WO2020081329A1

WO2020081329A1 - Methods and compositions for modulating pcsk9 and angptl3 expression

Info

Publication number: WO2020081329A1
Application number: PCT/US2019/055452
Authority: WO
Inventors: Alfica Sehgal; Brian Schwartz; Alla SIGOVA; Cynthia Smith; Gavin WHISSELL; Iris Grossman; Yuting Liu; Vaishnavi RAJAGOPAL; Yuchun GUO; David Bumcrot
Original assignee: Camp4 Therapeutics Corporation
Priority date: 2018-10-18
Filing date: 2019-10-09
Publication date: 2020-04-23

Abstract

Provided herein are methods for modulating PCSK9 or ANGPTL3 expression by administering compounds that affect PCSK9 or ANGPTL3 transcription factors or signaling pathways. Also provided are methods for treating subjects with diseases associated with high LDL-cholesterol by administering compounds that modulate PCSK9 or ANGPTL3 expression.

Description

METHODS AND COMPOSITIONS FOR MODULATING PCSK9 AND ANGPTL3 EXPRESSION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application 62/747,629, filed October 18, 2018, U.S. Application 62/789,469, filed January 7, 2019; U.S. Application 62/795,397, filed January 22, 2019; U.S. Application 62/805,516, filed February, 14, 2019; and International Patent Application PCT/US2019/026402, filed April 8, 2019, each of which is hereby incorporated in their entirety by reference.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled CTC_Ol6WO_Sequence_listing.txt, was created on October 9, 2019, and is 116,398 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The invention relates to methods and compositions for modulating PCSK9 expression. Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is associated with elevated cholesterol diseases. Circulating low-density lipoprotein (LDL) particles in the blood are bound by the LDL Receptor on a cell surface, which induces internalization of the LDL-LDLR complex. After releasing the LDL particle in the endosome, the LDLR recycles back to the plasma membrane. PCSK9 binds to the LDL-LDLR complex, inhibits release of the LDL-particle from the LDLR, and blocks recycling of internalized LDLR from the endosome back to the plasma membrane. This results in decreased surface expression of the LDLR and decreased metabolism of LDL- particles. This may result in hypercholesterolemia. Currently available treatments for blocking or decreasing PCSK9 include monoclonal antibodies such as evolocumab, bococizumab, and alirocumab. However, the shortcomings of the present therapeutics include the inability of patients to tolerate the treatments. Therefore, additional therapies are needed to modulate the expression of PCSK9. The present invention addresses these and other shortcomings.

[0004] Nonalcoholic fatty liver disease (NAFLD) is one of the most common hepatic disorders worldwide. In the United States, it affects an estimated 80 to 100 million people. NAFLD occurs in every age group but especially in people in their 40s and 50s. NAFLD is a buildup of excessive fat in the liver that can lead to liver damage resembling the damage caused by alcohol abuse, but this occurs in people who drink little to no alcohol. The condition is also associated with adverse metabolic consequences, including increased abdominal fat, poor ability to use the hormone insulin, high blood pressure and high blood levels of triglycerides.

[0005] In some cases, NAFLD leads to inflammation of the liver, referred to as non-alcoholic steatohepatitis (NASH). NASH is a progressive liver disease characterized by fat accumulation in the liver leading to liver fibrosis. About 20 percent of people with NASH will progress to fibrosis. NASH affects approximately 26 million people in the United States. With continued inflammation, fibrosis spreads to take up more and more liver tissue, leading to liver cancer and/or end-stage liver failure in most severe cases. NASH is highly correlated to obesity, diabetes and related metabolic disorders. Genetic and environmental factors also contribute to the development of NASH.

[0006] Currently, no drug treatment exists for NAFLD or NASH. The condition is primarily managed in early stages through lifestyle modification (e.g., physical exercise, weight loss, and healthy diet) which may encounter poor adherence. Losing weight addresses the conditions that contribute to nonalcoholic fatty liver disease. Weight-loss surgery is also an option for those who need to lose a great deal of weight. Anti-diabetic medication, vitamins or dietary supplements can be useful for controlling the condition. For those who have cirrhosis due to NASH, liver transplantation may be an option. This is the 3^rd most common reason for liver transplants in the US and is projected to become most common reason in three years.

[0007] Alcoholic liver disease (ALD) accounts for the majority of chronic liver diseases in Western countries. It encompasses a spectrum of liver manifestations of alcohol

overconsumption, including fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. Alcoholic liver cirrhosis is the most advanced form of ALD and is one of the major causes of liver failure, hepatocellular carcinoma and liver-related mortality causes. Restricting alcohol intake is the primary treatment for ALD. Other treatment options include supportive care (e.g., healthy diet, vitamin supplements), use of corticosteroids, and sometimes liver transplantation.

[0008] Therefore, there is a need for developing effective therapeutics for the treatment of NAFLD, NASH and/or ALD.

SUMMARY

[0009] In one aspect, provided herein are methods for modulating PCSK9 expression in a cell, comprising: contacting the cell with a compound that modulates a first target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, thereby modulating PCSK9 expression. [0010] In one aspect, provided herein are methods for modulating ANGPTL3 expression in a cell, comprising: contacting the cell with a compound that modulates a second target selected from the group consisting of mTOR, Transforming Growth Factor b receptor (TGF R) I, TGF receptor II, SMAD2, SMAD3, STAT1, NF-kB, BRIM, p53, and TCF7L2 thereby modulating ANGPTL3 expression. In some embodiments the contacting is done in vivo or ex vivo.

[0011] In some embodiments, the cell has a PNPLA3 mutation. In some embodiments, the PNPLA3 mutation is a gain of function mutation. In some embodiments, the mutation is the presence of a G allele at SNP rs738409. In some embodiments, the cell is homozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the cell is heterozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the mutation is an I148M mutation in the PNPLA3 protein. In some embodiments, the cell is homozygous for the mutant PNPLA3 protein carrying the I148M mutation. In some embodiments, the cell is heterozygous for the mutant PNPLA3 protein carrying the I148M mutation.

[0012] In some embodiments, the presence of the PNPLA3 mutation in the cell is determined using a method selected from the group consisting of a mass spectroscopy assay, an

oligonucleotide microarray analysis, an allele-specific hybridization assay, an allele-specific PCR assay, and a nucleic acid sequencing assay.

[0013] In some embodiments, the cell is a hepatocyte.

[0014] In some embodiments, the compound is a compound selected from Table 2 or Table 7.

[0015] In some embodiments, modulating PCSK9 or ANGPTL3 expression reduces PCSK9 or ANGPTL3 expression.

[0016] In some embodiments, the target is mTOR and the compound is selected from the group consisting of OSI-027, PF-04691502, WYE-125132, CC-223, Everolimus, Palomid 529 (P529), GDC-0349, Torin 1, PP242, WAY600, CZ415, INK128, TAK659, AZD-8055, Deforolimus, and JR-AB2-011. In some embodiments, the mTOR inhibitor is OSI-027. In some embodiments, the mTOR inhibitor inhibits mTORC2. In some embodiments, the mTORC2 inhibitor inhibits RICTOR. In some embodiments, the mTORC2 inhibitor is JR-AB2-011.

[0017] In some embodiments, the compound comprises a small interfering RNA (siRNA) directed against the first or the second target. In some embodiments, the siRNA targets a gene selected from the group consisting of RICTOR, mTOR, Deptor, AKT, mLST8, mSINl, and Protor.

[0018] In some embodiments, the first and/or second target is TGF RI, TGF RII, SMAD2, or SMAD3 and the compound is selected from the group consisting of LY2157299, LY-364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R-268712, SB-431542, EW-7197, LDN-212854, Halofuginone, ITD-l, LDN-214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83-01 sodium salt, A 83-01, LDN193189

(Hydrochloride), Oxymatrine, Kartogenin, SRI-011381 (hydrochloride), Halofuginone, SIS3, LY2157299.

[0019] In some embodiments, the first and/or second target is TGF RI, and the compound is LY2157299.

[0020] In some embodiments, the first and/or second target is NF-kB, and the compound is selected from the group consisting of SC75741, BAY 11-7082, JSH-23, and Neferine.

[0021] In some embodiments, the first and/or second target is BRIM, and the compound is selected from the group consisting of FL-411, ZL0420, ZEN-3411, and PLX51107.

[0022] In some embodiments, the first and/or second target is TP53, and the compound is selected from the group consisting of PK11007, Serdemetan, RITA, JNJ-26854165, and MI-773.

[0023] In some embodiments, the first and/or second target is TCF7L2, and the compound is selected from the group consisting of LY2090314, A 1070722, and AZD2858.

[0024] In some embodiments, the first and/or second target is STAT1 or STAT3 and the compound is selected from the group consisting of AG 18, Stattic, Alantolactone, Napabucasin, OPB-31121, OPB-51602, STAT3 inhibitor XIII, danvatirsen, WP1066, Chrysophanol, SMI-l6a, RG13022, TCS-PIM-l-4a, RG14620, Nifuroxazide, Dihydroisotanshinone I, STAT5-IN-1, Hispidulin, Tyrphostin AG 528, AG-1478, Tyrphostin AG 879, AG 555, Niclosamide,

PD158780, Piml/AKKl-IN-l, PD153035, NSC 74859, TCS PIM-l 1, AZD1208, CL-387785, EAI045, Artesunate, BIBX 1382, Icotinib, PD153035 (Hydrochloride), AS1517499, HJC0152 hydrochloride, Diosgenin, Fedratinib (SAR302503, TG101348), TP-3654, Morusin, Icotinib (Hydrochloride), PF-06459988, AEE788, AZD3759, CX-6258, Scutellarin, HO-3867, Pelitinib, Mubritinib, CP-724714, Dacomitinib, Cl 88-9, Sapitinib, Irbinitinib, Gefitinib (hydrochloride), AZ-5104, Olmutinib, Poziotinib, WZ4002, AZD-9291, CNX-2006, TAK-285, Lazertinib, CO- 1686, Neratinib, Canertinib (dihydrochloride), BMS-599626 (Hydrochloride), AZD-9291 (mesylate), Avitinib (maleate), SH-4-54, BP-1-102, CO-1686 (hydrobromide), Saikosaponin D.

[0025] In some embodiments, the first and/or second target is Myc and the compound is selected from the group consisting of Myc -targeting siRNA DCR-MYC and AVI -4126.

