucleic acid encoding thereof, an agent for the upregulation of endogenous APOA5, or a combination thereof.

Description

Atty. Docket No. UM-43304.601TREATMENT OF AORTIC ANEURYSMFIELD

[0001] The present disclosure provides methods for treating abdominal aortic aneurysm. Particularly, the present disclosure provides methods for treating abdominal aortic aneurysms with one or more inhibitors of APOC3, ANGPTL3, ANGPTL8, or a combination thereof, APOA5, an active fragment or variant thereof, or a nucleic acid encoding thereof, an agent for the upregulation of endogenous APOA5, or a combination thereof.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 682,075, filed August 12, 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled UM_43304_601_SequenceListing.xml (Size: 54,623 bytes; and Date of Creation: August 8, 2025) is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under HL109946 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Arterial aneurysms are permanent localized dilatations of the vessel, typically enlarging by at least 150% compared to a relatively normal diameter of the adjacent artery. An abdominal aortic aneurysm is characterized by abnormal focal dilation of the abdominal aorta and can affect 0.5% to 3% of the population. Most abdominal aortic aneurysms are incidentally identified during examinations for other unrelated pathologies or at time of rupture, and most individuals are asymptomatic. The rupture of an abdominal aortic aneurysm is life-threatening. More than 50% of patients die before they can reach medical assistance. Surgical repair (e.g., transabdominal, retroperitoneal, or endovascular approaches) of unruptured abdominal aortic aneurysms is recommended when the aneurysm diameter reaches a certain size, shows a rapid enlargement, or if it becomes symptomatic. Thus, there is a need for prophylactic and therapeutic interventions which repair or prevent enlargement of unruptured abdominal aortic aneurysms or prevent rupture.Atty. Docket No. UM-43304.601SUMMARY

[0006] Disclosed herein are methods for treating or preventing abdominal aortic aneurysm (AAA) in a subject in need thereof. In some embodiments, the subject does not have and / or is not receiving treatment for a disease or disorder caused or mediated by high levels of triglycerides. In some embodiments, the subject is having or had an AAA repair. In some embodiments, the subject is having or had endovascular repair of a AAA.

[0007] In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of one or more inhibitors of APOC3, ANGPTL3, ANGPTL8, or a combination thereof. In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of APOA5, an active fragment or variant thereof, or a nucleic acid encoding thereof, or an agent for the upregulation of endogenous APOA5.

[0008] In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of APOA5, an active fragment or variant thereof, or a nucleic acid encoding thereof, or an agent for the upregulation of endogenous APOA5. In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more inhibitors of APOC3, ANGPTL3, ANGPTL8, or a combination thereof.

[0009] In some embodiments, the one or more inhibitors of APOC3, ANGPTL3, ANGPTL8comprise gene silencing or downregulating oligonucleotides, a protein configured to bind APOC3, ANGPTL3, ANGPTL8, a small molecule inhibitor, or combinations thereof. In some embodiments, the one or more inhibitors of APOC3, ANGPTL3, ANGPTL8 comprise antibodies to one or more of APOC3, ANGPTL3, and ANGPTL8. In some embodiments, the one or more inhibitors of APOC3, ANGPTL3, and / or ANGPTL8 comprise antisense oligonucleotides (ASOs), interfering RNAs (siRNAs), guide RNAs, or combinations thereof. In some embodiments, the one or more inhibitors of APOC3, ANGPTL3, and / or ANGPTL8 further comprises one or more components of a gene editing system.

[0010] In some embodiments, the one or more inhibitors of APOC3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 1-10 and / or an antisense strand sequence comprising any of SEQ ID NOs: 11-20; the one or more inhibitors of ANGPTL3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 21-29 and / or an antisense strand sequence comprising any of SEQ ID NOs: 30-38; and / or the one or more inhibitors of ANGPTL8 comprise antisense oligonucleotidesAtty. Docket No. UM-43304.601(ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 39-48 and / or an antisense strand sequence comprising any of SEQ ID NOs: 49-58.

[0011] In some embodiments, the methods further comprise identifying an abdominal aortic aneurysm in the subject. In some embodiments, the identifying comprises an ultrasound, X-ray, computed tomography (CT / CAT / CTA) scan, and / or magnetic resonance imaging (MRI) of the abdomen.

[0012] In some embodiments, the methods further comprise monitoring the abdominal aortic aneurysm over time. In some embodiments, the monitoring comprises an ultrasound, X-ray, computed tomography (CT / CAT / CTA) scan, and / or magnetic resonance imaging (MRI) of the abdomen. In some embodiments, the method further comprises characterizing the abdominal aortic aneurysm by size and the monitoring comprises determining size change over time.

[0013] In some embodiments, the methods further comprise repair of the abdominal aortic aneurysm.

[0014] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1F show moderately increased TG levels accelerated AAA development. 12 weeks old male Apoa5 KO and littermate control mice placed on standard chow diet were implanted with mini pumps with Ang II (1,000 ng / kg / min) for 4 weeks to induce AAA. FIG. 1A and FIG. IB, blood TG and TC levels. Student’s / -test. FIG. 1C, Representative morphology of aortas from WT and Apoa5 KO mice. FIG. ID, AAA incidence rate (the diameter of suprarenal AA in surviving mice were > 1.2 mm, a 50% increase relative to that of normal suprarenal AA.). Chi-square test. FIG. IE, The maximal abdominal aortic diameters. Mann- Whitney test. FIG. IF, The systolic blood pressure.

[0016] FIGS. 2A-2F show severe increased TG levels aggravate AAA dissection. Male hAPOC3 Tg (heterozygous) and littermate control mice on standard chow diet were implanted mini pump with Ang II (1,000 ng / kg / min). AAA was evaluated 4 weeks after induction. FIG. 2A and FIG. 2B, blood TG and TC levels. Student’s / -test. FIG. 2C, Representative morphology of aortas from WT and hAPOC3 Tg mice. FIG. 2D, The maximal abdominal aortic diameters. Mann- Whitney test. FIG. 2E, The dissection rate. Chi-square test. FIG. 2F, The systolic blood pressure.

[0017] FIGS. 3A-3D show severe increased TG levels aggravate AAA development in female hAPOC3 Tg mice. On standard chow diet, female hAPOC3 Tg (heterozygous) and littermate control mice were implanted mini pump with Ang II (1,000 ng / kg / min). AAA was evaluated 4 weeks afterAtty. Docket No. UM-43304.601 induction. FIG. 3A and FIG. 3B, blood TG and TC levels. Student’s / -test. FIG. 3C, The maximal abdominal aortic diameters. Mann-Whitney test. FIG. 3D, Representative morphology of aortas from WT and hAPOC3 Tg mice.

[0018] FIGS. 4A-4D show increased TG levels promote aneurysm growth in PPE induced AAA model. The “PPE AAA” model involves peri- adventitial elastase application to the infrarenal aorta (PPE, elastase from porcine pancreas, ~44 units / ml, 30pl, 30mins; heat-inactivated elastase as control). On standard chow diet, hAPOC3 Tg (heterozygous) and littermate control mice were treated with PPE to induce AAA development. AAA was evaluated 2 weeks after induction. FIG. 4A and FIG. 4B, blood TG and TC levels. Student’s / -test. FIG. 4C, Representative morphology of aortas from WT and hAPOC3 Tg mice. FIG. 4D, The maximal abdominal aortic diameters. Mann- Whitney test.

[0019] FIGS. 5A-5D show palmitic acid affects extracellular matrix in HASMCs. FIG. 5A, Reactome pathway enrichment analysis of down-regulated genes in the palmitic acid incubated group. FIG. 5B, Top 5 downregulated Reactome pathways identified from Gene Set Enrichment Analysis (GSEA). FIG. 5C, Reactome pathway enrichment analysis of up-regulated genes in the palmitic acid incubated group. FIG. 5D, Top 5 upregulated Reactome pathways identified from Gene Set Enrichment Analysis (GSEA).

[0020] FIGS. 6A-6C show palmitic acid inhibits LOX maturation in HASMC. FIG. 6A, HASMC were starved for 24 h and then treated with PA at 250 pM for 24 h in the presence or absence of TGF0. Expression of LOX was determined by western blot. FIG. 6B, HASMC were infected with AdLacZ or AdLOX for 24 h and then treated with PA at 250 pM for 24 h. Expression of LOX was determined by western blot. FIG. 6C, Quantification analysis of mature LOX protein abundance (data from 5 independent experiments).

[0021] FIG. 7 shows palmitic acid inhibits LOX maturation in the abdominal aorta. Male C57BL / 6 mice were administrated with ethyl palmitate at 600 mg / kg for 5 days. The abdominal aortas were collected and the expression of LOX was determined by western blot.

[0022] FIGS. 8A-8F show administration of Angptl3 ASO decreased TG levels, AAA incidence rate and maximal diameter in hAPOC3 transgenic mice. Male hAPOC3 transgenic mice were randomly divided into control ASO or Angptl3 ASO groups. Three days before AAA induction, ASO was S.C. injected at 10 mg / kg and repeated at 3 mg / kg weekly. Those mice were implanted mini pump with Ang II (1,000 ng / kg / min). AAA was evaluated 4 weeks after induction. FIG. 8A, Before ASO injection, on days indicated after ASO injection, TG levels were determined. One-way ANOVA followed by Sidak post hoc analysis. FIG. 8B and 8C, AAA incidence and maximalAtty. Docket No. UM-43304.601 diameters, respectively. Mann-Whitney U test. FIG. 8D, total cholesterol (TC) concentrations on days 0, 3, and 28. One-way ANOVA followed by Sidak post hoc analysis. FIG. 8E and 8F, dissection rate and elastic fiber degradation score , respectively. Mann- Whitney U test. #, P < 0.05 of the comparison of Tg and WT group. *, P < 0.05 of the comparison of Ctr and ASO group.

[0023] FIG. 9 shows administration of Angptl3 ASO decreased the endogenous expression of Angptl3 in hAPOC3 transgenic mice. Male hAPOC3 transgenic mice were randomly divided into control ASO or Angpt!3 ASO groups. Three days before AAA induction, ASO was S.C. injected at 10 mg / kg and repeated at 3 mg / kg weekly. Those mice were implanted mini pump with Ang II (1,000 ng / kg / min). AAA was evaluated 4 weeks after induction. The mRNA level of Angptl3 in the liver were determined by qRT-PCR and the circulating ANGPTL3 levels were measured by ELISA kit.

[0024] FIG. 10 shows administration oiAngpt!3 ASO does not affect the expression of APOC3 in hAPOC3 transgenic mice.

[0025] FIG. 11 shows representative aortic trees (top) and representative H&E staining (bottom left) and Verhoeff-Van Gieson (VVG) staining (bottom right) of suprarenal abdominal aorta sections for hAPOC3 transgenic mice untreated or administered control ASO or Angptl3 ASO.

[0026] FIG. 12 shows administration of Angptl3 ASO reduced NEFA (Non-Esterified Fatty Acids) in APOC3 transgenic mice (left) and Apoe-deficient mice (right). Animals were treated as mentioned in FIG. 8.

[0027] FIG. 13 shows administration of Cas9-sgRNA Apoc3 decreases TG levels and the expression of Apoc3 in the liver in C57BL / 6 mice.

[0028] FIGS. 14A-14F show administration of Angptl3 ASO reduces plasma triglyceride (TG) (FIG. 14A) and total cholesterol (TC) (FIG. 14B) concentrations in ApoE-deficient mice. Systolic blood pressure (FIG. 14C) before and after Angll infusion and body weight (FIG. 14F) changes are also shown. The TG (FIG. 14D) and TC (FIG. 14E) concentrations of size exclusion chromatography resolved fractions from animals were determined by enzymatic assays. Fractions 8 to 11 contained VLDL, fractions 12 to 17 contained IDL and LDL, and fractions 22 to 25 contained HDL.

[0029] FIG. 15 shows administration of Angpll3 ASO inhibits AAA progression in ApoEdeficient mice.

[0030] FIGS. 16A-16K show impaired LPL activity accelerates abdominal aortic aneurysm development and aortic rupture.. FIG. 16A is a schematic of the design of the AAA study in Apoa5- deficient mice. Mice were fed a standard rodent laboratory diet. After 4 weeks of Angll infusion, aortas from surviving mice were harvested. FIG. 16B shows representative aortas of male wild-typeAtty. Docket No. UM-43304.601(n=17) and A / ?oa5-deficient (n= 18) mice after 4 weeks of Angll infusion. Plasma samples were collected on day 28 and subjected individually to analytical chemistry to measure triglycerides and total cholesterol. FIGS. 16C-16F, The triglycerides (FIG. 16C), total cholesterol (FIG. 16D), maximal aortic diameter (FIG. 16E), and AAA incidence (FIG. 16F) in male mice. FIGS. 16G-16K, triglycerides (FIG. 16G), total cholesterol (FIG. 16H), maximal aortic diameter (FIG. 161), AAA incidence (FIG. 16J), and systolic blood pressure (FIG. 16K) in female WT (n=10) and Apoa5- deficient (n=8) mice in the Angll-induced AAA study. Data are presented as circles and mean+SEM (FIGS. 16C-16E, 16G-16I, and 16K). Statistical analyses were conducted as follows: Fisher exact test for FIGS. 16F and 16J; Student t test for FIGS. 16C-16E and 16G-16H; Mann-Whitney U test for FIG. 161; and 2-way mixed-effects ANOVA followed by Sidak post hoc analysis for FIG. 16K. Scale bars=l mm (each interval) in FIG. 16B.

[0031] FIGS. 17A-17M show increased triglyceride concentrations accelerate abdominal aortic aneurysm development and dissection in human APOC3 transgenic mice. FIG. 17A is a schematic design of the abdominal aortic aneurysm study in human APOC3 transgenic mice. Twelve- to 16- week-old human APOC3 transgenic mice (n of males=15; n of females=24) or wild-type (WT) littermates (n of males=17, n of females=l) were infused with angiotensin II (1000 ng / kg / min) for 4 weeks to induce an abdominal aortic aneurysm. Systolic blood pressure was measured before and after angiotensin II infusion. The mice were fed a standard rodent laboratory diet. After 4 weeks, aortas from surviving mice were harvested. Plasma samples were collected on day 28 and subjected individually to analytical chemistry. FIGS. 17B-17D, plasma triglycerides (FIG. 17B), total cholesterol (FIG. 17C), and nonesterified fatty acids (FIG. 17D) in male mice. FIG. 17E shows representative aortas of male wild-type and human APOC3 transgenic mice after 4 weeks of angiotensin II infusion. Maximal aortic diameter (FIG. 17F) and abdominal aortic aneurysm incidence (FIG. 17G) in male mice. FIGS. 17H-17M show triglycerides (FIG. 17H), total cholesterol (FIG. 171), nonesterified fatty acids (FIG. 17J), maximal aortic diameter (FIG. 17K), abdominal aortic aneurysm incidence (FIG. 17L), and dissection rate (FIG. 17M) in female WT and human APOC3 transgenic mice after 4 weeks of angiotensin II infusion. Data are presented as circles and mean±SEM (FIGS. 17B-17D, 17F, and 17H-17K). Statistical analyses were conducted as follows: Mann-Whitney U test for FIGS. 17B, 17D, 17F, 17H, and 171; Student t test for FIGS. 17C, 17J, and 17K; and Fisher exact test for FIGS. 17L-17M;. Scale bars=l mm (each interval) in FIG. 17E. Angll indicates angiotensin II; hAPOC3 Tg, human APOC3 transgenic; and NEFA, nonesterified fatty acid.

[0032] FIGS. 18A-18N show palmitic acid inhibits lysyl oxidase maturation in human aortic smooth muscle cells and aortas. FIG. 18A shows two-sample mendelian randomization to detect theAtty. Docket No. UM-43304.601 causal effects of triglycerides on circulating total fatty acid, palmitic acid (PA; 16:0), stearic acid (18:0), palmitoleic acid (16: ln-7), and oleic acid (18: ln-9) levels. FIG. 18B shows untargeted metabolomics was applied to measure the ethyl palmitate levels in plasma from 14 to 18-week-old male human APOC3 transgenic (heterozygous) mice (n=7) and littermate controls (n=7). C through H, Human aortic smooth muscle cells were starved in OptiMEM -reduced serum medium for 24 hours, followed by incubation with PA (250 pM) or vehicle (BSA) for another 24 hours. Total RNA was extracted for RNA sequencing (n=3 / group) or qRT-PCR. FIG. 18C is a Heatmap of gene expression levels involved in elastic fiber formation and maturation. Color represents the Z score of the expression of each gene, with red indicating higher and blue indicating lower expression relative to the mean of the gene across samples. FIGS. 18D-18G, qRT-PCR analysis of LOX (lysyl oxidase) (FIG. 18D) and BMP1 (FIG. 18F) (n=8 / group, data from 4 independent experiments). FIG. 18E, LOX activity in the conditioned medium (n=6 / group, data from 2 independent experiments). FIG. / 18G, Correlation between liver APOC3 expression level and aortic BMP1 expression level among 131 donors in the GTEx project. Correlation coefficient was calculated using Pearson’s method. The fitted lines represent the linear regression, with shaded bands indicating the 95% CI. In FIGS. 18H- 181 human aortic smooth muscle cells were starved in OptiMEM for 24 hours and then incubated with PA (250 pM) or vehicle along with TGF-P (10 ng / mL) or vehicle for another 24 hours, or human aortic smooth muscle cells were transfected with adenovirus GFP (green fluorescent protein) or LOX (30 MOI) for 6 hours in a growth medium and then starved in OptiMEM for another 24 hours. Cells were incubated with PA (250 pM) or vehicle for an additional 24 hours. Conditioned medium was collected for Western blot analysis. Cell total DNA was used for normalization. FIG. 18H is a representative Western blot image of mature LOX in conditioned medium (n=5 / group). FIG. 181 shows quantification analysis of mature LOX protein abundance (n=5 / group). In FIGS. 18J and 18K, Human aortic smooth muscle cells were transfected with siBMPl at 1 nM or siCtr with RNAimax for 6 hours in OptiMEM-reduced serum medium and then transfected with adenovirus GFP or LOX (30 MOI) for another 23 hours in OptiMEM-reduced serum medium. The conditioned medium was used for Western blot analysis, and whole cells were used for RNA extraction or DNA extraction. FIG. 18J is qRT-PCR analysis of BMP1 (n=4 / group). FIG. 18K shows representative LOX expression in conditioned medium (top) and quantification analysis (bottom, n=6 / group). In FIG. 18L eight- week-old male C57BL / 6I mice were given saline, vehicle, or ethyl palmitate (600 mg / kg) for 5 consecutive days via intraperitoneal injection. On day 6, mice were euthanized, suprarenal abdominal aortas were isolated, and total protein was extracted and analyzed by Western blot to detect LOX abundance in suprarenal abdominal aortas, with corresponding quantifications forAtty. Docket No. UM-43304.601 mature LOX (n=5 / group). In FIG. 18M 12- to 16- week-old human APOC3 transgenic mice and littermate control mice were infused with saline or angiotensin II (1000 ng / kg / min) for 7 days. On day 8, mice were euthanized, suprarenal abdominal aortas were isolated, and total protein was extracted and analyzed by Western blot to detect LOX expression in suprarenal abdominal aortas, with corresponding quantifications for mature LOX (n=3 or 4 / genotyping / treatment). In FIG. 18N, 14- to 20- week-old human APOC3 transgenic mice and littermate control mice were infused with saline or angiotensin II (1000 ng / kg / min) for 7 days. On day 8, mice were euthanized, suprarenal abdominal aortas were isolated, and LOX activity was measured (n=4-6 / genotyping / treatment). Data are presented as dots and mean+SEM (FIGS. 18B, 18D-18F, and 18G-18N). Statistical analyses were conducted as follows: Student t test for FIGS. 18B and 18D-18F, Mann- Whitney U test followed by Bonferroni correction for FIGS. 181 and 18K, 2- way ANOVA followed by Sidak post hoc analysis for FIGS. 18J and 18N, and 1-way ANOVA followed by Sidak post hoc analysis for FIG. 18K.

[0033] FIGS. 19A-19K show lysyl oxidase overexpression inhibits abdominal aortic aneurysm formation and dissection in human APOC3 transgenic mice. FIG. 19A is a schematic design of the lysyl oxidase (LOX) overexpression study in human APOC3 transgenic mice. Twelve- to 16- week- old male human APOC3 transgenic mice and their littermate wild-type controls were transfected with 9x108 pfu adenovirus GFP (green fluorescent protein) or LOX to the suprarenal abdominal aorta and then infused with angiotensin II (1000 ng / kg / min) for 18 days to induce an abdominal aortic aneurysm. The surviving mice were used for the following analysis (wild-type, GFP: n=10; wild-type, LOX: n=12; transgenic, GFP, n=9; transgenic, LOX, n=10). FIG / 19B is representative aortic trees from the 4 groups. FIGS. 19C-19E show quantification of abdominal aortic aneurysm incidence (FIG. 19C), dissection rate (FIG. 19D), and maximal aorta diameter (FIG. 19E) among the 4 groups. FIGS. 19F-19J show systolic blood pressure (FIG. 19F), body weight (FIG. 19G), plasma triglycerides (FIG. 19H), total cholesterol (FIG. 191), and nonesterified fatty acids (FIG. 19J). FIG. 19K is representative Masson trichrome, Verhoeff-van Gieson, and H&E staining of suprarenal abdominal aorta sections. Data are presented as dots and mean±SEM (FIGS. 19E-19I). Statistical analyses were conducted as follows: Fisher exact test for FIGS. 19C-19D, 2-way ANOVA followed by Sidak post hoc analysis for FIGS. 19E and 19G-19J, and 2-way mixed-effects ANOVA followed by Sidak post hoc analysis for FIG. 19F. Scale bars=l mm (each interval) in FIG. 19B and 100 pm in FIG. 19K. Angll indicates angiotensin II; BW, body weight; hAPOC3 NEFA, nonesterified fatty acid; hAPOC3 Tg, human APOC3 transgenic; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; VVG, Verhoeff-van Gieson; and WT, wild-type.Atty. Docket No. UM-43304.601

[0034] FIGS. 20 A- 20 K show administration of the Angptl3 antisense oligonucleotide prevents abdominal aortic aneurysm formation in human APOC3 transgenic mice. FIG. 20A is a schematic design of the Angptl3 antisense oligonucleotide (ASO) study in human APOC3 transgenic (Tg) mice. Twelve- to 16-week-old male human APOC3 Tg mice were given one injection of the Angptl3 ASO (10 mg / kg) or scrambled ASO by subcutaneous administration. After 3 days, mice were infused with angiotensin II (1000 ng / kg / min) for 25 days. Three more injections (3 mg / kg) were conducted on days 7, 14, and 21. A wild-type group was included, receiving injections of scrambled ASO. At the end of the study, aortas, livers, and plasma were harvested (wild-type, n=l 1 ; Tg, ASO Ctr, n=8; Tg, ASO Angptl3, n=10). FIG. 20B shows the relative abundance of Angptl3 mRNA in livers of the 3 groups. FIG. 20C is plasma ANGPTL3 protein concentrations and FIG. 20D is nonesterified fatty acids at the end point. Quantification of abdominal aortic aneurysm incidence among the 3 groups is shown in FIG. 20E. FIG. 20F shows the relative abundance of Apoc3 in the liver. FIG. 20G shows the plasma mouse-specific APOC3 concentrations were measured by ELISA at the end point. FIG. 20H shows the relative liver abundance of human APOC3. FIG 201 shows plasma human APOC3 concentrations were determined by ELISA at the end point. FIG 201 shows body weight comparisons among the 3 groups before and at the end of the study. FIG 20K shows systolic blood pressure comparisons among the 3 groups before and at the end of the study. Data are presented as circles / dots and mean+SEM or mean only. Statistical analyses were conducted as follows: 1-way ANOVA followed by Sidak post hoc analysis for FIGS. 20B-20D and 20F-20G; Kruskal- Wallis test followed by Dunn’s post hoc analysis for FIGS. 20H-20I; Fisher exact test for FIG. 20E; and 2- way mixed-effects ANOVA followed by Sidak post hoc analysis for FIGS. 20I-20K. AAA indicates abdominal aortic aneurysm; Angll, angiotensin II; hAPOC3 Tg, human APOC3 transgenic; NEFA, non-esterified fatty acid; SBP, systolic blood pressure; VVF, Verhoeff-van Gieson; and WT, wildtype.