[0026] In some embodiments, the first and/or second first and/or second target is NR3C1 and the compound is selected from the group consisting of rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate, fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate,

1 lbeta hydrocortisone acetate, betamethasone, dexamethasone, budesonide, fluticasone propionate, beclomethasone dipropionate, betamethasone acetate/betamethasone phosphate, betamethasone acetate, triamcinolone acetonide, ciprofloxacin/hydrocortisone,

ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone

dipropionate/calcipotriene, fluticasone furoate, budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate, beclomethasone 17-monopropionate,

dexamethasone/lenalidomide/sorafenib, docetaxel/prednisone, carmustine/prednisone, cabazitaxel/prednisone, dexamethasone/lenalidomide, hydrocortisone/prednisone,

dexamethasone/thalidomide, cyclophosphamide/prednisone/vincristine,

hydrocortisone/mitoxantrone, mitoxantrone/prednisone, docetaxel/hydrocortisone,

cytarabine/dexamethasone, dexamethasone/pomalidomide, bortezomib/dexamethasone, cyclophosphamide/dexamethasone/thalidomide, bortezomib/dexamethasone/doxorubicin, bortezomib/dexamethasone/lenalidomide, bortezomib/dexamethasone/thalidomide, carfilzomib/dexamethasone/lenalidomide,

cyclophosphamide/daunorubicin/imatinib/prednisone/vincristine, bortezomib/prednisone, cyclophosphamide/dexamethasone/rituximab, cyclophosphamide/prednisone/rituximab, prednisone/thalidomide, octreotide/prednisone, bortezomib/dexamethasone/rituximab, L- asparaginase/prednisone/vincristine, cyclophosphamide/prednisone, dexamethasone/fludarabine phosphate/mitoxantrone, cyclophosphamide/etoposide/prednisone/rituximab/vincristine, cytarabine/dexamethasone/methotrexate,

cyclophosphamide/mitoxantrone/prednisone/vincristine,

cyclophosphamide/etoposide/prednisone/vincristine, dexamethasone/fludarabine

phosphate/mitoxantrone/rituximab, chlorambucil/mitoxantrone/prednisone,

cyclophosphamide/prednisone/rituximab/vincristine, methylprednisolone/rituximab, cyclophosphamide/mitoxantrone/prednisone/rituximab/vincristine,

chlorambucil/mitoxantrone/prednisone/rituximab, clocortolone, alclometasone,

prednisone/somatotropin, carfilzomib/dexamethasone/rituximab,

cyclophosphamide/prednisolone/vincristine,

cyclophosphamide/prednisolone/rituximab/vincristine,

cytarabine/dexamethasone/oxaliplatin/rituximab, everolimus/prednisone, cyclophosphamide/gemcitabine/prednisolone/rituximab/vincristine,

cyclophosphamide/epirubicin/prednisone/vincristine, dexamethasone/enzalutamide, abiraterone/prednisolone, dexamethasone/palonosetron, docetaxel/prednisolone,

cabazitaxel/prednisolone, prednisone/vincristine, carfdzomib/dexamethasone,

ciprofloxacin/fluocinolone acetonide, dexamethasone/vincristine,

glycopyrrolate/indacaterol/mometasone furoate, indacaterol/mometasone furoate,

fluticasone/salmeterol, betamethasone/clotrimazole, aprepitant/dexamethasone,

dexamethasone/netupitant, dexamethasone/olanzapine, aprepitant/dexamethasone/granisetron, aprepitant/dexamethasone/ondansetron, aprepitant/dexamethasone/palonosetron,

bortezomib/dexamethasone/pomalidomide, dexamethasone/imatinib/vincristine,

dexamethasone/imatinib, dexamethasone/rituximab/verapamil, dexamethasone/rituximab, dasatinib/dexamethasone, imatinib/prednisone, prednisone/rituximab, prednisolone/rituximab, 6- mercaptopurine/prednisone, 6-mercaptopurine/prednisone/thioguanine, relacorilant, miconazole, dexamethasone/granisetron, dexamethasone/ondansetron,

cyclophosphamide/epirubicin/prednisone/rituximab/vincristine, prednisolone acetate, crizotinib/prednisolone, fluticasone furoate/umeclidinium/vilanterol,

acetaminophen/diphenhydramine/prednisolone,

acetaminophen/diphenhydramine/methylprednisolone,

cimetidine/dexamethasone/diphenhydramine, dexamethasone/diphenhydramine/ranitidine, acetaminophen/cetirizine/prednisolone, cyclophosphamide/daunorubicin/prednisone, daunorubicin/etoposide/6-mercaptopurine/mitoxantrone/prednisolone/vindesine, L- asparaginase/daunorubicin/prednisone/vincristine, CORT125281,

cytarabine/dasatinib/dexamethasone/methotrexate,

cytarabine/dexamethasone/imatinib/methotrexate,

cyclophosphamide/daunorubicin/imatinib/prednisolone/vincristine,

methylprednisolone/mycophenolate mofetil, mycophenolate mofetil/prednisone,

infliximab/methylprednisolone, prednisone/tacrolimus, infliximab/prednisone,

cyclophosphamide/methylprednisolone, methylprednisolone/tacrolimus, methylprednisolone acetate, mometasone furoate, amcinonide, methylprednisolone succinate, betamethasone phosphate, fluocinonide, prednicarbate, hydrocortisone cypionate, hydrocortisone succinate, prednisolone phosphate, betamethasone valerate, betamethasone benzoate, fludrocortisone acetate, prednisolone tebutate, betamethasone dipropionate, hydrocortisone buteprate, alclometasone dipropionate, hydrocortisone butyrate, fluorometholone acetate, hydrocortisone valerate, loteprednol etabonate, hydrocortisone phosphate, methylprednisolone, halobetasol propionate, fhmisolide, and mifepristone.

[0027] In some embodiments, the first and/or second target is VDR and the compound is selected from the group consisting of calcipotriene, ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3 -hydroxypropoxy)calcitriol, betamethasone

dipropionate/calcipotriene, alfacalcidol, calcium carbonate/cholecalciferol, paricalcitol, doxercalciferol, cholecalciferol, calcitriol, calcifediol, and seocalcitol.

[0028] In some embodiments, the first and/or second target is ESR1 and the compound is selected from the group consisting of l7-alpha-ethinylestradiol, fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF- 4227, bazedoxifene, estradiol valerate/testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol/ethynodiol diacetate, estradiol acetate, esterified estrogens, estradiol cypionate/medroxyprogesterone acetate, estradiol/norethindrone acetate, estradiol

cypionate/testosterone cypionate, synthetic conjugated estrogens, B, etonogestrel, CC8490, MITO-4509, cyproterone acetate/ethinyl estradiol, ethinyl estradiol/etonogestrel, pipendoxifene, chlorotrianisene, icaritin, megestrol acetate/tamoxifen, sulindac/tamoxifen, sulindac/toremifene, raloxifene/sulindac, F18 l6-alpha-fluoroestradiol, ARN-810, Z-endoxifen, goserelin/tamoxifen, raloxifene/teriparatide, AZD9496, elacestrant, SRN-927, fulvestrant/palbociclib,

anastrozole/tamoxifen, fulvestrant/letrozole/tamoxifen, anastrozole/exemestane/fulvestrant, anastrozole/goserelin/tamoxifen, anastrozole/fulvestrant/tamoxifen, exemestane/fulvestrant, fulvestrant/letrozole, letrozole/tamoxifen, exemestane/tamoxifen,

exemestane/fulvestrant/letrozole/tamoxifen, anastrozole/exemestane/fulvestrant/tamoxifen, anastrozole/fulvestrant/goserelin/tamoxifen, exemestane/fulvestrant/tamoxifen,

exemestane/fulvestrant/goserelin/letrozole/tamoxifen, anastrozole/fulvestrant,

fulvestrant/pertuzumab/trastuzumab, pertuzumab/tamoxifen/trastuzumab, FSZ102, selective estrogen receptor modulator, desogestrel/ethinyl estradiol, drospirenone/ethinyl estradiol, ethinyl estradiol/norelgestromin, ethinyl estradiol/norethindrone, ethinyl estradiol/levonorgestrel, ethinyl estradiol/norgestrel, ethinyl estradiol/norgestimate, diethylstilbestrol, ospemifene, toremifene, tamoxifen, raloxifene, everolimus/tamoxifen, H3B-6545, arzoxifene, clomiphene, SAR439859, estramustine phosphate, diethylstilbestrol diphosphate, estradiol/levonorgestrel, tamoxifen/trastuzumab, fulvestrant/trastuzumab, everolimus/fulvestrant, TTC-352,

fulvestrant/ribociclib, 4-hydroxytamoxifen, dienestrol, acolbifene, estramustine, medroxyprogesterone acetate, desogestrel, danazol, trilostane, fluoxymesterone, norgestimate, progesterone, and S-equol.

[0029] In some embodiments, the modulation of the first and/or second target alters binding of the target to a PCSK9 or ANGPTL3 enhancer region.

[0030] In some embodiments, the alteration of binding reflects binding of the compound to the first and/or second target or binding of the compound to the enhancer and the alteration is selected from group consisting of an alteration in phosphorylation of the first and/or second target, an alteration in localization of the first and/or second target, an alteration in the expression level of the first and/or second target, an alteration in methylation of the first and/or second target, an alteration in acetylation of the first and/or second target, an alteration in ubiquitination of the first and/or second target, an alteration in glycosylation of the first and/or second target, an alteration in sumoylation of the first and/or second target, an alteration in stability of the first and/or second target, and an alteration in degradation of the first and/or second target.

[0031] In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced by at least about 30%, 50% or 70%. In some embodiments, the reduction is determined in a population of cells and the amount of reduction is determined by reference to a matched control cell population.

[0032] In one aspect, provided herein are methods for treating a disease comprising:

administering to a mammalian subject an effective amount of a compound that modulates a first target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, wherein said modulating of the target reduces PCSK9 expression and thereby treats the disease.

[0033] In one aspect, provided herein are methods for treating a disease comprising:

administering to a mammalian subject an effective amount of a compound that modulates a second target selected from the group consisting of mTOR, Transforming Growth Factor b receptor (TGF R) I, TGFp receptor II, SMAD2, SMAD3, STAT1, NF-kB, BRIM, p53, and TCF7F2, wherein said modulating of the target reduces ANGPTF3 expression and thereby treats the disease.

[0034] In some embodiments, the disease is a liver disease or a disease associated with a blood or serum ratio of high density lipoprotein (HDF)-cholesterol/ low density lipoprotein (FDF)- cholesterol of < 0.3, optionally wherein the disease is selected from the group consisting of: non- alcoholic faty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), and a high LDL-cholesterol associated disease.

[0035] In some embodiments, the high LDL-cholesterol associated disease occurs in a subject having a PCSK9-activating (GOF) mutation, a marked elevation of low density lipoprotein particles in the plasma, primary hypercholesterolemia, or heterozygous Familial

Hypercholesterolemia (heFH).

[0036] In some embodiments, the subject has a PNPLA3 mutation. In some embodiments, the PNPLA3 mutation is a gain of function mutation. In some embodiments, the mutation is the presence of a G allele at SNP rs738409. In some embodiments, a cell from the subject is homozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the cell is heterozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the mutation is an I148M mutation in the PNPLA3 protein. In some embodiments, the cell is homozygous for the mutant PNPLA3 protein carrying the I148M mutation. In some embodiments, the cell is heterozygous for the mutant PNPLA3 protein carrying the I148M mutation.

[0037] In some embodiments, the presence of the PNPLA3 mutation in the cell is determined using a method selected from the group consisting of a mass spectroscopy assay, an

[0038] In some embodiments, the subject is human.