[0035] FIGS. 21A-21D show administration of the Angptl3 antisense oligonucleotide prevents abdominal aortic aneurysm formation in Apoe-defi cient mice. FIG. 21 A is a schematic of the design of the Angptl3 antisense oligonucleotide study in male Apoe-deficient mice. Ten-week-old male Apoe-deficient mice were given a subcutaneous injection of the Angptl3 antisense oligonucleotide (10 mg / kg, n=14 in the beginning and 13 at the end point) or scrambled antisense oligonucleotide (n=14 in the beginning and 12 at the end point). After 3 days, mice were infused with angiotensin II (1000 ng / kg / min) for 25 days. Three more injections were administered on days 7, 14, and 21. At the end of the study, aortas, livers, and plasma were harvested. FIG. 21B shows a graph of the relative abundance of Angptl3 mRNA in the liver. FIGS. 21C and 21D, show plasma ANGPTL3 proteinAtty. Docket No. UM-43304.601 concentrations (FIG. 21C) and nonesterified fatty acids (FIG. 21D) at the end point. Data are presented as circles / dots or mean+SEM. Statistical analyses were conducted as follows: Student t test for FIGS. 21B and 21C; Mann-Whitney U test for FIG. 21D. ASO, antisense oligonucleotide;NEFA, non-esterified fatty acid; TC, total cholesterol; and TG, triglyceride.

[0036] FIGS. 22A-22C show knockdown efficiencies of target human APOC3, ANGPTL3, and ANGPTL8. siRNAs against human APOC3, ANGPTL3, ANGPTL8 were designed and the knockdown efficiency was evaluated in HepG2 cells, a human liver cancer cell line commonly used in research. HepG2 cells were transfected with siRNA at 1 nM for 48 hours and the mRNA levels of APOC3 (FIG. 22A), ANGPTL3 (FIG. 22B), or ANGPTL8 (FIG. 22C) were determined by qRT- PCR (n = 3 ). .DETAILED DESCRIPTION

[0037] The present disclosure provides methods for treating or preventing abdominal aortic aneurysm (AAA) in a subject. As shown herein, knockdown, silencing or downregulation of any of APOC3, ANGPTL3, and ANGPTL8, resulted in decreased triglyceride levels, AAA incidence rate and maximal diameters, whereas decreasing or knocking out of APOA5 increased AAA incidences.

[0038] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0039] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0040] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.Atty. Docket No. UM-43304.601

[0041] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, molecular biology, immunology, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] “Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. The nucleic acid, whether DNA or RNA may comprise non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”). Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or doublestranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0043] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein” are used interchangeably herein.

[0044] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,Atty. Docket No. UM-43304.601 a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual- variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25( 11 ): 1290- 1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.

[0045] “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, singlechain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0046] As used herein, “treat,” “treating” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the progression. As such, “treating” means carrying out the methods described herein on a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.Atty. Docket No. UM-43304.601

[0047] As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0048] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., a human or a non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0049] As used herein, the terms “providing,” “administering,” “introducing,” are used interchangeably herein and refer to the placement into a subject by a method or route which results in at least partial localization to a desired site. Administration can be by any appropriate route which results in delivery to a desired location in the subject.

[0050] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Treating and / or Preventing Abdominal Aortic Aneurysm (AAA)

[0051] The present disclosure provides methods for treating or preventing abdominal aortic aneurysm (AAA) in a subject.

[0052] The abdominal aorta spans from the diaphragm to the aortoiliac bifurcation, and is the most common site for aortic aneurysms. The methods disclosed herein are suitable for use with anyAtty. Docket No. UM-43304.601 location of abdominal aortic aneurysm. The methods are also suitable for treating an aneurysm of any size.

[0053] In some embodiments, the subject does not have and / or is not receiving treatment for a disease or disorder caused or mediated by high levels of triglycerides or hypertriglyceridemia. Exemplary diseases caused or mediated by high levels of triglycerides include, for example, atherosclerosis, cardiovascular or heart disease, pancreatitis, stroke, metabolic syndrome, type 2 diabetes, non-alcoholic fatty liver disease, kidney diseases, and gallstones.

[0054] In some embodiments, the subject is having or had a surgical repair of an AAA. Aneurysms are often repaired using open (e.g., transperitoneal or retroperitoneal) surgical procedures. Surgical methods for repairing AAAs, for example, require opening the abdominal region from the breastbone to the pelvic bone, clamping the aorta to control bleeding, dissecting the aorta to remove the aneurysmal section, and attaching a prosthetic graft to replace the diseased artery. Minimally invasive surgical techniques that implant prosthetic grafts across aneurysmal regions of the aorta have been developed as an alternative to open surgery for certain patient groups. Endovascular aortic repairs (“EVAR”), for example, generally use small incisions in the groin. Using X-ray guidance and specially-designed instruments, the aneurysm is repaired by inserting a stent or graft inside the aorta. In some embodiments, the subject had or is having an endovascular aortic repair. In some embodiments, the subject had or is having an open surgical repair.

[0055] Accordingly, the methods may be utilized following repair (e.g., hours, days, weeks, months after repair) of the abdominal aortic aneurysm or prior to repair (e.g., hours, days, weeks, or months to the repair) of the abdominal aortic aneurysm.

[0056] In some embodiments, the methods further comprise identifying an abdominal aortic aneurysm in the subject. The identification of an abdominal aortic aneurysm is usually made with ultrasound, but a computed tomography (CT / CAT / CTA) scan can determine the exact location, size, and involvement of other vessels, and provide better imagining depending on location of the aneurysm. Magnetic resonance angiography and X-ray can be used as well to delineate the aneurysm. In some embodiments, the identifying comprises an ultrasound, X-ray, computed tomography (CT / CAT / CTA) scan, and / or magnetic resonance imaging (MRI) of the abdomen [0057| In some embodiments, the methods may further comprise monitoring the abdominal aortic aneurysm, e.g., for changes in size or shape. Any of the methods above for identifying the abdominal aortic aneurysm can also be used for the monitoring, including but not limited to ultrasound, X-ray, computed tomography scan, and / or MRI. For example, monitoring the aneurysm can include: surveillance imaging every 6, 12, or 36 months, depending on size of aneurysm, assessment of distalAtty. Docket No. UM-43304.601 leg or foot pulses (e.g., femoral and popliteal pulses); abdominal palpitations examining for pulsatile abdominal mass; assessment of symptoms of abdominal aortic aneurysm (e.g., abdominal, flank, or back pain, gastrointestinal (GI) or renal manifestations); and investigations of other possible associated aneurysms (e.g., iliac artery aneurysm).

[0058] In some embodiments, the methods further comprise characterizing the abdominal aortic aneurysm by size. As described above, the methods may be used to treat an abdominal aortic aneurysm of any size. In some embodiments, the aneurysm is less than about 3 cm. In some embodiments, the aneurysm is less than about 4.5 cm. In some embodiments, the aneurysm is less than about 5.5 cm. In some embodiments, the aneurysm is greater than about 5.5 cm. In some embodiments, the aneurysm is greater than about 3 cm.

[0059] In some embodiments, the methods further comprise repairing the abdominal aortic aneurysm. Depending on the patient, the repair may include open surgical repair or minimally invasive surgical techniques such as endovascular repair.

[0060] In some embodiments, the methods stop or slow down the growth of the abdominal aortic aneurysm. In some embodiments, the methods decrease the size of the abdominal aortic aneurysm. Accordingly, the methods may delay the need for repair of the abdominal aortic aneurysm. In some embodiments, the methods prevent recurrence of an abdominal aortic aneurysm following repair. In some embodiments, the methods prevent or delay rupture of the abdominal aortic aneurysm. a) Inhibitors of APOC3, ANGPTL3, and ANGPTL8

[0061] In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of one or more inhibitors of APOC3 (Apolipoprotein C3), ANGPTL3 (angiopoietin-like 3), Angiopoietin-like 8 (ANGPTL8), or a combination thereof. APOC3, ANGPTL3, and ANGPTL8 inhibitors include those compounds which target or inhibit the activity and / or expression of APOC3, ANGPTL3, and ANGPTL8. Suitable APOC3, ANGPTL3, and ANGPTL8 inhibitors include, but are not limited to, gene silencing or downregulating oligonucleotides (e.g., an siRNA, an antisense oligonucleotide, a short-hairpin RNA, a miRNA, a dicer-substrate RNA, a DNAzyme, a guide RNA, a small circular RNA, an aptamer targeting the gene or messenger RNA), protein configured to bind APOC3, ANGPTL3, or ANGPTL8 (e.g., an antibody (e.g., a monoclonal, polyclonal, murine, chimeric, humanized, or human antibody targeting)), a small molecule inhibitor of APOC3, ANGPTL3, or ANGPTL8, or combinations thereof.Atty. Docket No. UM-43304.601

[0062] In some embodiments, the gene silencing or downregulating oligonucleotides are part of a gene editing system. The gene editing system may be used to modulate (e.g., repress) a target gene (e.g., APOC3, ANGPTL3, or ANGPTL8), introduce one or more nucleotide substitutions, addition, or deletions into a target gene (e.g., to insert a mutation, disrupt target gene expression, or alter a promoter region for a target gene), or delete a target gene. For example, the gene editing system may encode a zinc-finger nuclease, a homing endonuclease, a TALEN (transcription activator-like effector nuclease), a NgAgo (agronaute endonuclease), a SGN (structure-guided endonuclease), or an RNA-guided endonuclease or components of a CRISPR-Cas system. Thus, the inhibitors may further include one or more components of a gene editing system.

[0063] A “CRISPR-Cas system” refers collectively to transcripts and other elements involved in the expression of and / or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, Cas protein, a cr (CRISPR) sequence (e.g., crRNA or an active partial crRNA), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. The CRISPR-Cas system can be an engineered system for use in activation, repression, or deletion of a target gene (e.g., CRISPRi, CRISPRa). CRISPR-Cas editing technology is described in detail in, for example, U.S. Patent Nos. 8,546,553, 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445;8,889,356; 8,889,418; 8,895,308; 8,9066,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,115,348; 9,149,049; 9,493,844; 9,567,603; 9,637,739; 9,663,782; 9,404,098; 9,885,026; 9,951,342; 10,087,431 ; 10,227,610; 10,266,850; 10,601,748; 10,604,771 ; and 10,760,064; and U.S. Patent Application Publication Nos. US2010 / 0076057; US2014 / 0113376; US2015 / 0050699;US2015 / 0031134; US2014 / 0357530; US2014 / 0349400; US2014 / 0315985; US2014 / 0310830; US2014 / 0310828; US2014 / 0309487; US2014 / 0294773; US2014 / 0287938; US2014 / 0273230; US2014 / 0242699; US2014 / 0242664; US 2014 / 0212869; US2014 / 0201857; US2014 / 0199767; US2014 / 0189896; US2014 / 0186919; US2014 / 0186843; and US2014 / 0179770, each incorporated herein by reference.

[0064] For example, the gene editing system may comprise one or more Cas proteins (e.g., Cas9), or other RNA-guided nucleases, to work in conjunction with one or more guide RNAs directed to the target gene (e.g., APOC3, ANGPTL3, or ANGPTL8). RNA sequences employed in CRISPR / Cas systems are referred to collectively as “guide RNA” (gRNA) or single guide RNA (sgRNA). Thus, the terms “guide RNA,” “single guide RNA,” and “synthetic guide RNA,” are used interchangeably herein and may refer to a nucleic acid sequence comprising a tracrRNA and a pre-crRNA array containing a guide sequence. The terms “guide sequence,” “guide,” and “spacer,” are usedAtty. Docket No. UM-43304.601 interchangeably herein and refer to the nucleotide sequence within a guide RNA that specifies the target gene.

[0065] In some embodiments, the Apoc3 inhibitor comprises antisense oligonucleotides (ASOs), interfering RNAs (siRNAs), guide RNAs, for use with CRISPR-Cas or other genetic modifying systems, and / or antibodies (see, for example, Khetarpal SA, Nat Med. 2017 Sep;23(9):1086-1094).

[0066] In select embodiments, the APOC3 inhibitor comprises an siRNA. In some embodiments, the APOC3 siRNA comprises a sense strand sequence having 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 1-10 and / or an antisense strand sequence having 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 11-20. In some embodiments, the Apoc3 siRNA comprises a sense strand sequence having 1, 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 1-10 and / or an antisense strand sequence having 1 , 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 11-20. In some embodiments, the APOC3 siRNA comprises a sense strand sequence having any of SEQ ID NOs: 1-10 and / or an antisense strand sequence having any of SEQ ID NOs: 11-20.

[0067] In some embodiments, the ANGPTL3 inhibitor comprises antisense oligonucleotides (ASOs) (e.g., Vupanorsen (ANGPTL3-LRX), a second-generation ligand-conjugated ASO targeting the ANGPTL gene mRNA coding sequence), interfering RNAs (siRNAs), guide RNAs, for use with CRISPR-Cas or other genetic modifying systems and / or antibodies (Evinacumab).

[0068] In select embodiments, the ANGPTL3 inhibitor comprises an siRNA. In some embodiments, the ANGPTL3 siRNA comprises a sense strand sequence having 10 or more (e.g., 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 21-29 and / or an antisense strand sequence having 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 30-38. In some embodiments, the Angptl3 siRNA comprises a sense strand sequence having 1, 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 21-29 and / or an antisense strand sequence having 1, 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 30-38. In some embodiments, the Angptl3 siRNA comprises a sense strand sequence having any of SEQ ID NOs: 21-29 and / or an antisense strand sequence having any of SEQ ID NOs: 30-38.[0069| In some embodiments, the Angptl8 inhibitor comprises antisense oligonucleotides (ASOs), interfering RNAs (siRNAs), guide RNAs, for use with CRISPR-Cas or other genetic modifying systems and / or antibodies (e.g., REGN3776).

[0070] In select embodiments, the ANGPTL8 inhibitor comprises an siRNA. In some embodiments, the ANGPTL8 siRNA comprises a sense strand sequence having 10 or more (e.g., 10,Atty. Docket No. UM-43304.60111, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 39-48 and / or an antisense strand sequence having 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) contiguous nucleotides as in any of SEQ ID NOs: 49-58. In some embodiments, the Angptl8 siRNA comprises a sense strand sequence having 1, 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 39-48 and / or an antisense strand sequence having 1, 2, 3, 4, 5, 6, or 7 substitutions as compared to any of SEQ ID NOs: 49-58. In some embodiments, the ANGPTL8 siRNA comprises a sense strand sequence having any of SEQ ID NOs: 39-48 and / or an antisense strand sequence having any of SEQ ID NOs: 49-58. siRNA SequencesAtty. Docket No. UM-43304.601*Each sense and antisense sequence includes, in addition to the above recited sequences, a two-nucleotide 3' overhang of dcoxythymidincs (dTdT). The dTdT docs not bind to the target mRNA, acts to increase stability and functionality.10071] In some embodiments, the gene silencing or downregulating oligonucleotides (e.g., an siRNA, an antisense oligonucleotide, a short-hairpin RNA, a miRNA, a dicer-substrate RNA, a DNAzyme, a guide RNA, a small circular RNA, an aptamer targeting the gene or messenger RNA) comprise at least one chemical modification or chemically modified base or nucleoside. The chemical modifications may comprise any modification which is or is not present in naturally occurring forms of adenosine, guanosine, uridine, thymidine, or cytidine ribonucleosides or deoxyribonucleosides. For example, an engineered target nucleic acid may include both naturally occurring and non-naturally occurring modifications. Chemical modifications may be located in any portion of the engineered target nucleic acid and the engineered target nucleic acid may contain any percentage of modified nucleosides (1-100%). A particular modification may be used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1 -methyl-pseudouridine) or on a per base level. In some embodiments, the at least one chemical modification comprises a modified uridine residue. In some embodiments, the at least one chemical modification comprises a modified thymidine residue. In some embodiments, the at least one chemical modification comprises a modified cytosine residue. In some embodiments, the at least one chemical modification comprises a modified adenine residue. In some embodiments, the at least one chemical modification comprises a modified guanine residue.

[0072] Exemplary nucleosides having a modified uracil include pseudouridine ( ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5 -iodo-uridine or 5-bromo-uridine), 3-methyl-uridine, 5-methyl-uridine, 5 -methoxy -uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5- aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5- methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl- uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio- uridine, l-taurinomethyl-4-thio-pseudouridine, 1 -methylpseudouridine, 5-methyl-2-thio-uridine, 1- methyl-4-thio-pseudouridine, 4-thio-l -methyl-pseudouridine, 3 -methyl-pseudouridine, 2-thio-l-Atty. Docket No. UM-43304.601 methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy -pseudouridine, 4-methoxy-2-thio-pseudouridine, 1 -methylpseudouridine, 3 -(3 -amino- 3- carboxypropyl)uridine, l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine , 5- (isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, a-thio-uridine, 2’-O- methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl-pseudouridine, 2-thio-2’-O-methyl-uridine, 5- methoxycarbonylmethyl-2’ -O-methyl-uridine, 5-carbamoylmethyl-2’-O-methyl-uridine, 5- carboxymethylaminomethyl-2’-O-methyl-uridine, 3,2’-O-dimethyl-uridine, 5- (isopentenylaminomethyl)-2’ -O-methyl-uridine, 1 -thio-uridine, deoxythymidine, 2’-F-ara-uridine, 2’ -F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l -E- propenylamino)uridine. In some embodiments, the modified uridine residue is selected from the group consisting of: pseudouridine, 1 -methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5 -methyluridine, 2-thio-l -methyl- 1 -deaza-pseudouridine, 2- thio-l-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'- 0-methyl uridine.

[0073] Exemplary nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5- methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1-deaza-pseudoisocy tidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O-methyl- cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O-dimethyl-cytidine, 5- formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1 -thio-cytidine, 2 ’-F-aracy tidine, 2’-F- cytidine, and 2’-OH-aracytidine. In some embodiments, the at least one chemical modification comprises 5 -methylcytosine (base) or 5 -methylcytidine (nucleoside).

[0074] Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-Atty. Docket No. UM-43304.601 deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio- N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl-adenosine, N6,2’-O-dimethyl- adenosine, N6,N6,2’-O-trimethyl-adenosine, l,2’-O-dimethyl-adenosine, 2’-O-ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0075] Exemplary nucleosides having a modified guanine include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7 -deaza- guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7- aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1 -methyl-guanosine, N2-methyl-guanosine, N2,N2- dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7- methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl- 6-thio-guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2,7-dimethyl-2’-O- methyl-guanosine, 2’-O-methyl-inosine, l,2’-O-dimethyl-inosine, and 2’-O-ribosylguanosine (phosphate).

[0076] In some embodiments, the nucleosides of the gene silencing or downregulating oligonucleotides may be linked together using one or more modified intemucleoside linkages. Representative phosphorus-containing intemucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates, and phosphorodithioates. Modified intemucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotideAtty. Docket No. UM-43304.601 b) Upregulation of APOA5

[0077] In some embodiments, the methods comprise increasing the level of APOA5 (Apolipoprotein A5). In some embodiments, the methods comprise administering to the subject an effective amount of APOA5, an active fragment or variant thereof, or a nucleic acid encoding APOA5, or an active fragment or variant thereof. In some embodiments, the methods comprise administering an agent for the upregulation of endogenous APOA5. For example, the methods may comprise administering a site directed transcriptional activator or system thereof (e.g., TALEs, CRISPRa) for endogenous APOA5. Other agents which may be used to increase endogenous APOA5 include insulin, fibrates (such as fenofibrate), and dietary factors such as polyunsaturated fatty acids (PUFAs), particularly omega-3 fatty acids.

[0078] APOA5 is a 366 amino acid protein (~39 kDa) with lipid-binding properties that has several amphipathic helices and an N-terminal signal peptide. In some embodiments, the APOA5is wild-type APOA5. In some embodiments, the APOA5 is truncated APOA5. For example, APOA5 with an internal deletion of amino acids 170-208 (WALLQGLQSRVVHHTGRFKELFHPYAESLVSGIGRHVQE; SEQ ID NO: 59). In some embodiments, the APOA5 is human APOA5.

[0079] The APOA5, or active fragment or variant thereof, may further comprise an epitope tag (e.g., 3xFLAG tag, a poly-histidine tag, an HA tag, a Myc tag, and the like). The epitope tags may be at the N-terminus, a C-terminus, or a combination thereof of the corresponding protein.

[0080] In some embodiments, the APOA5, or active fragment or variant thereof, may be fused with one or more (e.g., two, three, four, or more) protein transduction domains or PTDs, also known as a CPP, cell penetrating peptide. A protein transduction domain is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to a terminus of the APOA5, or active fragment or variant thereof, (e.g., N-terminus, C- terminus, or both). In some embodiments, the PTD is inserted internally at a suitable insertion site. Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV- 1 TAT comprising); a polyarginine sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9,Atty. Docket No. UM-43304.60110, or 10-50 arginine residues); a VP22 domain; a Drosophila Antennapedia protein transduction domain; a truncated human calcitonin peptide; polylysine; transportan, and the like. c) Dosage and Administration

[0081] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient amount that is delivered to a subject, either in a single dose or as part of a series, which is effective for inducing a response in the subject. This amount varies depending upon the health and physical condition of the subject to be treated, the selected inhibitor and formulations thereof, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by one of skill in the art through routine trials.

[0082] The route and regimen of administration will vary depending upon the subject and is to be determined by the skilled practitioner. For example, the inhibitors disclosed herein may he administered parentally, e.g., in intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to a subject in need thereof.

[0083] The administration may comprise an initial dose and at least one subsequent dose. The subsequent doses will be adequately spaced at such times where the levels of inhibitor fall below a desired level. Subsequent does may be the same or different dosage, and comprise the same or different formulations. The specific dose level may depend upon a variety of factors including the activity of the inhibitor, the age, body weight, general health, and diet of the subject, time of administration, and route of administration. For prophylaxis purposes, the amount in each dose is an amount which induces a protective response without significant adverse side effects.

[0084] The administration of any of the above inhibitors or APOA5 can be in the form of a composition comprising excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0085] Excipients and carriers may include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and itsAtty. Docket No. UM-43304.601 derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, preservatives, and antioxidants. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The route or administration and the form of the composition usually dictates the type of carrier to be used.

[0086] The compositions may be formulated for any appropriate manner of administration, and thus administered, including for example, oral, nasal, intraocular, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, or via inhalation. Techniques and formulations may generally be found in “Remington’s Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic or pharmaceutical compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0087] The agents (e.g., APOC3, ANGPTL3, ANGPTL8 inhibitors) or a composition thereof can be evaluated for efficacy and toxicity using known methods. For example, the toxicology may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity in an animal model, such as mice, rats, rabbits, dogs, or monkeys, may be determined using known methods. The efficacy may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and / or regime.[0088| It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the aneurysm and the route of administration.Atty. Docket No. UM-43304.601Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0089] A wide range of additional therapies may be used in conjunction with the methods of the present disclosure. The additional therapy may be administration of an additional active agent or may be a second therapy not connected to administration of another agent. Such additional therapies include, but are not limited to, blood pressure lowering medications, cholesterol lowering medications, and lifestyle changes (e.g., healthy eating, loss of weight, reduce alcohol consumption, increase exercise, smoking cessation). d) Nucleic Acids

[0090] The present disclosure also provides for DNA segments encoding the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides (e.g., antisense oligonucleotides (ASOs), interfering RNAs (siRNAs), guide RNAs)) disclosed herein, vectors containing these segments and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0091] The nucleic acid encoding the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein may be any nucleic acid including DNA, RNA, or combinations thereof. In some embodiments, the nucleic acid encoding the proteins comprises a messenger RNA or a vector.