[0039] In some embodiments, the compound is a compound selected from Table 2 or Table 7.

[0040] In some embodiments, the target is mTOR and the compound is selected from the group consisting of OSI-027, PF-04691502, WYE-125132, CC-223, Everolimus, Palomid 529 (P529), GDC-0349, Torin 1, PP242, WAY600, CZ415, INK128, TAK659, AZD-8055, Deforolimus, and JR-AB2-011. In some embodiments, the mTOR inhibitor is OSI-027. In some embodiments, the mTOR inhibitor inhibits mTORC2. In some embodiments, the mTORC2 inhibitor inhibits RICTOR. In some embodiments, the mTORC2 inhibitor is JR-AB2-011.

[0041] In some embodiments, the compound comprises a small interfering RNA (siRNA) directed against the first or the second target.

[0042] In some embodiments, the siRNA targets one or more genes selected from the group consisting of RICTOR, mTOR, Deptor, AKT, mLST8, mSINl, and Protor.

[0043] In some embodiments, the administration of the compound capable of modulating the expression of the PCSK9 or ANGPTL3 gene does not induce hyperinsulinemia in the subject. [0044] In some embodiments, the administration of the compound capable of modulating the expression of the PCSK9 or ANGPTL3 gene does not induce hyperglycemia in the subject.

[0045] In some embodiments, the first and/or second target is TGF RI, TGF RII, SMAD2, or SMAD3 and the compound is selected from the group consisting of LY2157299, LY-364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R-268712, SB-431542, EW-7197, LDN-212854, Halofuginone, ITD-l, LDN-214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83-01 sodium salt, A 83-01, LDN193189

[0046] In some embodiments, the first and/or second target is TGF RI, and the compound is LY2157299.

[0047] In some embodiments, the first and/or second target is NF-kB, and the compound is selected from the group consisting of SC75741, BAY 11-7082, JSH-23, and Neferine.

[0048] In some embodiments, the first and/or second target is BRIM, and the compound is selected from the group consisting of FL-411 , ZL0420, ZEN-3411 , and PLX51107

[0049] In some embodiments, the first and/or second target is TP53, and the compound is selected from the group consisting of PK11007, Serdemetan, RITA, JNJ-26854165, and MI-773.

[0050] In some embodiments, the first and/or second target is TCF7L2, and the compound is selected from the group consisting of LY2090314, A 1070722, and AZD2858

[0051] In some embodiments, the first and/or second target is STAT1 or STAT3 and the compound is selected from the group consisting of AG 18, Stattic, Alantolactone, Napabucasin, OPB-31121, OPB-51602, STAT3 inhibitor XIII, danvatirsen, WP1066, Chrysophanol, SMI-l6a, RG13022, TCS-PIM-l-4a, RG14620, Nifuroxazide, Dihydroisotanshinone I, STAT5-IN-1, Hispidulin, Tyrphostin AG 528, AG-1478, Tyrphostin AG 879, AG 555, Niclosamide,

PD158780, Piml/AKKl-IN-l, PD153035, NSC 74859, TCS PIM-l 1, AZD1208, CL-387785, EAI045, Artesunate, BIBX 1382, Icotinib, PD153035 (Hydrochloride), AS1517499, HJC0152 hydrochloride, Diosgenin, Fedratinib (SAR302503, TG101348), TP-3654, Morusin, Icotinib (Hydrochloride), PF-06459988, AEE788, AZD3759, CX-6258, Scutellarin, HO-3867, Pelitinib, Mubritinib, CP-724714, Dacomitinib, Cl 88-9, Sapitinib, Irbinitinib, Gefitinib (hydrochloride), AZ-5104, Olmutinib, Poziotinib, WZ4002, AZD-9291, CNX-2006, TAK-285, Lazertinib, CO- 1686, Neratinib, Canertinib (dihydrochloride), BMS-599626 (Hydrochloride), AZD-9291 (mesylate), Avitinib (maleate), SH-4-54, BP-1-102, CO-1686 (hydrobromide), Saikosaponin D. [0052] In some embodiments, the first and/or second target is MY C and the compound is selected from the group consisting of MYC-targeting siR A DCR-MYC and AVI-4126.

[0053] In some embodiments, the first and/or second target is NR3C1 and the compound is selected from the group consisting of rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate, fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate, l lbeta hydrocortisone acetate, betamethasone, dexamethasone, budesonide, fluticasone propionate, beclomethasone dipropionate, betamethasone acetate/betamethasone phosphate, betamethasone acetate, triamcinolone acetonide, ciprofloxacin/hydrocortisone, ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone dipropionate/calcipotriene, fluticasone furoate, budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate,

beclomethasone 17 -monopropionate, dexamethasone/lenalidomide/sorafenib,

docetaxel/prednisone, carmustine/prednisone, cabazitaxel/prednisone,

dexamethasone/lenalidomide, hydrocortisone/prednisone, dexamethasone/thalidomide, cyclophosphamide/prednisone/vincristine, hydrocortisone/mitoxantrone,

mitoxantrone/prednisone, docetaxel/hydrocortisone, cytarabine/dexamethasone,

dexamethasone/pomalidomide, bortezomib/dexamethasone,

cyclophosphamide/dexamethasone/thalidomide, bortezomib/dexamethasone/doxorubicin, bortezomib/dexamethasone/lenalidomide, bortezomib/dexamethasone/thalidomide, carfilzomib/dexamethasone/lenalidomide,

cyclophosphamide/mitoxantrone/prednisone/vincristine,

cyclophosphamide/etoposide/prednisone/vincristine, dexamethasone/fludarabine

phosphate/mitoxantrone/rituximab, chlorambucil/mitoxantrone/prednisone,

cyclophosphamide/prednisone/rituximab/vincristine, methylprednisolone/rituximab, cyclophosphamide/mitoxantrone/prednisone/rituximab/vincristine, chlorambucil/mitoxantrone/prednisone/rituximab, clocortolone, alclometasone,

prednisone/somatotropin, carfilzomib/dexamethasone/rituximab,

cyclophosphamide/prednisolone/vincristine,

cyclophosphamide/prednisolone/rituximab/vincristine,

cytarabine/dexamethasone/oxaliplatin/rituximab, everolimus/prednisone,

cyclophosphamide/gemcitabine/prednisolone/rituximab/vincristine,

cabazitaxel/prednisolone, prednisone/vincristine, carfilzomib/dexamethasone,

ciprofloxacin/fluocinolone acetonide, dexamethasone/vincristine,

glycopyrrolate/indacaterol/mometasone furoate, indacaterol/mometasone furoate,

fluticasone/salmeterol, betamethasone/clotrimazole, aprepitant/dexamethasone,

bortezomib/dexamethasone/pomalidomide, dexamethasone/imatinib/vincristine,

acetaminophen/diphenhydramine/prednisolone,

acetaminophen/diphenhydramine/methylprednisolone,

cytarabine/dasatinib/dexamethasone/methotrexate,

cytarabine/dexamethasone/imatinib/methotrexate,

cyclophosphamide/daunorubicin/imatinib/prednisolone/vincristine,

methylprednisolone/mycophenolate mofetil, mycophenolate mofetil/prednisone,

infliximab/methylprednisolone, prednisone/tacrolimus, infliximab/prednisone,

cyclophosphamide/methylprednisolone, methylprednisolone/tacrolimus, methylprednisolone acetate, mometasone furoate, amcinonide, methylprednisolone succinate, betamethasone phosphate, fluocinonide, prednicarbate, hydrocortisone cypionate, hydrocortisone succinate, prednisolone phosphate, betamethasone valerate, betamethasone benzoate, fludrocortisone acetate, prednisolone tebutate, betamethasone dipropionate, hydrocortisone buteprate, alclometasone dipropionate, hydrocortisone butyrate, fluorometholone acetate, hydrocortisone valerate, loteprednol etabonate, hydrocortisone phosphate, methylprednisolone, halobetasol propionate, flunisolide, and mifepristone.

[0054] In some embodiments, the first and/or second target is VDR and the compound is selected from the group consisting of calcipotriene, ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3 -hydroxypropoxy)calcitriol, betamethasone

[0055] In some embodiments, the first and/or second target is ESR1 and the compound is selected from the group consisting of l7-alpha-ethinylestradiol, fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF- 4227, bazedoxifene, estradiol valerate/testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol/ethynodiol diacetate, estradiol acetate, esterified estrogens, estradiol cypionate/medroxyprogesterone acetate, estradiol/norethindrone acetate, estradiol

exemestane/fulvestrant/goserelin/letrozole/tamoxifen, anastrozole/fulvestrant,

fulvestrant/ribociclib, 4-hydroxytamoxifen, dienestrol, acolbifene, estramustine,

medroxyprogesterone acetate, desogestrel, danazol, trilostane, fluoxymesterone, norgestimate, progesterone, and S-equol.

[0056] In some embodiments, modulating the first and/or second target alters binding of the target to a PCSK9 or ANGPTL3 enhancer region.

[0057] In some embodiments, the alteration of binding reflects binding of the compound to the first and/or second target or binding of the compound to the enhancer and the alteration is selected from group consisting of an alteration in phosphorylation of the first and/or second target, an alteration in localization of the first and/or second target, an alteration in the expression level of the first and/or second target, an alteration in methylation of the first and/or second target, an alteration in acetylation of the first and/or second target, an alteration in ubiquitination of the first and/or second target, an alteration in glycosylation of the first and/or second target, an alteration in sumoylation of the first and/or second target, an alteration in stability of the first and/or second target, and an alteration in degradation of the first and/or second target.

[0058] In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced in the liver of the subject. In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatocytes of the subject. In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatic stellate cells of the subject. In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatocytes and hepatic stellate cells of the subject.

[0059] In some embodiments, the method further comprises assessing or having assessed a hepatic triglyceride content in the subject. In some embodiments, the assessing or having assessed step comprises using a method selected from the group consisting of liver biopsy, liver ultrasonography, computer-aided tomography (CAT) and nuclear magnetic resonance (NMR).

In some embodiments, the assessing or having assessed step comprises proton magnetic resonance spectroscopy (¹H-MRS). In some embodiments, the subject is eligible for treatment based on a hepatic triglyceride content greater than 5.5% volume/volume.

[0060] In some embodiments, the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a matched control population. In some embodiments, the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a pre-treatment baseline measurement.

[0061] In one aspect, provided herein are methods for identifying a compound that reduces PCSK9 or ANGPTL3 gene expression comprising providing a candidate compound; assaying the candidate compound for at least two of activities selected from the group consisting of: mTOR inhibitory activity, mTORC2 inhibitory activity, PI3K inhibitory activity, RI3Kb inhibitory activity, DNA-PK inhibitory activity, ability to induce hyperinsulinemia, ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity; and identifying the candidate compound as the compound based on results of the two or more assays that indicate the candidate compound has two or more desirable properties.