[0092] In certain embodiments, engineering the nucleic acid for use in eukaryotic cells may involve codon-optimization. It will be appreciated that changing native codons to those most frequently used in mammals allows for maximum expression of the system proteins in mammalian cells (e.g., human cells). Such modified nucleic acid sequences are commonly described in the art as “codon-optimized,” or as utilizing “mammalian-preferred” or “human-preferred” codons. In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g.,Atty. Docket No. UM-43304.60165%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the codons encoded therein are mammalian preferred codons.

[0093] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.

[0094] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[0095] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that the nucleic acid encoding the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein may be removed from the cells under certain conditions.

[0096] A variety of viral constructs may be used to deliver the proteins (e.g., APOA5 or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein to theAtty. Docket No. UM-43304.601 targeted cells and / or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(l):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0097] In one embodiment, a DNA segment encoding the proteins (e.g., APOA5or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification of the protein produced by the recombinant vector. Accordingly, the proteins can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0098] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0099] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerateAtty. Docket No. UM-43304.601 kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focusforming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1 -alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0100] Moreover, inducible and tissue specific expression of a nucleic acid or protein can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various ubiquitous as well as tissue-specific promoters and tumor- specific are commercially available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein or RNA operably linked thereto.

[0101] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell withinAtty. Docket No. UM-43304.601 the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0102] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5 ’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly- expressed genes like a-globin or P-globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0103] When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0104] The present proteins, nucleic acids, and compositions comprising the proteins and / or nucleic acids described herein may be delivered by any suitable means. In certain embodiments, they are delivered in vivo, as described above.

[0105] As described above, the proteins or nucleic acids may be introduced into cells by methods known in the art. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a mammalian cell (e.g., a cell of a non-human primate or a human cell). Accordingly, provided herein are cells comprising the disclosed proteins or nucleic acids encoding thereof.

[0106] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-Atty. Docket No. UM-43304.601 precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0107] Any of the vectors comprising a nucleic acid sequence that encodes proteins (e.g., APOA5or active fragment or variant thereof; antibodies, or fragments thereof, to APOC3, ANGPTL3, ANGPTL8; Cas proteins or other nucleases) or nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing or downregulating oligonucleotides) disclosed herein is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the vector can be delivered by any method appropriate for introducing nucleic acids into a cell.

[0108] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.3. Examples[0109| The following are examples of the present invention and are not to be construed as limiting.Example 1

[0110] Combining cis-pQTL and GWAS findings, Mendelian randomization (MR) analyses were performed to assess the causal effects of circulating proteins on AAA risk and 41 out of 2,698 circulating proteins were significantly associated with AAA after adjusting for multiple tests. AmongAtty. Docket No. UM-43304.601 them, genetically determined circulating AP0C3 (OR=1.84) and AP0A5 (OR=0.61) had the most significant effects on AAA risk. Seven of the 41 circulating proteins, including APOC3, AP0A5, LPL, APOE, PLTP (phospholipid transfer protein), PCSK9 (proprotein convertase subtilisin / kexin type 9), and LPA (lipoprotein(A)), play an important role in lipid metabolism, especially in VLDL and chylomicron metabolism. Previous GWAS findings demonstrated that 42 lead variants at the AAA risk loci are associated with major lipid fractions. After prioritizing the role of five major lipoprotein components (HDL-C, LDL-C, TG, AP0A1, and APOB) as risk factors for AAA, TG was the top-ranked risk factor for AAA with a marginal inclusion probability of 0.81 (P=0.007). These genetic and proteomic findings indicate that TG-related metabolism may play a substantial role in AAA pathogenesis.

[0111] To further identify genetic associations between AAA risk loci and circulating metabolic traits, the data from a recently published GWAS study of 233 circulating metabolites (Kaqalainen, M.K., et al. Nature 628, 130-138 (2024)) was utilized to perform a two-sample MR analysis. The NMR-measured metabolites included 213 lipid traits (lipids, lipoproteins, and fatty acids) and 20 non-lipid traits (amino acids, ketone bodies, and glucose / glycolysis-related metabolites).Lipoproteins are classified by particle sizes (XXL, XL, L, M, S, and XS) and then subgrouped by the components (particle number, phospholipids, cholesterol, cholesterol ester, free cholesterol, and TG). For each metabolite, the inverse variance-weighted (IVW) method was used as the primary MR approach and MR-Egger analysis and weighted median-based regression methods as sensitivity analyses. TG-rich lipoproteins, including VLDL, IDL (intermediate density lipoprotein), and LDL, and all the components in TRL particles, were positively associated with AAA risk, while HDL and glucose were negatively associated with AAA risk, consistent with population findings. Interestingly, TG component of HDL was positively associated with AAA risk compared to other components in HDL particles, such as cholesterol and phospholipids. Concomitantly, total fatty acid and saturated fatty acid, the metabolites of triglycerides, were also positively associated with AAA risk. Lowering triglycerides and related metabolites could provide a therapeutic pathway for reducing the risk of AAA.Example 2

[0112] The angiotensin II (Ang II) infusion mouse models are very popular due to similarities to human AAA, but largely depend on hypercholesterolemia background. Studies were performed without western diet to minimize the effects of hypercholesterolemia. Male Apoa5 knockout mice showed a 2-3-fold increase in TG levels but no difference in total cholesterol (TC) levels. In the Ang II infusion AAA model, moderately increased TG levels accelerated AAA development, showingAtty. Docket No. UM-43304.601 increased AAA incidence rate and larger maximal abdominal aorta diameter. There was no difference in blood pressure before and after Angll infusion between ApouJ-deficient mice and their littermate controls. This phenotype was not observed in Ap««5-dellcienl female mice, which had only slightly increased TG concentrations compared with male Apou5-deficient mice (142+51 mg / dL versus 303+146 mg / dL, P=0.006). In addition, female mice demonstrate markedly lower incidences of AAA than males due to sex hormones or sex chromosomes.

[0113] Using size exclusion chromatography, increased TG concentrations were found in VLDL and intermediate-density lipoprotein in the Apoa5-deficient mice compared with their littermate wild-type (WT) controls, whereas cholesterol concentrations remained similar. Plasma nonesterified fatty acids (NEFAs), which are primarily released during TG hydrolysis, were also significantly elevated in the Apoa5-deficient mice. AAA was evaluated after a 4-week Angll infusion. The hypertriglyceridemic Apou5-deficient mice had an increased AAA incidence and larger maximal abdominal aorta diameter (FIGS. 16B, and 16E-16F). Slightly increased TG concentrations in Apoa5-deficient mice promoted AAA formation but did not trigger aneurysm rupture and animal death.

[0114] Human APOC3 transgenic (hAPOC3 Tg) mice and age / sex-matched WT littermates were fed a standard rodent laboratory diet (FIG. 17A). Compared with controls, male hAPOC3 Tg mice had an about 8-fold increase in plasma TG concentrations (FIG. 17B) and an about 1-fold elevation in plasma TC concentrations (FIG. 17C) and NEFAs ( FIG. 17D). A low incidence of AAA was observed in WT littermates (2 of 17). However, there was a dramatically increased AAA incidence and a larger maximal diameter of the suprarenal aorta in hAPOC3 Tg mice (11 of 13 surviving animals) as well as a higher dissection rate (8 of 13 surviving animals), and another 2 mice died from AAA rupture (FIGS. 17E-17G). Heterozygous hAPOC3 transgenic mice showed 9-fold increase in TG levels with mildly increased TC level (less than 250 mg / dL). After Ang II induction, the AAA incidence rate and maximal diameter were dramatically increased in hAPOC3 Tg mice. More seriously, aorta dissection happened in all AAA cases in hAPOC3 Tg mice, and two hAPOC3 Tg mice (13.3%) died from a ruptured aneurysm. There was no difference in systolic blood pressure (SBP) between hAPOC3 Tg mice and controls. A similar phenotype was observed in female mice, which had a 5 -fold increase in TG concentrations compared with control mice, indicating that dramatically increased TG concentrations accelerated AAA development, surpassing the protective effects of female hormones.

[0115] The effects of increased TG concentrations on AAA development were also evaluated in a PPE (porcine pancreatic elastase) -induced AAA model, in which AAA progression is independent ofAtty. Docket No. UM-43304.601 hypercholesterolemia. Based upon a 50% increase in the maximal abdominal aorta diameter as AAA on day 14 post-operative, the incidence of AAA was 100% for both male and female hAPOC3 Tg and littermate control mice. Male hAPOCS Tg mice showed significantly larger maximal aortic diameters than littermate controls, demonstrating that increased TG concentrations accelerated AAA growth. Even though female hAPOC3 Tg mice had lower TG concentrations than males (634 ± 36 mg / dL v.s. 946 ± 258 mg / dL, p = 0.0005), the increased TG concentrations in females were sufficient to cause larger aortic diameters.

[0116] Serum from Apoa5-deficient or hAPOC3 Tg mice, as well as VLDL and HDL particles, did not affect primary human aortic smooth muscle cell (HASMC) viability. Overexpression of APOC3 in hAPOC3 Tg mice caused elevated plasma TG concentrations accompanied by increased plasma NEFA concentrations (FIG. 17D). The high availability of fatty acids in the liver further increases TG synthesis, resulting in VLDL assembly and secretion. Using MR, the causal association between genetically determining plasma TG level and circulating total fatty acid concentrations was confirmed (FIG. 18A). Palmitic acid (PA; 16:0), stearic acid (18:0), palmitoleic acid (16: ln-7), and oleic acid (18: ln-9) are major saturated and monounsaturated fatty acids that affect cellular signaling and metabolic pathways. PA is the most common saturated fatty acid in the human body, typically accounting for 20% to -30% of the total fatty acids. MR analysis found that increased TGs causally increased the PA level but not the other 3 fatty acids (FIG. 18 A), suggesting that PA serves as an important metabolite in mediating the AAA-promoting effects of high TG or high TG-related mutations. Using untargeted metabolomics, plasma ethyl palmitate levels (a lipid-soluble form of PA) were elevated in hAPOC3 Tg mice (FIG. 18B). Bulk RNA-seq analysis was performed to compare PA-incubated HASMCs with BSA-treated (vehicle control) cells. Compared with the BSA- incubated group, PA-incubated HASMCs showed an apparent reduction in ECM assembly pathways, including collagen chain trimerization, elastic fiber formation and maturation, and increased expression of the inflammation-related genes.

[0117] TGF-|3 and lysyl oxidase (LOX) regulate the cross-linking of collagen and elastin in the ECM. LOX was unexpectedly upregulated compared with the most downregulated genes in the elastic fiber formation and maturation pathways. In primary HASMCs, PA incubation induced the transcription of LOX, showing increased mRNA abundance by qRT-PCR (FIG. 18D). However, PA treatment paradoxically decreased LOX activity in the conditioned medium of HASMCs (FIG. 18E), indicating that PA may impair LOX protein maturation or posttranslational modification. LOX is initially synthesized as a preproprotein, which undergoes several posttranslational modifications for its maturation and functional activation. Impairments in LOX activity, whether because of geneticAtty. Docket No. UM-43304.601 mutations or LOX inhibitors, can lead to AAA formation and rupture. RNA-seq data indicated that PA incubation downregulated the expression of BMP-1 (bone morphogenetic protein 1), ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motif 2), and AD AMTS 14, which are critical enzymes controlling LOX activation by the proteolytic removal of the propeptide region. PA incubation downregulated BMP-1 and ADAMTS2 transcription in HASMCs (FIG. 18F).Immunoblot analyses demonstrated that incubation with TGF-(3 increased the abundance of the mature form of LOX both in the conditioned medium and cell lysates of HASMCs. However, the presence of PA blocked its effect, resulting in a less mature LOX (FIGS. 18H-18I). LOX OE increased the mature form of LOX in HASMCs, which was suppressed by incubation with PA, indicating that PA interferes with the maturation process of LOX in HASMCs.