[0062] In some embodiments, the desirable properties are selected from the group consisting of: mTOR inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of ability to induce hyperinsulinemia, lack of ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In some embodiments, the mTOR inhibitory activity comprises inhibition of mTORC2 activity. In some embodiments, the mTOR inhibitory activity is mTORCl and mTORC2 inhibitory activity. In some embodiments, the PI3K inhibitory activity is RI3Kb inhibitory activity. In some embodiments, the assaying step comprises assaying for at least three of the activities. In some embodiments, the assaying step comprises assaying for at least four of the activities. In some embodiments, the assaying step comprises assaying for at least five of the activities.

[0063] In some embodiments, the at least two assays of step (b) comprise assays for mTOR inhibitory activity and PI3K inhibitory activity. In some embodiments, the at least two assays of step (b) comprise assays for mTORC2 inhibitory activity and RI3Kb inhibitory activity. In some embodiments, the at least three assays of step (b) comprise assays for mTOR inhibitory activity, PI3K inhibitory activity, and ability to induce hyperinsulinemia. In some embodiments, the at least four assays of step (b) comprise mTOR inhibitory activity, PI3K inhibitory activity, ability to induce hyperinsulinemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity.

[0064] In some embodiments, the assay is a biochemical assay. In some embodiments, the assay is a cellular assay. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a wild type cell.

[0065] In some embodiments, the cell comprises the G allele at SNP rs738409 of a PNPLA3 gene or a mutant 1148M PNPLA3 protein. In some embodiments, the cell is homozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the cell is heterozygous for the PNPLA3 G allele at SNP rs738409. In some embodiments, the cell is homozygous for the mutant PNPLA3 protein carrying the I148M mutation. In some embodiments, the cell is heterozygous for the mutant PNPLA3 protein carrying the I148M mutation.

[0066] In some embodiments, assaying the PCSK9 or ANGPTL3 gene expression comprises a method selected from the group consisting of: mass spectroscopy, oligonucleotide microarray analysis, allele-specific hybridization, allele-specific PCR, and nucleic acid sequencing.

[0067] In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is reduced by at least about 30%, 50% or 70%. In some embodiments, the reduction is determined in a population of cells and the amount of reduction is determined by reference to a matched control cell population.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0068] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0069] FIG. 1 shows gene circuitry mapping of the PCSK9 gene. The top panel shows the HiChIP chromatin mapping, the bottom panel shows a comparison of the HiChIP, ChIP-seq, ATAC-seq, and RNA-seq mapping of the PCSK9 gene.

[0070] FIG. 2 shows a diagram of the known and newly identified PCSK9 transcription factors.

[0071] FIG. 3 shows the relative PCSK9/house keeper mRNA levels in primary human hepatocytes after treatment with the indicated compounds.

[0072] FIG. 4 shows the relative PCSK9/house keeper mRNA in mouse hepatocytes after treatment with the indicated compounds.

[0073] FIG. 5 shows the relative PCSK9/house keeper mRNA levels in mice after treatment with the indicated compound.

[0074] FIG. 6 shows the relative PCSK9/house keeper mRNA levels in mice after treatment with the indicated compounds.

[0075] FIG. 7 shows the relative PCSK9/house keeper mRNA levels in mice after treatment with the indicated compound.

[0076] FIG. 8A shows the effect of O SI-027, PF-04691502, and LY2157299 on PCSK9 liver mRNA levels in vivo at 6 hrs post dosing. FIG. 8B shows the effect of OSI-027 on PCSK9 liver mRNA levels in vivo at 6 hrs post dosing in individual mice. FIG. 8C shows the effect of PF- 04691502 on PCSK9 liver mRNA levels in vivo at 6 hrs post dosing in individual mice. FIG. 8D shows the effect of LY2157299 on PCSK9 liver mRNA levels in vivo at 6 hrs post dosing in individual mice. FIG. 8E shows the effect of OSI-027 on ANGPTL3 liver mRNA levels in vivo at 6 hrs post dosing in individual mice. FIG. 8F shows the effect of PF-04691502 on ANGPTL3 liver mRNA levels in vivo at 6 hrs post dosing in individual mice. FIG. 8G shows the effect of LY2157299 on ANGPTL3 liver mRNA levels in vivo at 6 hrs post dosing in individual mice.

[0077] FIG. 9A shows the effect of OSI-027 on PCSK9 liver mRNA levels in vivo at 4 hrs post dosing in individual mice. FIG. 9B shows the effect of PF-04691502 on PCSK9 liver mRNA levels in vivo at 4 hrs post dosing in individual mice. FIG. 9C shows the effect of CH5132799 and VS-5584 on PCSK9 liver mRNA levels in vivo at 4 hrs post dosing in individual mice. FIG. 9D shows the effect of OSI-027 on ANGPLT3 liver mRNA levels in vivo at 4 hrs post dosing in individual mice. FIG. 9E shows the effect of PF-04691502 on ANGPTL3 liver mRNA levels in vivo at 4 hrs post dosing in individual mice.

[0078] FIG. 10 shows the relative PCSK9 mRNA expressed in hepatocytes after treatment with 3 mM of the indicated compound.

[0079] FIG. 11 shows the relative PCSK9 mRNA expressed in hepatocytes after treatment with 1 mM of the indicated compound.

[0080] FIG. 12 shows the relative PCSK9 mRNA expressed in hepatocytes after treatment with 0.3 pM of the indicated compound.

[0081] FIG. 13 shows the relative PCSK9 mRNA expressed in hepatocytes after treatment with 0.1 pM of the indicated compound.

[0082] FIG. 14 shows the relative PCSK9 mRNA after treatment with each indicated compound in a time course.

[0083] FIG. 15 shows the relative PCSK9 mRNA levels in PNPLA3 homozygous I148M primary human hepatocytes after treatment with the indicated compounds.

[0084] FIG. 16 shows the relative PCSK9 mRNA levels after treatment with OSI-027 or PF- 04691502 in the left (L), medial (M), or right (R) kidney sections in mice engrafted with human hepatocytes homozygous for mutant PNPLA3 I148M protein.

[0085] FIG. 17A shows the serum glucose levels in mice after OSI-027 or PF-04691502 treatment. FIG. 17B shows the serum insulin levels in mice after OSI-027 or PF-04691502 treatment.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0086] Terms used in the claims and specification are defined as set forth below unless otherwise specified. [0087] The term“analog,” as used herein, refers to a compound that is structurally related to the reference compound and shares a common functional activity with the reference compound.

[0088] The term“biologic,” as used herein, refers to a medical product made from a variety of natural sources such as micro-organism, plant, animal, or human cells.

[0089] The term“boundary,” as used herein, refers to a point, limit, or range indicating where a feature, element, or property ends or begins.

[0090] The terms“chromatin” and“chromosome” are used interchangeably herein to refer to a complex of genomic DNA and proteins that bind the genomic DNA.

[0091] The term“compound,” as used herein, refers to a single agent or a pharmaceutically acceptable salt thereof, or a bioactive agent or drug.

[0092] The term“derivative,” as used herein, refers to a compound that differs in structure from the reference compound, but retains the essential properties of the reference molecule.

[0093] The term“downstream neighborhood gene,” as used herein, refers to a gene downstream of primary neighborhood gene that may be located within the same insulated neighborhood as the primary neighborhood gene.

[0094] The term“drug,” as used herein, refers to a substance or compound other than food intended for use in the diagnosis, cure, alleviation, or treatment of disease and intended to affect the structure or any function of the body.

[0095] The term“enhancer,” as used herein, refers to a regulatory DNA segment and associated proteins that, when bound by one or more transcription factors, enhances or suppresses the transcription of an associated gene.

[0096] The term“genomic signaling center,” i.e., a“signaling center,” as used herein, refers to regions within insulated neighborhoods that include regions capable of binding context-specific combinatorial assemblies of signaling molecule s/signaling proteins that participate in the regulation of the genes within that insulated neighborhood or among more than one insulated neighborhood.

[0097] The term“genomic system architecture,” as used herein, refers to the organization of an individual’s genome and includes chromosomes, topologically associating domains (TADs), and insulated neighborhoods.

[0098] The term“herbal preparation,” as used herein, refers to herbal medicines that contain parts of plants, or other plant materials, or combinations as active ingredients.

[0099] The term“insulated neighborhood,” (IN) as used herein, refers to chromosome structure formed by the looping of two interacting sites in the chromosome sequence that may comprise CCCTC-Binding factor (CTCF) co-occupied by cohesin and affect the expression of genes in the insulated neighborhood as well as those genes in the vicinity of the insulated neighborhoods.

[00100] The term“insulator,” as used herein, refers to regulatory elements that block the ability of an enhancer to activate a gene when located between them and contribute to specific enhancer-gene interactions.

[00101] The term“master transcription factor,”, as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene and establish cell-type specific enhancers. Master transcription factors recruit additional signaling proteins, such as other transcription factors to enhancers to form signaling centers.

[00102] The term“minimal insulated neighborhood,” as used herein, refers to an insulated neighborhood having at least one neighborhood gene and associated regulatory sequence region or regions (RSRs) which facilitate the expression or repression of the neighborhood gene such as a promoter and/or enhancer and/or repressor regions, and the like.

[00103] The term“modulate,” as used herein, refers to an alteration (e.g., increase or decrease) in the expression of the target gene and/or activity of the gene product. In some aspects, modulation of a target gene expression is determined by measuring the target gene expression after administration of a modulating compound and comparing the gene expression to the expression of the target gene in the absence of treatment with the compound.

[00104] The term“neighborhood gene,”, as used herein, refers to a gene localized within an insulated neighborhood.

[00105] The term“penetrance,” as used herein, refers to the proportion of individuals carrying a particular variant of a gene (e.g., mutation, allele or generally a genotype, whether wild type or not) that also exhibits an associated trait (phenotype) of that variant gene and in some situations is measured as the proportion of individuals with the mutation who exhibit clinical symptoms thus existing on a continuum.

[00106] The term“polypeptide,” as used herein, refers to a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. [00107] The term“primary neighborhood gene,” as used herein, refers to a gene which is most commonly found within a specific insulated neighborhood along a chromosome.

[00108] The term“primary downstream boundary,” as used herein, refers to the insulated neighborhood boundary located downstream of a primary neighborhood gene.

[00109] The term“primary upstream boundary,” as used herein, refers to the insulated neighborhood boundary located upstream of a primary neighborhood gene.

[00110] The term“promoter,” as used herein, refers to a DNA sequence that defines where transcription of a gene by RNA polymerase begins and defines the direction of transcription indicating which DNA strand will be transcribed.

[00111] The term“regulatory sequence regions,” as used herein, include but are not limited to regions, sections or zones along a chromosome whereby interactions with signaling molecules occur in order to alter expression of a neighborhood gene.

[00112] The term“repressor,” as used herein, refers to any protein that binds to DNA and therefore regulates the expression of genes by decreasing the rate of transcription.

[00113] The term“secondary downstream boundary,” as used herein, refers to the downstream boundary of a secondary loop within a primary insulated neighborhood.