[0118] To investigate how PA influences LOX maturation, using small interfering RNA- mediated gene silencing of BMP1 , ADAMTS2, or ADAMTS 14 in HASMCs, BMP1 knockdown led to dramatically decreased LOX maturation in the conditioned medium, suggesting that BMP-1 is the primary enzyme regulating LOX maturation in HASMCs. Additionally, BMP1 knockdown increased LOX transcription, likely as a compensatory effect; however, it was insufficient to compensate for the loss of mature LOX. In the presence of PA or knockdown of BMP1 , LOX maturation was largely impaired (FIG. 18H and 18K), showing dramatically reduced mature bands. Overexpression of LOX restored mature LOX levels, suggesting that LOX OE may prevent the pro-AAA effects of high TG and PA levels. Among the >1800 secreted proteins in the liver, APOC3 ranked 52nd in terms of its impact on aortic gene expression. Among the 131 donors from the GTEx project, liver APOC3 RNA expression showed a strong negative association with BMP1 RNA expression in the aorta (FIG.18G), whereas the associations with ADAMTS2 and ADAMTS14 were much weaker. In contrast, liver APOC3 expression was positively associated with aortic LOX expression. The tissue crosstalk analysis suggests that liver-secreted APOC3 regulates aortic gene expression, including BMP1 and LOX, essential for vascular integrity, potentially influencing aortic ECM remodeling and contributing to AAA pathogenesis.

[0119] To investigate the effects of increased circulating palmitate concentrations on the maturation of LOX in aortas, ethyl palmitate, which can be hydrolyzed to free palmitate in rodents, was administered. The mature LOX abundance in suprarenal abdominal aortas was decreased significantly after administration of ethyl palmitate compared with the vehicle or saline control aortas (FIG. 18L). There was no difference in the abundance of mature LOX and LOX activity in suprarenal abdominal aortas between hAPOC3 Tg and littermate control mice without Angll infusion (FIG. 18M and 18N). An upregulation of mature LOX and LOX activity was observed upon AngllAtty. Docket No. UM-43304.601 infusion in control mice, suggesting a protective and compensatory mechanism to enhance elastic fiber assembly to protect against the effects of AngIL However, this response was absent in hAPOC3 Tg mice, indicating that increased TG or palmitate inhibited LOX maturation and LOX activity in the aortas, which may contribute to AAA development and rupture.

[0120] These findings suggest that TG and palmitate promote AAA development by disrupting essential LOX maturation. To validate this mechanism in vivo, an adenoviral vector was used to overexpress LOX in the suprarenal abdominal aorta of both WT and hAPOC3 Tg mice, followed by an 18-day Angll infusion (FIG. 19A). Local adenovirus application successfully increased the expression of LOX or GFP (green fluorescent protein; as control) in the target region. LOX overexpression did not affect the rupture rate or induce AAA formation in WT mice (FIGS. 19B- 19E). Strikingly, none of the hAPOC3 Tg mice developed AAA when LOX was overexpressed locally alongside Angll induction compared with a 67% AAA incidence and a 44% dissection rate in the GFP-overexpressing control group. LOX overexpression significantly reduced the maximal suprarenal abdominal aorta diameter (FIG. 19E) without affecting systolic blood pressure (FIG. 19F), body weight (FIG. 19G), TG (FIG. 19H), TC (FIG. 191), and NEFA levels (FIG. 19J). Histological analysis revealed that hAPOC3 Tg mice exhibited substantial collagen disorganization and elastic fiber degradation compared with WT mice, indicating impaired LOX function (FIG.19K). Notably, LOX OE restored these structural features and reinforced vascular wall integrity even in the presence of elevated TG levels. Taken together, these results established that impaired LOX activation is a key mechanism by which elevated TG levels contribute to AAA development.Example 3

[0121] Fenofibrate and niacin, commonly used for treating hypertriglyceridemia, were administered but only resulted in a 14% to 17% reduction of plasma TG concentration in hAPOC3 Tg mice. In hAPOC3 Tg mice, administration of Angptl3 ASO dramatically suppressed hepatic Angptl3 mRNA abundance by 64% and the plasma ANGPTL3 concentrations by 81 %. Consequently, administration of the Angptl3 ASO significantly reduced concentrations of NEFA, triglycerides (from 1119 + 98 mg / dL to 586 + 114 mg / dL) and total cholesterol (from 283 + 103 mg / dL to 117 + 58 mg / dL). Consistent with the above findings, increased TG concentrations in hAPOC3 Tg mice accelerated AAA development during Angll infusion.

[0122] An antisense oligonucleotide (ASO) against Angptl3 mRNA (5’- GACATGTTCTTCACCTCCTC-3’ (SEQ ID NO: 60)) was administrated to hAPOC3 Tg mice, showing significantly decreased TG levels, AAA incidence rate, maximal diameters, elastinAtty. Docket No. UM-43304.601 degradation, and dissection. The elastase-induced AAA model is the second most used rodent AAA model. Consistent to Ang II model, hAPOC3 Tg mice had accelerated AAA development. Additionally, ASO against Angptl3 reduced NEFA (Non-Esterified Fatty Acids), free fatty acids levels in hAPOC3 Tg mice. qRT-PCR and ELISA analysis showed no significant changes in the levels of mouse Apoc3 and human APOC3. There were not significant changes in the TG and TC metabolism-related genes in the liver, such as Apoc2, Apoa5, Apoe, Apob, Mttp, Fas, Srebpl, Ldlr, and Pcsk9. Angptl3 ASO administration did not significantly change body weight or systolic blood pressure. Overall, administration of Angptl3 ASO protected against hypertriglyceridemia accelerated AAA development in hAPOC3 Tg mice.

[0123] To further explore whether triglyceride lowering can inhibit AAA development in mice without dramatically increasing triglyceride concentrations, the effects of AngptlS ASO on AAA development were evaluated in Apoe-deficient mice. Apoe-deficient mice were randomly divided into control ASO or Angptl3 ASO groups. Similar to the observation in hAPOC3 Tg mice, administration of the Angptl3 ASO dramatically reduced hepatic Angptl3 mRNA expression and the circulating levels of ANGPTL3. Administration of the Angptl3 ASO significantly decreased TG concentrations levels by 50%, NEFA by 31%, and slightly decreased TC concentrations by 8 % in Apoe-deficient mice fed a standard rodent laboratory diet. Angptl3 ASO administration largely inhibited AAA development, as evidenced by significantly decreased AAA incidence and maximal diameters. Size exclusion chromatography to separate lipoprotein classes found decreased TG concentrations, mainly in VLDL and IDL, while cholesterol concentrations were slightly reduced in VLDL and HDL. Blood pressure measurements and body weight recordings revealed no significant difference between control ASO and Angptl3 ASO-administered mice. AAA incidence in Apoe- deficient mice was 83% in the control group.Materials and Methods

[0124] Antibody against LOX (Cat: Al 1504) was purchased from Abclonal. Antibodies against (3-actin (Cat: 3700) and GFP (Cat:2956) were purchased from Cell Signaling Technology (CST, Danvers, MA. Angiotensin II (Angll, Cat: 4006473) was purchased from Bacham. Bovine serum albumin (BSA, Cat: A7030-100G) was purchased from Sigma. Palmitic acid was purchased from Sigma (Cat: P0500). BMP1 siRNA (Assay ID: 105352), ADAMTS2 siRNA (Assay ID: 105359), AD AMTS 14 siRNA (Assay ID: 105394), and siRNA control were obtained from Invitrogen.

[0125] C57BL / 6J mice (Stock No: 000664), human APOC3 transgenic (hAPOC3 Tg, Stock No: 006907) mice, and Apoe-deficient mice (Stock No: 002052) were purchased from The JacksonAtty. Docket No. UM-43304.601Laboratory. Apou5-deficient mouse was purchased from MMRRC (#011467-UCD; Davis, CA), and was backcrossed with C57BL / 6J inbred mice for at least 8 generations. hAPOCd heterozygous mice were bred with C57BL / 6J mice to generate hAPOCd heterozygous and littermate hAPOC3 wild-type control mice. For studies involving A / we-deficient mice or C57BL / 6J mice, animals were purchased from The Jackson Laboratory and acclimatized for at least 1 week at the University of Michigan before any procedures were initiated. All animal procedures were conducted in accordance with protocols approved by the University of Michigan Institutional Animal Care & Use Committee.

[0126] Causal effects of circulating proteins on AAA risk Genetic variants that affect protein concentrations in a ‘cis’ manner can serve as valuable tools for guiding therapeutic targeting as they mimic the beneficial or harmful effects observed by pharmacological modification. 717 cis-pQTLs compiled by Zheng, et al (Nature Genetics 52, 1122-1131 (2020)) from 5 pQTL studies and conducted Mendelian Randomization (MR) analyses to assess their causal effects on AAA risk. Five later published large-scale genome-wide association studies (GWAS) of plasma proteins were incorporated to broaden the investigation in the present study. Briefly, the cis-pQTLs of circulating proteins from a total of 10 genome-proteome-wide association studies were retrieved, including tierl cis instrument variables reported by Zheng et.al, as well as sentinel cis-pQTLs from five subsequent publications (Ferkingstad, E., et al. Nat Genet 53, 1712-1721 (2021); Zhang, J., et al. Nat Genet 54, 593-602 (2022); Folkersen, L., et al. Nat Metab 2, 1135-1148 (2020); Pietzner, M., et al. Science 374, eabjl541 (2021); Sun, B.B., el al. Nature 622, 329-338 (2023)). Sex chromosome variants and variants with minor allele frequency of less than 0.01 were excluded. Next, associations of the selected variants with AAA were retrieved from our previous large-scale GWAS meta-analysis involving 39,221 individuals with AAA and over 1 million controls. Data on exposure and outcome were then harmonized to ensure that the effect of an SNP on exposure and the outcome corresponded with the same allele. After standardizing protein target names to Ensemble gene ID, variant-protein pairs from different studies were integrated accordingly. For proteins reported by Zheng et al., the same cis-pQTLs were employed. For proteins reported only in later studies, multiple statistics could exist for the same variant-protein pair due to factors such as multiple probes or different protein isoforms. In these cases, the statistic with the highest F-statistics, an indication of instrument strength, were selected. In the same way, the variant with the highest F value for proteins with multiple available variants was selected. Ultimately, 2,698 circulating proteins were assigned unique and optimal instrumental variables. These were used in two-sample MR analyses to assess causal effects on AAA using the MR-Wald ratio method. The false discovery rate was controlled by the Bonferroni correction.Atty. Docket No. UM-43304.601

[0127] Multi-instrument variable (multi-IV) MR was applied for the circulating proteins that passed multiple tests. The available summary statistics for each of the proteins from 3 pQTL studies, which have the largest sample sizes and protein profile numbers, were retrieved. We selected the instrumental variables (IVs) and performed the MR analysis separately based on the summary statistics from each study. Briefly, IVs were selected by retrieving independent genetic variants (r2<0.001, kb = 10,000, based on individuals with European ancestors from the 1000 Genomes Project) of the corresponding protein targets at a genome-wide significance level (P<5xl0-8). Three MR methods, including inverse variance weighted (IVW) MR, weighted median-based regression, and MR-Egger analysis, were then applied. The inverse variance-weighted (IVW) MR provides the highest precision while assuming that all SNPs are valid instrumental variables. It offers an unbiased estimate when no horizontal pleiotropy is present or when horizontal pleiotropy is balanced. To account for potential pleiotropy, weighted median-based regression and MR-Egger analysis were also applied. The weighted median estimates are almost as precise as IVW estimates, but they require that at least half of the MR instrument weights on the exposure be valid. The MR-Egger analysis, though having lower precision, can detect and correct for pleiotropy, allowing for causal inference even if all genetic variants have pleiotropic effects. A consistent effect across all three methods is unlikely to be a false positive, resulting in increased robustness.