[00114] The term“secondary upstream boundary,” as used herein, refers to the upstream boundary of a secondary loop within a primary insulated neighborhood.

[00115] The term“signaling center,” as used herein, refers to a defined region of a living organism that interacts with a defined set of biomolecules, such as signaling proteins or signaling molecules (e.g., transcription factors) to regulate gene expression in a context-specific manner.

[00116] The term“signaling molecule,” as used herein, refers to any entity, whether protein, nucleic acid (DNA or RNA), organic small molecule, lipid, sugar or other biomolecule, which interacts directly, or indirectly, with a regulatory sequence region on a chromosome.

[00117] The term“signaling transcription factor,” as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene and also act as cell -cell signaling molecules.

[00118] The term“super-enhancers,” as used herein, refers to are large clusters of transcriptional enhancers that drive expression of genes that define cell identity.

[00119] The term“topologically associating domains,” (TADs) as used herein, refers to structures that represent a modular organization of the chromatin and have boundaries that are shared by the different cell types of an organism. [00120] The term“transcription factors,” as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene.

[00121] The term“upstream neighborhood gene,” as used herein, refers to a gene upstream of a primary neighborhood gene that may be located within the same insulated neighborhood as the primary neighborhood gene.

[00122] The term“small molecule,” as used herein, refers to a low molecular weight drug, i.e. <5000 Daltons organic compound that may help regulate a biological process.

[00123] The term“therapeutic agent,” as used herein, refers to a substance that has the ability to cure a disease or ameliorate the symptoms of the disease.

[00124] The term“therapeutic or treatment outcome,” as used herein, refers to any result or effect (whether positive, negative or null) which arises as a consequence of the perturbation of a GSC or GSN. Examples of therapeutic outcomes include, but are not limited to, improvement or amelioration of the unwanted or negative conditions associated with a disease or disorder, lessening of side effects or symptoms, cure of a disease or disorder, or any improvement associated with the perturbation of a GSC or GSN.

[00125] The term“therapeutic or treatment liability,” as used herein, refers to a feature or characteristic associated with a treatment or treatment regime which is unwanted, harmful or which mitigates the therapies positive outcomes. Examples of treatment liabilities include for example toxicity, poor half-life, poor bioavailability, lack of or loss of efficacy or

pharmacokinetic or pharmacodynamic risks.

[00126] The term“ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a high LDL-cholesterol disease state or fatty liver disease, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[00127] The term“in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[00128] The term“in vivo” refers to processes that occur in a living organism.

[00129] The term“ex vivo” refers to processes that occur outside a living organism.

[00130] The term“mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[00131] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[00132] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[00133] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et ah, infra).

[00134] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et ah, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

[00135] The term“sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[00136] The term“therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a

“prophylactically effective amount” as prophylaxis can be considered therapy.

[00137] The term“about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. An exemplary error range is plus or minus 5%. Reference to“about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. [00138] It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise.

Features and Properties of the PCSK9 gene

[00139] In some embodiments, methods of the present invention involve modulating the expression of the Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) gene. PCSK9 may also be referred to as Subtilisin/Kexin-Like Protease PC9, NARC-l, NARC1, PC9, Convertase Subtilisin/Kexin Type 9 Preproprotein, Hypercholesterolemia, Autosomal Dominant 3, or Neural Apoptosis Regulated Convertase 1. PCSK9 has a cytogenetic location of lp32.3 and the genomic coordinate are on Chromosome 1 on the forward strand at position 54,070,017-54,996,888. PCSK9 has a NCBI gene ID of 255738, Uniprot ID of Q8NBP7 and Ensembl Gene ID of ENSG00000169174. The nucleotide sequence of PCSK9 on chromosome 1 is shown in SEQ ID NO: 1. The protein sequence of PCSK9 is shown in SEQ ID NO: 2.

[00140] In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the PCSK9 gene. The present inventors have identified the insulated neighborhood containing the PCSK9 gene in primary human hepatocytes. The insulated neighborhood that contains the PCSK9 gene is on chromosome 1 on the forward strand at position 55,039,476-55,064,853. The number of signaling centers within the insulated neighborhood is 26. The insulated neighborhood contains PCSK9 and 15 other genes, namely TCEANC2, CDCP2, CYB5RL, MRPL37, SSBP3, ACOT11, FAM151A, MROH7, TTC4, PARS2, TTC22, LEXM, DHCR24, TMEM61, and BSND. The chromatin marks, or chromatin-associated proteins, identified at the insulated neighborhood include H3K27Ac and SMC1A. Transcription factors and signaling pathway proteins involved in the insulated neighborhood include mTOR, NR5A2, SMAD2, SMAD3, STAT3, YY1, HNF4A, HNF1A, ONECUT1, MYC, NR1H4, NR3C1, RXRA, VDR, CREB1, ESR1. Any components of these signaling centers and/or signaling molecules, or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of PCSK9.

Features and Properties of the ANGPTL3 gene

[00141] In some embodiments, methods of the present invention involve modulating the expression of the Angiopoietin Like 3 (ANGPTL3) gene. ANGPTL3 may also be referred to as Angiopoietin-Related Protein 3, Angiopoietin 5, ANGPT5, or ANG-5. ANGPTL3 has a cytogenetic location of lp32.3 and the genomic coordinate are on Chromosome 1 on the forward strand at position 62,597,487-62,606,305. ANGPTL3 has a NCBI gene ID of 27329, Uniprot ID of Q9Y5C1 and Ensembl Gene ID of ENSG00000132855. The nucleotide sequence of

ANGPTL3 on chromosome 1 is shown in SEQ ID NO: 3. The protein sequence of ANGPTL3 is shown in SEQ ID NO: 4.

[00142] In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the ANGPTL3 gene. The present inventors have identified the insulated neighborhood containing the ANGPTL3 gene in primary human hepatocytes. The insulated neighborhood that contains the ANGPTL3 gene is on chromosome 1 on the forward strand at position 62,597,487-62,606,305. The number of signaling centers within the insulated neighborhood is 9. The insulated neighborhood contains ANGPTL3 and 3 other genes, namely DOCK7, AL 138847.2 and AC103923.1. Transcription factors and signaling pathway proteins involved in the insulated neighborhood include HNF4A, RXRA, YY1, TEAD1, HNF1, P300, CREB1, mTOR, SMAD2, SMAD3, SMAD4, STAT1, STAT3, NF-KB, BRD4, TP53, TCF7L2, and JUN. Any components of these signaling centers and/or signaling molecules, or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ANGPTL3.

Methods of modulating PCSK9 and ANGPTL3 expression

[00143] This invention employs certain methods employed in PCT/US2018/055087, filed on October 9, 2018, and published on April 11, 2019, co-owned by the applicant, and incorporated by reference herein in its entirety for all purposes.

[00144] Provided herein are methods for modulating PCSK9 expression in a cell comprising contacting the cell with a compound that modulates a target selected from the group consisting of mTOR, MYC, NR1H4, NR3C1, NR5A2, RXRA, VDR, CREB1, ESR1, SMAD2, SMAD3, STAT3, UΎ1, HNF4A, HNF1A, and ONECUT1, thereby modulating PCSK9 expression. In some embodiments, the compound modulates a target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, thereby modulating PCSK9 expression.

[00145] Modulation of PCSK9 expression may be a direct or indirect effect of the methods as described herein. Indirect effects include upstream effects on a signaling pathway that results in modulation of PCSK9 expression. Modulation of PCSK9 expression may affect one or a combination of transcription factors. [00146] Modulation of a chromatin binding protein, such as a transcription factor, can include one or more of: phosphorylation, de -phosphorylation, methylation, de-methylation, acetylation, de -acetylation, ubiquitination, de-ubiquitination, glycosylation, de-glyosylation, sumoylation, de-sumoylation, stability, and degradation. The net effect of such modulation is to alter the function of the chromatin binding protein. Such alteration can include one or more of: increased or decreased binding to DNA, increased or decreased binding to one or more chromatin binding proteins, increased or decreased stability of the chromatin binding protein, or change in sub- cellular localization of the chromatin binding protein.

[00147] Gene circuitry mapping can be used to make novel connections between signaling pathways and genome-wide regulation of transcription, allowing for identification of draggable targets that are predicated to up- or down-regulate expression of disease-associated genes. The inventors have applied this gene circuitry mapping to identify dragging signaling pathways to reduce PCSK9 and ANGPTL3 transcription as therapeutic targets. Gene mapping utilizes four approaches: HiChIP, ATAC-Seq, ChIP-seq, and RNA-seq.

[00148] HiChIP is a technique that defines chromatin domains (insulated neighborhoods) and DNA-DNA interactions, such as enhancer-promoter interactions. ATAC-seq identifies open chromatin regions and activate enhancers. ChIP-seq reveals binding of transcription factors to DNA, modified histones, and chromatin-binding proteins genome wide. RNA-seq quantifies transcript levels of every gene.

PCSK9 Gene Circuitry MavOins

[00149] Using these gene mapping techniques showed PCSK9 is insulated from neighboring domains and highlighted key enhancers that are likely to regulate expression. The gene mapping results are shown in FIG. 1. The top panel shows the results of the HiChIP mapping, while the bottom panel shows a comparison of the results with the additional mapping techniques.

[00150] The gene circuitry mapping approach predicted multiple pathways with potential to regulate PCSK9 expression. The ChIP-seq assay identified 16 new transcription factors, in addition to the previously reported transcription factors that bind the PCSK9, as shown in FIG.

2. In addition, new signaling pathways associated with PCSK9 gene expression were identified. The newly identified transcription factors or signaling pathways are mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3.

[00151] PCSK9 transcription factors or signaling pathways identified via the gene circuitry mapping and previously known PCSK9 transcription factors or signaling pathways are listed in

Table 1A. Table 1A. PCSK9 Transcription Factors and Signaling Pathways

[00152] The gene mapping techniques were repeated with the ANGPTL3 gene.

[00153] The gene circuitry mapping approach predicted multiple pathways with potential to regulate ANGPTL3 expression. The ChIP-seq assay identified new transcription factors, in addition to the previously reported transcription factors that bind the ANGPTL3 gene. In addition, new signaling pathways associated with ANGPTL3 gene expression were identified. The newly identified transcription factors or signaling pathways are mTOR, SMAD2, SMAD3, SMAD4, STAT1, STAT3, NF-kB, BRIM, TP53, TCF7L2, and JUN.

[00154] ANGPTL3 transcription factors or signaling pathways identified via the gene circuitry mapping and previously known ANGPTL3 transcription factors or signaling pathways are listed in Table IB.

[00155] Table IB. ANGPTL3 Transcription Factors and Signaling Pathways

[00156] In some embodiments, compositions and methods of the present invention may be used to modulate expression of the PCSK9 or ANGPTL3 gene in a cell or a subject. Changes in gene expression may be assessed at the RNA level or protein level by various techniques known in the art and described herein, such as RNA-seq, qRT-PCR, Western Blot, or enzyme-linked immunosorbent assay (ELISA). Changes in gene expression may be determined by comparing the level of PCSK9 or ANGPTL3 expression in the treated cell or subject to the level of expression in an untreated or control cell or subject.