[0128] Prioritizing the role of major lipids and lipoproteins as risk factors for AAA To prioritize the role of circulating lipid and lipoproteins in AAA risk, Mendelian Randomization Bayesian Model Averaging (MR-BMA) analyses were applied by jointly considering 5 correlated exposures including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein Al (ApoAl), apolipoprotein B (ApoB), and triglycerides (TG). After retrieving independent genetic variants (r2<0.001, kb - 10,000, based on individuals with European ancestors from the 1000 Genomes Project) associated with any major lipoprotein-related trait (total cholesterol, LDL-C, HDL-C, or TG) at a genome-wide significance level (P<5xl0-8) in the Global Lipids Genetics Consortium GWAS (2013), influential variants were removed based on the Cook’ s distance and outliers based on the q-statistic. The genetic associations of the selected variants with LDL-C, HDL-C, ApoAl, ApoB, and TG were from the UK biobank study, and their association with AAA was from the previous GWAS analysis. Full details of the MR-BMA methodology can be found elsewhere (Zuber, V., et al. Nature Communications 11, 29 (2020)). Briefly, MR-BMA evaluates multiple potential causal models incorporating various subsets of exposures. For each exposure, a Marginal Inclusion Probability (MIP) is computed, indicating the likelihood of a metabolite being included in the true causal model across iterations (z). The followingAtty. Docket No. UM-43304.601 parameters were used: z - 1,000 iterations, prior probability set to 0.1, and prior variance (oA2) to 0.25. An empirical permutation procedure was used to calculate P-values, which were adjusted for multiple tests using the Benjamini-Hochberg false discovery rate (FDR) procedure.

[0129] Causal effects of circulating metabolites on AAA risk The genetic instruments for circulating metabolites were retrieved from Karjalainen, M.K., et al. (Nature 628, 130-138 (2024)), which conducted GWAS analysis on 233 NMR-measured metabolites. The inverse variance weighted (IVW) MR was used as the primary method, which provided the highest precision while assuming that all SNPs are valid instrumental variables. To address potential pleiotropy, MR-Egger analysis and weighted median-based regression were performed as sensitivity analyses. Consistent findings across all three methods bolstered robustness and minimized the risk of false positives. For illustration, the diameters of 14 lipoprotein subclass particles are plotted based on mean values from 5,651 participants in the Northern Finland Birth Cohort 1966 (NFBC66).

[0130] Causal effects of plasma triglycerides levels on circulating fatty acid concentrationsGenetic instruments and their associations with plasma TG were determined by retrieving independent genetic variants (r2<0.001, kb = 10,000, based on individuals with European ancestors from the 1000 Genomes Project) associated with TG at a genome-wide significance level (P<5xl0-8) in the Global Lipids Genetics Consortium GWAS (2013). Their associations with circulating total fatty acid level were retrieved from the NMR-based GWAS. Their associations with circulating levels of palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:ln-7), and oleic acid (18:ln-9) were retrieved from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. The inverse variance weighted (IVW) MR was used as the primary method, which provided the highest precision while assuming that all SNPs are valid instrumental variables. To address potential pleiotropy, MR-Egger analysis and weighted median-based regression were performed as sensitivity analyses.

[0131] Antisense Oligonucleotides Directed to Murine AngptI3 For the ASO-mediated TG lowering murine studies, chimeric 20-mer phosphorothioate antisense oligonucleotides (ASOs) directed to murine Angptl3 mRNA (5’-GACATGTTCTTCACCTCCTC-3’ (SEQ ID NO: 60) or control ASO (5’-CCTTCCCTGAAGGTTCCTCC- 3’ (SEQ ID NO: 61)) were produced by BOC Sciences. The ASOs contain 2’-O-methoxyethyl (2’-M0E) groups at positions 1-5 and 16-20 and have been modified by the addition of a covalently bonded triantennary N-acetyl galactosamine (GalNAc).

[0132] Induction of abdominal aortic aneurysmAtty. Docket No. UM-43304.601

[0133] AAA is defined as a > 50% increase in suprarenal (Angll-induced model) and infrarenal (PPE model) abdominal aortic diameter. In the Angll model, dissection was defined in the suprarenal aortic region as a transmural break of the media layers that leads to exit of blood to provoke adventitial dissection, which is characterized by the presence of vascular hematoma or remodeled thrombi that are visible during tissue harvesting or sectioning. Aortic rupture is defined as death resulting from aortic rupture (either thoracic or abdominal) during Angll infusion. Most of the animal models used in the present study utilized Angll infusion in which animals were infused subcutaneously via a mini-pump (Alzet, model 2004 or model 2002). Tail-cuff-based systolic blood pressure measurements were applied before pump implantation and before tissue harvesting.

[0134] In the Apoa5 knockout experiment, 10-12-week-old A / wa5-deficient mice and the littermate controls were fed on a standard rodent laboratory diet (LabDiet, 5L0D) and infused subcutaneously with Angll ( 1 ,500 ng / kg / min) for 4 weeks to induce AAA. In an independent cohort, the male mice were infused with Angll (1,000 ng / kg / min) for 2 weeks. The plasma was collected to measure the total cholesterol (TC), TG, non-esterified fatty acids and was used to run size exclusion chromatography.

[0135] In the hAPOC3 overexpression experiment, 12-16-week-old hAPOC3 Tg (heterozygous) mice and littermate controls were fed on a standard rodent laboratory diet (LabDiet, 5L0D) and infused subcutaneously with Angll (1,000 ng / kg / min) for 4 weeks to induce AAA.

[0136] For investigating the abundance of mature LOX in the abdominal suprarenal aorta at the initial stage of AAA formation, 12-14-week-old hAPOC3 Tg (heterozygous) mice and littermate controls were infused subcutaneously with Angll (1,000 ng / kg / min) or saline for 7 days.

[0137] For the local LOX overexpression experiment, a high-concentration F-127 gel solution was prepared by dissolving 1 g F-127 in 2 mL PBS overnight and stored at 4°C. 12-16 week-old male hAPOC3 Tg (heterozygous) mice and littermate controls were anesthetized via intraperitoneal injection of a ketamine (90 mg / kg) and xylazine (5 mg / kg) mixture. The perivascular adipose tissue was carefully detached from the suprarenal abdominal aorta. Next, 9xl08pfu of adenovirus expressing GFP or LOX were mixed with the prepared F-127 gel solution at a 1 : 1 ratio (v:v) and injected immediately into the region between the suprarenal abdominal aorta and the surrounding adipose tissue. After a 20-minute incubation period, the abdominal cavity was closed, and the mice were implanted with Angll ( 1 ,000 ng / kg / min) for 18 days to induce AAA. All experiments were conducted using mice fed a standard rodent 181 laboratory diet (LabDiet, 5L0D).

[0138] In a prevention study using Angptl3 ASO in the hAPOCd Tg mice, 12-16-week-old male hAPOC3 Tg (heterozygous) were subcutaneously injected with Angptl3 ASO (10 mg / kg) or controlAtty. Docket No. UM-43304.601ASO. After 3 days, the mice were infused subcutaneously with Angll (1,000 ng / kg / min) for 25 days to induce AAA, and additional ASO injections were performed on days 7, 14, and 21, with the dose decreased to 3 mg / kg. A littermate WT group was also included with control ASO injection. All experiments were performed in mice with a standard rodent laboratory diet.

[0139] In a prevention study using Angptl3 ASO in Apoe-deficient mice, 10-week-old male Apoe- deficient mice purchased from The Jackson Laboratory were subcutaneously injected with Angptl3 ASO (10 mg / kg) or control ASO for 3 days, and infused subcutaneously with Angll (1,000 ng / kg / min) for 25 days to induce AAA and additional ASO injections were performed on day 7, 14, and 21, with the dose decreased to 3 mg / kg. All animals were fed a standard rodent laboratory diet.

[0140] The peri- adventitial elastase application-induced (PPE-induced) AAA model was performed as described previously. For the PPE-induced AAA model in hAPOC3 Tg mice, 8-week- old Tg (heterozygous) mice and their littermate controls were anesthetized via intraperitoneal injection of a ketamine (90 mg / kg) and xylazine (5 mg / kg) mixture. The infrarenal abdominal aorta was isolated and surrounded by sterile gauze soaked in 30 pL of elastase (41 U / ml, Sigma, E1250). After a 30-minute incubation period, the gauze was removed, and the abdominal cavity was washed once with sterile saline before suturing. Mice were harvested 14 days after the PPE exposure.

[0141] Unless specified, no fasting procedures were conducted prior to sample collection. Mice were euthanized with CO2 and infused with 8-10 ml of sterile saline. The suprarenal abdominal aorta (in the Angll model) or infrarenal abdominal aorta (in the PPE model) was isolated and measured promptly. The maximum abdominal aortic diameter was measured using a digital caliper by at least two individuals who were blinded to the experimental groups, and the mean value was used as the result.

[0142] Histology analysis At termination, the suprarenal abdominal aorta (in the Angll model) was carefully dissected, fixed in neutral buffered formalin (10% v / v), embedded in paraffin, and sectioned into 5 pm thick slices. The H&E and Verhoeff-van Gieson (VVG) stainings were performed by the University of Michigan ULAM Pathology Core. Elastic fiber degradation was graded as follows: 1, <25% degradation; 2, 25-50% degradation^, for 50-75% degradation; and 4, for >75% degradation, or dissection.[0143| Measurement of plasma lipid, human APOC3, mouse APOC3, mouse ANGPTL3 level, and free fatty acid Whole blood was collected either from the facial vein during the experiment or through the heart at the time of tissue harvesting into EDTA-containing anticoagulant blood collection tubes without prior fasting unless specified. The blood was then centrifuged at 4°CAtty. Docket No. UM-43304.601 at 1,500 g for 20 minutes, and the supernatant plasma was collected and either measured or immediately stored at -80 °C.

[0144] Plasma total cholesterol (TC), TG, and non-esterified fatty acids (NEFA, or free fatty acids) concentrations were measured using enzymatic kits (FUJIFILM Wako Diagnostics). Plasma APOC3 concentrations were determined using species-specific ELISA kits for human APOC3 protein (Cat: abl54131, Abeam) and mouse APOC3 protein (Cat: LS-F22680, LS Bioscience). Plasma mouse ANGPTL3 levels were determined using the ANGPTL3 ELISA kit (Cat: MANL30, R&D Systems).

[0145] Untargeted metabolomics in APOC3 Tg mice Whole blood was collected from 14- to 18-week-old male hAPOC3 Tg (heterozygous) mice and their littermate controls via cardiac puncture at the time of tissue harvesting. Blood was drawn into heparin-containing anticoagulant collection tubes and the centrifuged at 4°C at 1,500 g for 20 minutes. The supernatant plasma was collected and immediately stored at -80°C. Untargeted metabolomics analyses were performed by the Metabolomics Core at the University of Michigan. All animals were fed a standard rodent laboratory diet (LabDiet, 5L0D).

[0146] Size Exclusion Chromatographic Resolution of Lipoproteins Aliquots of plasma from 2 animals in the same group were pooled at a 1 : 1 ratio and centrifuged at 4°C, 10,000 g for 10 min. The supernatant was then analyzed using a Waters HPLC system equipped with a Superose 6, 10 / 300 GL column (GE Healthcare, Piscataway, NJ). Samples were eluted with PBS at a flow rate of 0.5 ml / min and monitored at 220 nm. After removing the first 8 minutes’ fractions, 44 more fractions (500 ul per fraction) were collected. The TG and TC contents in each fraction were measured using enzymatic kits (FUJIFILM Wako Diagnostics).

[0147] HASMC culture and treatment Human aortic smooth muscle cells (HASMCs, CC-2571) were purchased from Lonza (Walkersville, MD). HASMCs were cultured in SMC Growth Medium 2 (Promo Cell, Germany) at 37°C with 5% CO2 in a humidified incubator. Cells were passaged at a 1:3 ratio, and passages 4 to 6 were used for experiments. Prior to incubation, HASMCs at 80-90% confluence were incubated in Opti-MEM™ Reduced Serum Medium for 24 hours. For the palmitic acid (PA) incubation study, a 10% BSA (10% wt / vol) solution was prepared by dissolving fatty acid free BSA (Sigma) in sterile saline and stored at 4°C. PA was dissolved in saline at a 50 mM stock concentration and stored at -20°C. For each experiment, PA was thawed at 70°C and dissolved in 10% BSA to 5 mM. This solution was then added to Opti-MEM serum-reduced medium to obtain a final concentration of 250 pM. The control group mixed with saline instead of PA to maintain consistent BSA concentration. After the indicated hours of incubation, cells were harvested for RNAAtty. Docket No. UM-43304.601 extraction and whole-cell protein extraction. The conditioned medium was centrifuged at 300 g at 4°C for 5 mins and stored immediately at -80°C.