[00157] In some embodiments, modulation of PCSK9 or ANGPTL3 expression is a reduction in PCSK9 or ANGPTL3 expression in a cell or a subject. In some embodiments, compositions and methods of the present invention cause reduction in the expression of a PCSK9 or

ANGPTL3 gene as measured in a cell-based assay of cells exposed to the compound at a level corresponding to the plasma level achieved at steady state in a subject dosed with the effective amount as compared to cells exposed to a placebo. In some embodiments, the cells are homozygous for the wild type PNPLA3 gene. In some embodiments, the cells are heterozygous for the wild type and the mutant 1148M PNPLA3 gene and protein. In some embodiments, the cells are homozygous for the mutant I148M PNPLA3 gene and protein.

[00158] In some embodiments, compositions and methods of the present invention cause reduction in the expression of a PCSK9 or ANGPTL3 gene on average in a population administered the compound as compared to control subjects administered a placebo.

[00159] In some embodiments, compositions and methods of the present invention cause reduction in the expression of a PCSK9 or ANGPTL3 gene in a subject as compared to pre dosing PCSK9 or ANGPTL3 gene expression levels in the subject.

[00160] In some embodiments, the expression of the PCSK9 or ANGPTL3 gene is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, from about 25% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, more than 80%, or even more than 90%, 95% or 99% as compared to the PCSK9 or ANGPTL3 expression in an untreated cell, untreated subject, or untreated population. In some embodiments, the administration of a compound reduces the expression of the PCSK9 or ANGPTL3 gene in a cell in vivo or in vitro by at least about 1%,

5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, or 99% as compared to the PCSK9 or ANGPTL3 expression in an untreated cell, untreated subject, or untreated population. In some embodiments, the reduced expression is in a cell in a subject. [00161] In some embodiments, the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a matched control population. In some embodiments, the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a pre-treatment baseline measurement. In some embodiments, the reduction is determined in a population of cells and the amount of reduction is determined by reference to a matched control cell population.

Pathways and modulators

[00162] In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates a first target protein selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates a protein selected from the group consisting of MYC, NR1H4, NR3C1, NRTA2, RXRA, VDR, CREB1, ESR1, MTOR, SMAD2, SMAD3, STAT3, YY1, HNF4A, HNF1A, and ONECUT1. In some embodiments, modulation of PCSK9 expression is a reduction or decrease in PCSK9 expression. In some embodiments, modulation of PCSK9 expression is an increase in PCSK9 expression.

[00163] In some embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates a second target protein selected from the group consisting of mTOR, SMAD2, SMAD3, SMAD4, STAT1, STAT3, NF-kB, BRD4, TP53, TCF7F2, and JU . In some embodiments, modulation of ANGPTF3 expression is a reduction or decrease in ANGPTF3 expression. In some embodiments, modulation of ANGPTF3 expression is an increase in ANGPTF3 expression.

[00164] In some embodiments, the PCSK9 or ANGPTF3 modulating compound comprises an mTOR pathway inhibitor. The mTOR pathway comprises two signaling complexes, mTORCl and mTORC2. The mTORCl complex comprises mTOR, mFST8, PRAS40, Deptor, and Raptor. In contrast, the mTORC2 complex comprises mTOR, mFST8, mSINl, Protor, Deptor, and RICTOR. Activation of the mTORCl complex results in phosphorylation of p70^S6K (also called S6 Kinase, S6K or S6) and 4E-BP1, resulting in downstream gene transcription (e.g., expression) and translation. Activation of the mTORC2 complex results in phosphorylation and activation of the AKT, SGK1, NDRG1, and PKC proteins. mTORC2 phosphorylates AKT at serine 473 and Threonine 308. AKT also activates the mTORCl complex. Direct or indirect inhibition includes, but is not limited to, inhibiting the catalytic activity of the mTOR kinase or inhibiting binding of substrate to the kinase. [00165] In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell (e.g., hepatocyte) modulates the mTOR and/or PI3K signaling pathway. In certain embodiments, the compound that modulates the mTOR and/or PI3K signaling pathway is an inhibitor of the mTOR and/or PI3K signaling pathway. In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell modulates a protein selected from the group consisting of mTOR, PI3K, AKT, PDK1, DNA-PK, 4E-BP1, PKC, 6SK1, and SGK1. In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell modulates a protein selected from the group consisting of mTOR, PDK1, and PI3K. In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell modulates mTOR. In certain embodiments, the compound that modulates the activity of mTOR to modulate the expression of PCSK9 or ANGPTL3 in a cell is selected from the group consisting of OSI-027, PF-04691502, WYE-125132 (WYE-132), JR- AB2-011, Apitolisib (GDC-0980, RG7422), AZD8055, BGT226 (NVP-BGT226), CC-223, Chrysophanic Acid, CZ415, Dactolisib (BEZ235, NVP-BEZ235), Everolimus (RAD001), GDC- 0349, Gedatolisib (PF-05212384, PKI-587), GSK1059615, INK 128 (MLN0128), KU-0063794, LY3023414, MHY1485, Omipalisib (GSK2126458, GSK458), OSI-027, Palomid 529 (P529), PF-04691502, PI-103, PP121, Rapamycin (Sirolimus), Ridaforolimus (Deforolimus, MK-8669), SF2523, Tacrolimus (FK506), Temsirolimus (CCI-779, NSC 683864), Torin 1, Torin 2,

Torkinib (PP242), Vistusertib (AZD2014), Voxtalisib (SAR245409, XL765) Analogue, Voxtalisib (XL765, SAR245409), WAY-600, WYE-354, WYE-687, XL388, or Zotarolimus (ABT-578). In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell is OSI-027 or PF-04691502.

[00166] In some embodiments, the mTOR inhibitor comprises an mTORC 1 and mTORC2 inhibitor. In some embodiments, the mTOR inhibitor comprises an mTORC2 inhibitor. In some embodiments, the mTORC2 inhibitor comprises a RICTOR inhibitor.

[00167] Any appropriate method to measure inhibition of mTOR activity may be used. Such methods are well known in the art and include ELISAs or Western Blotting to measure the phosphorylation of mTOR substrates, such as S6K, AKT, SGK1, PKC, NDRG1, and/or 4EBP1, or any other mTOR substrate known in the art. ELISA kits for phosphorylated mTOR substrates are available from a variety of manufacturers, including MilliporeSigma, Cell Signaling, and Abeam. Antibodies for phosphorylated mTOR substrates are available from a variety of manufacturers, including Cell Signaling, Abeam, and Santa Cruz Biotech. [00168] In some embodiments, the PCSK9 or ANGPTL3 modulating compound comprises an mTOR pathway inhibitor that does not inhibit phosphoinositide 3-kinases (PI3K, also known as phosphatidylinositol 3-kinase). PI3Ks are intracellular signaling molecules that phosporylate phosphatidylinositols (Pis). The PI3K family is divided into 3 classes based on primary structure, regulation and lipid substrate specificity: Class I, Class II, and Class III. Class I PI3Ks are heterodimeric molecules comprising a regulatory subunit and a catalytic subunit. They catalyze the phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PI(4.5)Pi) into phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) in vivo. Class IA PI3Ks comprise a pl 10a/b/d catalytic subunit and a r85a/b, r55a/g, or r50a regulatory subunit. RI3Ka, RI3Kb, and PI3K5 are all Class IA PI3Ks. Class IB PI3Ks comprise a pl 10g catalytic subunit and a plOl regulatory subunit. RI3Kg is a Class 1B PI3K. Class II PI3Ks comprise catalytic subunits only, termed C2a, C2b, and C2y, which lack aspartic acid residues and catalyze the production of PI(3)P from PI and PI(3.4)Pi from PI(4)P. Class III PI3Ks are heterodimers of a catalytic subunit, Vps34, and regulator subunits (Vspl5/pl50). Class III PI3Ks catalyze the production of only PI(3)P from PI.

[00169] Inhibitors that do not inhibit the PI3K pathway include mTOR inhibitors that do not directly or indirectly inhibit class I, class II, or class III PI3K proteins. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit class I, class II, or class III PI3K enzymatic activity. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit class I, class II, or class III PI3K protein stability or class I, class II, or class III PI3K gene expression. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit the catalytic subunits of the class I, class II, or class III PI3K proteins, or the regulatory subunits of the class I, class II, or class III PI3K proteins. Direct or indirect inhibition includes, but is not limited to, inhibiting the catalytic activity of the PI3 kinase or inhibiting binding of substrate to the kinase.

[00170] Methods of assessing PI3K activity in cells are known in the art and include ELISAs to measure the phosphorylation of PI3K substrates, such as PI, (PI(4,5)P2), or PI(3,4)P2. In addition, methods of assessing purified PI3K activity are also well known in the art and include monitoring of radioactive or fluorescent g-ATR into PI3K substrates or ratiometric fluorescence superquenching (Stankewicz C, et al, Journal of Biomolecular Screening 11(4); 2006). Any appropriate method to measure PI3K activity may be used.

[00171] In some embodiments, the PCSK9 or ANGPTL3 modulating compound comprises an mTOR pathway inhibitor that does not inhibit DNA-PK. DNA-PK is a member of the phosphatidylinositol 3-kinase-related kinases (PIKK) protein family, which is sometimes referred to as Class IV PI3K. DNA-PK is a heterodimer formed by the catalytic subunit DNA- PKcs and the autoimmune antigen Ku. DNA-PK phosphorylates p53, Akt/PKB, and CHK2, among other protein targets. Inhibitors that do not inhibit DNA-PK include inhibitors that do not directly or indirectly inhibit DNA-PK. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit DNA-PK enzymatic activity. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit DNA-PK protein stability or gene expression. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit the catalytic or regulatory subunits of DNA-PK. Direct or indirect inhibition includes, but is not limited to, inhibiting the catalytic activity of the DNA-PK kinase or inhibiting binding of substrate to the kinase.

[00172] In some embodiments, the PCSK9 or ANGPTL3 modulating compound comprises an mTOR pathway inhibitor that does not inhibit PIP4K2C. PIP4K2C is a subunit of type-2 phosphatidylinositol-5-phosphate 4-kinase that converts phosphatidylinositol-5-phosphate (PI(5)P) to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Inhibitors that do not inhibit PIP4K2C include inhibitors that do not directly or indirectly inhibit PIP4K2C. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit PIP4K2C enzymatic activity. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit PIP4K2C protein stability or gene expression. In some embodiments, the mTOR inhibitors do not directly or indirectly inhibit the catalytic or regulatory subunits of PIP4K2C. Direct or indirect inhibition includes, but is not limited to, inhibiting the catalytic activity of the PIP4K2C kinase or inhibiting binding of substrate to the kinase.

[00173] In some embodiments, the PCSK9 or ANGPTL3 modulating compound does not induce hyperinsulinemia in the subject. Hyperinsulinemia is a higher than normal fasting insulin level in a subject’s blood plasma. Reference ranges for hyperinsulinemia generally recite normal insulin levels under fasting conditions (8 hour fast) as less than 25 pU/L or less than 174 pmol/L. 30 minutes after a meal or glucose administration, a normal insulin level is 30-230 pU/L or 208-1597 pmol/L. One hour after a meal or glucose administration, a normal insulin level is 18-276 pU/L or 125-1917 pmol/L. Two hours after a meal or glucose administration, a normal insulin level is 16-166 pU/L or 111-1153 pmol/L. In some embodiments, hyperinsulinemia is an insulin level greater than 25 pU/L after an 8 hour fast. In some embodiments, hyperinsulinemia is an insulin level greater than 170 pU/L two hours after a meal or glucose administration. [00174] In some embodiments, the PCSK9 or ANGPTL3 modulating compound does not induce hyperglycemia in the subject. Hyperglycemia is a higher than normal amount of glucose in a subject’s blood plasma. Reference ranges for hyperglycemia generally recite blood sugar levels higher than 11.1 mmol/L or 200 mg/dL. A non-diabetic normal glucose level is generally considered to be under 140 mg/dL two hours after a meal. However, even consistent blood sugar levels between 5.6 and 7 mmol/l (100-126 mg/dL) can be considered slightly hyperglycemic. In some embodiments, a blood sugar level higher than 130 mg/dL after an 8 hour fast is a hyperglycemic level. In some embodiments, a blood sugar level higher than 180 mg/dL two hours after a meal is a hyperglycemic level.

[00175] In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell (e.g., hepatocyte) inhibits the STAT pathway. In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell modulates the activity of STAT1 and/or STAT3. In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates the activity of STAT1. In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates the activity of STAT3. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of STAT3. In certain embodiments, the compound that modulates the activity of STAT1 and/or STAT3 to modulate the expression of PCSK9 or ANGPTL3 in a cell is selected from the group consisting of AG 18, Stattic, Alantolactone, Napabucasin, OPB-31121, OPB-51602, STAT3 inhibitor XIII, danvatirsen, WP1066, Chrysophanol, SMI-l6a, RG13022, TCS-PIM-l-4a, RG14620, Nifuroxazide, Dihydroisotanshinone I, STAT5-IN-1, Hispidulin, Tyrphostin AG 528, AG-1478, Tyrphostin AG 879, AG 555, Niclosamide, PD158780,

Piml/AKKl-IN-l, PD153035, NSC 74859, TCS PIM-l 1, AZD1208, CL-387785, EAI045, Artesunate, BIBX 1382, Icotinib, PD153035 (Hydrochloride), AS1517499, HJC0152 hydrochloride, Diosgenin, Fedratinib (SAR302503, TG101348), TP-3654, Morusin, Icotinib (Hydrochloride), PF-06459988, AEE788, AZD3759, CX-6258, Scutellarin, HO-3867, Pelitinib, Mubritinib, CP-724714, Dacomitinib, Cl 88-9, Sapitinib, Irbinitinib, Gefitinib (hydrochloride), AZ-5104, Olmutinib, Poziotinib, WZ4002, AZD-9291, CNX-2006, TAK-285, Lazertinib, CO- 1686, Neratinib, Canertinib (dihydrochloride), BMS-599626 (Hydrochloride), AZD-9291 (mesylate), Avitinib (maleate), SH-4-54, BP-1-102, CO-1686 (hydrobromide), Saikosaponin D.

[00176] In certain embodiments, the compound that modulates the expression of PCSK9 in a cell (e.g., hepatocyte) modulates the Myc pathway. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of Myc. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell is selected from the group consisting of Myc -targeting siRNA DCR-MYC and AVI-4126.

[00177] NR3C1 is also known as the glucocorticoid receptor (GR or GCR) and is a cytosolic protein. Cortisol and other glucocorticoids bind NR3C1 and activate NR3C1, resulting in translocation of the NR3C1 protein into the nucleus where it binds target DNA sequences called glucocorticoid-responsive elements (GREs) and regulates the expression of target genes. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell (e.g., hepatocyte) modulates the NR3C1 transcription factor. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of NR3C1. In certain embodiments, the compound that modulates the activity of NR3C1 to modulate the expression of PCSK9 in a cell is selected from the group consisting of rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate, fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate,

ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone

dipropionate/calcipotriene, fluticasone furoate, budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate, beclomethasone l7-monopropionate.

[00178] In certain embodiments, the compound that modulates the expression of PCSK9 in a cell (e.g., hepatocyte) modulates the Vitamin D Receptor (VDR) transcription factor. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of VDR. In certain embodiments, the compound that modulates the activity of VDR to modulate the expression of PCSK9 in a cell is selected from the group consisting of

calcipotriene, ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3- hydroxypropoxy)calcitriol, betamethasone dipropionate/calcipotriene, alfacalcidol, calcium carbonate/cholecalciferol, paricalcitol, doxercalciferol, cholecalciferol, calcitriol, calcifediol, and seocalcitol.

[00179] In certain embodiments, the compound that modulates the expression of PCSK9 in a cell (e.g., hepatocyte) modulates the Estrogen Receptor 1 (ESR1) transcription factor. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of ESR1. In certain embodiments, the compound that modulates the activity of ESR1 to modulate expression of PCSK9 in a cell is selected from the group consisting of l7-alpha- ethinylestradiol, Fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF-4227, bazedoxifene, estradiol valerate, testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol, estradiol acetate, esterified estrogens, estradiol cypionate, medroxyprogesterone acetate, norethindrone acetate, testosterone cypionate, synthetic conjugated estrogens, etonogestrel, CC8490, MITO-4509, cyproterone acetate, pipendoxifene, chlorotrianisene, icaritin, megestrol acetate, tamoxifen, sulindac, toremifene, raloxifene, F18 l6-alpha-fluoroestradiol, ARN-810, Z-endoxifen, goserelin, teriparatide, AZD9496, elacestrant, SRN-927, palbociclib, anastrozole, letrozole, exemestane, pertuzumab, trastuzumab, LSZ102, selective estrogen receptor modulator, desogestrel, drospirenone, norelgestromin, norethindrone, levonorgestrel, norgestrel, norgestimate, diethylstilbestrol, ospemifene, toremifene, Everolimus, H3B-6545, arzoxifene, clomiphene, SAR439859, estramustine phosphate, diethylstilbestrol diphosphate, TTC-352, ribociclib, 4- hydroxytamoxifen, dienestrol, acolbifene, estramustine, medroxyprogesterone acetate, danazol, trilostane, fluoxymesterone, norgestimate, progesterone, and S-equol.

[00180] In certain embodiments, the compound that modulates the expression (e.g., transcription) of PCSK9 or ANGPTL3 in a cell (e.g., hepatocyte) modulates the TGF signaling pathway. There are three types of TGF family receptors; type I, type II, and type III. There are seven TGF type I receptors, termed actavin-like receptors (ALK1-7), five type II receptors, and one type III receptor. TGF superfamily ligands bind to a TGF type II receptor that recruits and phosphorylates a TGF type I receptor. This results in phosphorylation of receptor-regulated SMAD proteins (R-SMAD) such as SMAD2 and SMAD3, which then bind SMAD4 (coSMAD) and form a complex. The R-SMAD/SMAD4 complexes then translocate to the nucleus and act as transcription factors that regulate gene expression. The specific protein TGF RI is also known as AT5, ACVRLK4, ALK-5, ALK5, ESS1, LDS1, LDS1A, LDS2A, MSSE, and SKR4.

[00181] In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell (e.g., hepatocyte) modulates the TGF pathway. In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell modulates the activity of the TGF pathway. In certain embodiments, the compound that modulates the TGF pathway modulates SMAD2, SMAD3, or SMAD4 activity. In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates the activity of SMAD2. In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates the activity of SMAD3. In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell modulates the activity of SMAD4. In certain

embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of SMAD2. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of SMAD3. In certain embodiments, the compound that modulates the expression of PCSK9 in a cell modulates the activity of SMAD4. In certain embodiments, the compound that modulates SMAD2, SMAD3, or SMAD4 activity to modulate the expression of PCSK9 or ANGPTL3 in a cell is selected from the group consisting of Oxymatrine, Kartogenin, SRI-011381 (hydrochloride), Halofuginone, SIS3, LY2157299, LY- 364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R- 268712, SB-431542, EW-7197, LDN-212854, Halofuginone, ITD-l, LDN-214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83-01 sodium salt, A 83-01, and LDN193189

(Hydrochloride), Disitertide, and SB-431542.

[00182] In certain embodiments, the compound that modulates the expression of PCSK9 or ANGPTL3 in a cell (e.g., hepatocyte) modulates the activity of TGF RI (ALK5). In certain embodiments, the compound that modulates the activity of TGF RI to modulate the expression of PCSK9 or ANGPTL3 in a cell is selected from the group consisting of LY2157299, LY- 364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R- 268712, SB-431542, EW-7197, LDN-212854, Halofuginone, ITD-l, LDN-214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83-01 sodium salt, A 83-01, and LDN193189

(Hydrochloride).

[00183] In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell (e.g., hepatocyte) modulates the activity of NF-kB. In certain embodiments, the compound that modulates the activity of NF-kB to modulate the expression of ANGPTF3 in a cell is selected from the group consisting of SC75741, BAY 11-7082, JSH-23, and Neferine.

[00184] In certain embodiments, the compound that modulates the expression of ANGPTF3 in a cell (e.g., hepatocyte) modulates the activity of BRD4. In certain embodiments, the compound that modulates the activity of BRD4 to modulate the expression of ANGPTF3 in a cell is selected from the group consisting of FF-411, ZF0420, ZEN-3411, and PFX51107.

[00185] In certain embodiments, the compound that modulates the expression of ANGPTF3 in a cell (e.g., hepatocyte) modulates the activity of TP53. In certain embodiments, the compound that modulates the activity of TP53 to modulate the expression of ANGPTF3 in a cell is selected from the group consisting of PK11007, Serdemetan, RITA, J J-26854165, and MI- 773.

[00186] In certain embodiments, the compound that modulates the expression of ANGPTL3 in a cell (e.g., hepatocyte) modulates the activity of TCF7L2. In certain embodiments, the compound that modulates the activity of TCF7L2 to modulate the expression of ANGPTL3 in a cell is selected from the group consisting of LY2090314, A 1070722, and AZD2858.

[00187] In certain embodiments, the compound modulates the expression of PCSK9 or ANGPTL3 in the cell by decreasing the expression of PCSK9 or ANGPTL3 by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In certain embodiments, the compound modulates the expression of PCSK9 or ANGPTL3 in the cell by decreasing the expression of PCSK9 or ANGPTL3 from about 25% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, from about 80% to about 100%, from about 100% to about 125%, from about 100 to about 150%, from about 150% to about 200%, from about 200% to about 300%, from about 300% to about 400%, from about 400% to about 500%, or more than 500%. In some embodiments, the compound decreases the expression of PCSK9 or ANGPTL3 in the cell by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 12 fold, about 15 fold, about 18 fold, about 20 fold, about 25 fold, or more than 30 fold.