[0148] Preparation of adenovirus. The full-length human LOX cDNA encoding LOX was subcloned into the pCRXGW / TOPO entry vector (Invitrogen). After sequencing, the LR recombination reaction was carried out between the entry clone pCRXGW / TOPO / LOX and the destination vector pAd / CMV / V5-DEST according to the manufacturer’s protocol (Invitrogen). The Ad293 cells were transfected with PacI linearized recombinant adenoviruses for packaging. After amplification, the recombinant adenoviruses were purified by CsCh density gradient ultracentrifugation. Adenovirus titration was performed using the Adeno-X qPCR Titration Kit (Clontech). AdLacZ was used as a control.

[0149] In vivo palmitic acid incubation Ethyl palmitate (Tokyo Chemical Industry) was dissolved with lecithin (1.6% wt / vol; Thermo Scientific, 413102500) and glycerol (3.3% vol / vol) in water to produce a mixture containing ethyl palmitate (600 mM), lecithin (1.2% wt / vol), and glycerol (2.5% vol / vol), as reported previously (Daugherty A, et al., J Clin Invest. 2000;105:1605-12). The lecithin-glycerol-water solution was used as the control. 8-week-old C57BL / 6J mice fed a normal rodent laboratory diet were intraperitoneally administered either ethyl palmitate or vehicle daily for 5 consecutive days (600 mg / kg). Prior to tissue harvest, mice were fasted for 16 hours, and the last injection was conducted 6 hours before euthanasia. At the time of tissue harvesting, mice were euthanized with CO2 and infused with 8-10 ml of sterile saline. The suprarenal abdominal aorta was isolated and snap-frozen for protein extraction.

[0150] RNA extraction, RT-qPCR, and RNA sequencing analyses

[0151] RNA extraction, reserve transcription, and qPCR Total RNA was extracted from the liver or cultured HASMCs using RNeasy Mini kit (74106, QIAGEN, Hilden, Germany). cDNA samples were synthesized using oligo(dT) primers and the SuperScript III First-Strand Synthesis System (18080051, Invitrogen). For qRT-PCR analysis, cDNA reverse transcribed from 40 ng RNA was used. Relative mRNA expression was determined using the 2-A Ctmethod, with Ppia being the internal control.

[0152] RNA sequencing and quantification About 1 pg of RNA extracted from cultured HASMCs was submitted to the Advanced Genomics Core at the University of Michigan for RNA-seq analysis. cDNA libraries were prepared using the Illumina NEBNext Ultra RNA Library Prep Kit, and sequencing was conducted on the NovaX 10B 300 cycle to generate 150-base pair paired-end reads. Cutadapt (v2.3) was employed to remove potential low-quality sequences and adapter remnants. The quality of the trimmed data was assessed using FastQC (vO.11.8), and Fastq Screen (vO. 13.0) wasAtty. Docket No. UM-43304.601 utilized to screen for various types of contamination. For alignment, reads were mapped to the reference genome GRQ138 (ENSEMBL), using STAR v2.7.8a (Dobin et al., 2013) and assigned count estimates to genes with RSEM vl .3.3 (Li and Dewey, 2011), which provides count values and Transcripts Per Million (TPM) values.

[0153] Bioinformatic analysis Differentially expressed gene analysis was conducted using the R DESeq2 package (vl.40. 1). Differentially expressed gene was defined as Ilog2foldchangel > 1 and a False Discovery Rate (FDR) < 0.05. Biological process annotations for gene sets were analyzed using the enrichPathway function from the R clusterProfiler package (v4.8.1). Gene Set Enrichment Analysis (GSEA) analysis was conducted using the GSEA function from the R clusterProfiler package utilizing the ranking list of all genes sorted by fold change.

[0154] Protein extraction and Western blotting Total protein from tissues or cells was extracted using RIPA buffer, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. Membranes were blocked in TBST (Tris-buffered saline with Tween-20) containing nonfat dry milk (5% wt / vol) at room temperature for 30 minutes and then incubated with primary antibodies ((3-actin, LOX) at 4°C overnight. After three washes with TBST, membranes were incubated with secondary antibodies (Li-Cor Biosciences, Lincoln, NE) at a 1:10,000 dilution for 30 minutes at room temperature. The membranes were then washed 3 more times with TBST and scanned using the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE). Band intensities were quantified using the LI-COR Image Studio Software.

[0155] Western blotting of conditioned medium 30 pL of conditioned medium was mixed with 10 pL of 4X SDS-loading buffer and boiled at 95 °C for 5 mins, then used for Western blotting as described above. Whole-cell DNA was extracted using the Proteinase K digestion-based method, and the dsDNA concentration was used for normalization.

[0156] Statistical analyses Analyses of RNA sequencing and genetic data were performed using R language (v4.3.0). Statistical analyses of other data were conducted using GraphPad Prism 10. Most of the data are presented as mean ± standard error of the mean (SEM). The distribution of data was assessed using the Kolmogorov-Smirnov test (for n > 4) or the Shapiro-Wilk test (for n < 4). For normally distributed data, Student’s t-tests were used to compare two groups, one-way analysis of variance (ANOVA) was used to compare multiple groups, and the Sidak method was applied for multiple comparison correction. For non-normally distributed data, Mann- Whitney U tests were used to compare two groups, Kruskal- Wallis tests were employed to compare multiple groups, and the Dunn method was used for multiple comparisons correction. A P-value less than 0.05 was considered statistically significant.Atty. Docket No. UM-43304.601

[0157] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0158] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

Atty. Docket No. UM-43304.601CL IMSWhat is claimed is:

1. A method for treating or preventing abdominal aortic aneurysm (AAA) in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of one or more inhibitors of APOC3, ANGPTL3, ANGPTL8, or a combination thereof, wherein the subject does not have and / or is not receiving treatment for a disease or disorder caused or mediated by high levels of triglycerides and / or wherein the subject is having or had an AAA repair.

2. The method of claim 1, wherein the subject is having or had endovascular repair of an AAA.

3. A method for treating or preventing abdominal aortic aneurysm (AAA) in a subject in need thereof, comprising: identifying an abdominal aortic aneurysm in the subject; and administering to the subject a therapeutically effective amount of one or more inhibitors of AP0C3, ANGPTL3, ANGPTL8, or a combination thereof.

4. The method of claim 3, wherein the identifying comprises an ultrasound, X-ray, computed tomography scan, and / or magnetic resonance imaging (MRI) of the abdomen.

5. The method of any of claims 1-4, wherein the one or more inhibitors of APOC3, ANGPTL3, ANGPTL8 comprise: gene silencing or downregulating oligonucleotides; a protein configured to bind AP0C3, ANGPTL3, and / or ANGPTL8; a small molecule inhibitor; or combinations thereof.

6. The method of claim 5, wherein the one or more inhibitors of AP0C3, ANGPTL3, ANGPTL8 comprise antibodies to one or more of AP0C3, ANGPTL3, and ANGPTL8.

7. The method of claim 5 or 6, wherein the one or more inhibitors of AP0C3, ANGPTL3, ANGPTL8 comprise: antisense oligonucleotides (ASOs); interfering RNAs (siRNAs); guide RNAs; or combinations thereof.

8. The method of any of claims 5-7, wherein: the one or more inhibitors of AP0C3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 1-10 and / or an antisense strand sequence comprising any of SEQ ID NOs: 11-20;Atty. Docket No. UM-43304.601 the one or more inhibitors of ANGPTL3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 21-29 and / or an antisense strand sequence comprising any of SEQ ID NOs: 30-38; and the one or more inhibitors of ANGPTL8 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 39-48 and / or an antisense strand sequence comprising any of SEQ ID NOs: 49-58.

9. The method of claim 7 or 8, wherein the one or more inhibitors of APOC3, ANGPTL3, ANGPTL8 further comprises one or more components of a gene editing system.

10. The method of any of claims 1-9, further comprising administering to the subject a therapeutically effective amount of APOA5, an active fragment or variant thereof, or a nucleic acid encoding APOA5, an active fragment or variant thereof, or an agent for the upregulation of endogenous APOA5.

11. A method for treating or preventing abdominal aortic aneurysm (AAA) in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of APOA5, an active fragment or variant thereof, or a nucleic acid encoding APOA5, an active fragment or variant thereof, or an agent for the upregulation of endogenous APOA5, wherein the subject does not have and / or is not receiving treatment for a disease or disorder caused or mediated by high levels of triglycerides and / or wherein the subject is having or had an AAA repair.

12. The method of claim 11, wherein the subject is having or had endovascular repair of an AAA.

13. A method for treating or preventing abdominal aortic aneurysm (AAA) in a subject in need thereof, comprising: identifying an abdominal aortic aneurysm in the subject; and administering to the subject a therapeutically effective amount of: APOA5, an active fragment or variant thereof, or a nucleic acid encoding APOA5, an active fragment or variant thereof, or an agent for the upregulation of endogenous APOA5.

14. The method of claim 13, wherein the identifying comprises an ultrasound, X-ray, computed tomography (CT / CAT / CTA) scan, and / or magnetic resonance imaging (MRI) of the abdomen.

15. The method of any of claims 11-14, wherein the method comprises administering a site directed transcriptional activator, or system thereof, for endogenous APOA5.Atty. Docket No. UM-43304.60116. The method of any of claims 11-15, further comprising administering to the subject a therapeutically effective amount of one or more inhibitors of AP0C3, ANGPTL3, ANGPTL8, or a combination thereof.

17. The method of claim 16, wherein: the one or more inhibitors of APOC3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 1-10 and / or an antisense strand sequence comprising any of SEQ ID NOs: 11-20; the one or more inhibitors of ANGPTL3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 21-29 and / or an antisense strand sequence comprising any of SEQ ID NOs: 30-38; and the one or more inhibitors of ANGPTL8 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 39-48 and / or an antisense strand sequence comprising any of SEQ ID NOs: 49-58.

18. The method of any of claims 1-17, wherein the method further comprises monitoring the abdominal aortic aneurysm over time.

19. The method of claim 18, wherein the monitoring comprises an ultrasound, X-ray, computed tomography scan, and / or magnetic resonance imaging (MRI) of the abdomen.

20. The method of claim 18 or 19, wherein the method further comprises characterizing the abdominal aortic aneurysm by size and the monitoring comprises determining size change over time.

21. The method of any of claims 1-20, further comprising repairing the abdominal aortic aneurysm.

22. A composition comprising: one or more inhibitors of APOC3, ANGPTL3, and ANGPTL8; APOA5, an active fragment or variant thereof, or a nucleic acid encoding thereof; an agent for the upregulation of endogenous APOA5; or any combination thereof for use in treating an abdominal aortic aneurysm (AAA) in a subject in need thereof.

23. The composition of claim 22, wherein the subject does not have and / or is not receiving treatment for a disease or disorder caused or mediated by high levels of triglycerides and / or wherein the subject is having or had an AAA repair.

24. The composition of claim 23, wherein the subject is having or had endovascular repair of a AAA.

25. The composition of any of claims 22-24, wherein:Atty. Docket No. UM-43304.601 the one or more inhibitors of AP0C3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 1-10 and / or an antisense strand sequence comprising any of SEQ ID NOs: 11-20; the one or more inhibitors of ANGPTL3 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 21-29 and / or an antisense strand sequence comprising any of SEQ ID NOs: 30-38; and the one or more inhibitors of ANGPTL8 comprise antisense oligonucleotides (ASOs) having a sense strand sequence comprising any of SEQ ID NOs: 39-48 and / or an antisense strand sequence comprising any of SEQ ID NOs: 49-58.

Images