Methods of treating cholesterol and/or liver diseases

[00188] In one aspect, provided herein are methods for treating a disease comprising administering to a mammalian subject an effective amount of a compound that modulates a target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, wherein said modulating of said target reduces PCSK9 expression and thereby treats the disease.

[00189] In another aspect, provided herein are methods for treating a disease comprising administering to a mammalian subject an effective amount of a compound that modulates a second target selected from the group consisting of mTOR, Transforming Growth Factor b receptor (TGF R) I, TGFp receptor II, SMAD2, SMAD3, STAT1, NF-kB, BRIM, p53, and TCF7L2, wherein said modulating of the target reduces ANGPTL3 expression and thereby treats the disease. [00190] In some embodiments, the disease is a liver disease or a disease associated with a blood or serum ratio of high density lipoprotein (HDL)-cholesterol/ low density lipoprotein (LDL)-cholesterol of < 0.3, optionally wherein the disease is selected from the group consisting of: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), and a high LDL-cholesterol associated disease.

[00191] The ratio of HDL-cholesterol and LDL-cholesterol is HDL and LDL levels can be determined by any appropriate lipid panel or assay known in the art. Such panels and assays are generally known to one of skill in the art.

[00192] Ratios of HDL-cholesterol to LDL-cholesterol are determined after measuring both HDL and LDLs cholesterol and comparing the levels of HLD to LDL. An HDL/LDL cholesterol ratio of greater than 0.3 is generally considered a healthy ratio. An HDL/LDL cholesterol ratio of less than or equal to (<) 0.3 is generally considered an unhealthy ratio. In some embodiments, the compound that modulates PCSK9 or ANGPTL3 expression is administered to a subject with an HDL/LDL ratio of less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.10, less than 0.5, less than 0.1, less than 0.5, and less than 0.01. In some embodiments, the compound that modulates PCSK9 or ANGPTL3 expression is administered to a subject with an HDL/LDL ratio of between about 0.01-0.3, between about 0.01-0.5, between about 0.5-0.1, between about 0.1-0.15, between about 0.15-0.2, between about 0.2-0.25, and between about 0.25-0.3.

[00193] In some embodiments, the low-density lipoprotein (LDL) cholesterol disease is a liver disease such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and/or alcoholic liver disease (ALD).

[00194] In some embodiments, the high LDL-cholesterol associated disease occurs in a subject having a PCSK9-activating (GOF) mutation, a marked elevation of low density lipoprotein particles in the plasma, primary hypercholesterolemia, or heterozygous Familial Hypercholesterolemia (heFH).

[00195] PCSK9 mutations, resulting in PCSK9 gain of function and loss of function mutations, are described in“Loss- and Gain-of-fimction PCSK9 Variants”, Benjannet S, et al J Biol Chem. 2012 Sep 28; 287(40): 33745-33755 and“ Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease” Abifadel M, et al, Hum Mutat. 2009 Apr;30(4):520-9. doi: l0.l002/humu.20882, both of which are hereby incorporated by reference in their entirety. PCSK9 gain of functions mutations include, but are not limited to, L108R, D374Y, D374H, D374W, D374M, D374F, D374E, D374K, and D374L.

[00196] Hypercholesterolemia is characterized by high levels of cholesterol in the blood. Subjects with high levels of cholesterol can develop a form of heart disease called coronary artery disease. When excess cholesterol in the blood is deposited on the walls of blood vessels, the abnormal buildup of cholesterol forms plaques that narrow and harden the blood vessels and arteries. This build up causes chest pain and increases a person’s risk of having a heart attack.

[00197] Familial Hypercholesterolemia (FH) is an inherited genetic disorder that results in high cholesterol levels and heart disease, heart attacks, or strokes. Patients with HF have elevated serum low-density lipoprotein (LDL) cholesterol levels. Heterozygous HF (heHF) is more common that homozygous HF (HoHF). HeHF is thought to have a prevalence of 1 in 500 in the western world. HF genetics and diagnosis are discussed in“Familial

hypercholesterolemia: A review,” Varghese MJ et al, Ann Pediatr Cardiol. 2014 May-Aug; 7(2): 107-117, hereby incorporated by reference in its entirety.

Liver diseases and PNPLA3 mutations

[00198] A polymorphic variation rs738409 C/G of PNPLA3, encoding for the isoleucine to methionine substitution at residue 148 (I148M), has been linked to NAFLD, hepatic steatosis and nonalcoholic steatohepatitis (NASH) as well as its pathobiological sequelae fibrosis, cirrhosis, and hepatocellular cancer (Krawczyk M et al, Semin Liver Dis. 2013 Nov;33(4):369- 79, which is hereby incorporated by reference in its entirety). The nucleotide sequence of PNPLA3 on chromosome 22 is shown in SEQ ID NO: 5. The protein sequence of PNPLA3 is shown in SEQ ID NO: 6. The rs738409 C/G allele in PNPLA3 was first reported to be strongly associated with increased hepatic fat levels (P = 5.9 x 10 ¹⁰) and with hepatic inflammation (P = 3.7 x 10 ⁴) (Romeo et al., Nat Genet. 2008 Dec;40(l2): 1461-5, which is hereby incorporated by reference in its entirety). Research suggests that the altered protein leads to increased production and decreased breakdown of fats in the liver. PNPLA3 I148M enhances steatosis by impairing the liberation of triglycerides from lipid droplets (Trepo E et al, J Hepatol. 2016 Aug;65(2):399- 412, which is hereby incorporated by reference in its entirety). Recent data also suggests that PNPLA3 I148M protein evades degradation and accumulates on lipid droplets (BasuRay et al, Hepatology. 2017 Oct;66(4): 1111-1124, which is hereby incorporated by reference in its entirety). I148M variant is associated with NAFLD in both adults and in children, but is predominant in women, not in men. The specific mechanism of the PNPLA3 I148M variant in the development and progression of NAFLD is still not clear. However, it is thought that the PNPLA3 I148M variant may promote the development of fibrogenesis by activating the hedgehog signaling pathway, which, in turn, leads to the activation and proliferation of hepatic stellate cells, and excessive generation and deposition of intrahepatic extracellular matrix (Chen LZ, et al, World J Gastroenterol. 2015 Jan 21; 21(3): 794-802, which is hereby incorporated by reference in its entirety).

[00199] The I148M variant has also been correlated with alcoholic liver disease and clinically evident alcoholic cirrhosis (Tian et al, Nature Genetics 42, 21-23 (2010), which is hereby incorporated by reference in its entirety). Moreover, it has been identified as a prominent risk factor for hepatocellular carcinoma in patients with alcoholic cirrhosis (Nischalke et al, PLoS One. 2011 ;6(l l):e27087, which is hereby incorporated by reference in its entirety).

[00200] The I148M variant also influences insulin secretion levels and obesity. In obese subjects the body mass index and waist are higher in carriers of the variant allele (Johansson LE et al, Eur J Endocrinol. 2008 Nov;l59(5):577-83, which is hereby incorporated by reference in its entirety). The I148M carriers display decreased insulin secretion in response to oral glucose tolerance test. I148M allele carriers are seemingly more insulin resistant at a lower body mass index.

[00201] In some embodiments, the methods for identifying a subject for modulation of expression of the PCSK9 or ANGPTL3 genes includes the step of determining whether the subject has the mutation PNPLA3-I148M. Specifically, the genetic marker is a G allele at SNP rs738409 (c.444 C-G). The G allele frequency varies by ethnicity and is estimated to be about 0.57 in Latino, 0.38 in East Asian, 0.23 in European, 0.22 in South Asian, and 0.14 in African populations.

[00202] Genotyping for the PNPLA3-1148M variant may be carried out via any suitable methods known in the art. For example, a biological sample is obtained from the subject, and genomic DNA is isolated. The biological sample may be any material that can be used to determine a DNA profile such as blood, semen, saliva, urine, feces, hair, teeth, bone, tissue and cells. The gene variant may then be detected by methods such as, but not limited to, mass spectroscopy, oligonucleotide microarray analysis, allele-specific hybridization, allele-specific PCR, and/or sequencing. See U.S. Patent No. 8,785,128, which is hereby incorporated by reference in its entirety.

[00203] Alternatively, the gene variant may also be detected by detecting the mutant PNPLA3 protein, e.g., with an antibody or any other binding molecules. An antibody binding assay, such as a Western blot or ELISA, may be performed. The mutant protein can also be detected using protein mass spectroscopy methods, including mass spectroscopy (MS), tandem mass spectroscopy (MS/MS), liquid chromatography-mass spectrometry (LC-MS) gas

chromatography-mass spectrometry (GC-MS), or high-performance liquid chromatography (HPLC) mass spectroscopy (LC-MS or LC-MS/MS). Any appropriate mass analyzer may be used, including, but not limited to, time-of-flight [TOF], orbitraps, quadruples and ion traps.

[00204] In some embodiments, the subject may have been biopsied or otherwise sampled prior to the diagnosis described herein. In that case, detection of the genetic marker of PNPLA3- I148M, whether DNA-based or protein-based, may be performed using the biopsy sample or any other biological sample already obtained from the subject.

[00205] In some embodiments, the presence of a PNPLA3 gene variant may be determined or already have been determined in the subject. Such determination or prior determination may be performed by a commercial or non-commercial third-party genetic test or genotyping kit.

Commercial genotyping kits are available from a variety of vendors, including 23andMe, AncestryDNA, HelixDNA, Vitagene DNA Test, National Geographic DNA Test Kit: Geno2.0, and DNA Consultants. Determination or prior determination of the presence of a PNPLA3 gene variant may also be determined by a healthcare provider. In some embodiments, a biological sample is obtained from the subject and a dataset comprising the genomic or proteomic data from the biological sample is obtained.

[00206] In some embodiments, the methods for identifying a subject for the PCSK9 or ANGPTL3 treatment may further include a step of measuring hepatic triglyceride in the subject. As a non-limiting example, the hepatic triglyceride content may be measured using proton magnetic resonance spectroscopy (¹H-MRS). Proton magnetic resonance spectroscopy allows for accurate, quantitative noninvasive assessment of tissue fat content.

[00207] In some embodiments, the methods for identifying a subject for the PCSK9 or ANGPTL3 treatment may further include a step of determining if the subject has or is predisposed to having a PNPLA3 -related disorder (e.g., NAFLD, NASH, and/or ALD). Such disorders may be assessed using conventional clinical diagnosis. For example, fatty liver or hepatic steatosis may be determined inter alia using computer-aided tomography (CAT) scan or nuclear magnetic resonance (NMR), such as proton magnetic resonance spectroscopy. Diagnosis is generally clinically defined as having hepatic triglyceride content greater than 5.5% volume/volume. Indicators of predisposition to fatty liver may include obesity, diabetes, insulin resistance, and alcohol ingestion. [00208] In some embodiments, the methods may further include performing a liver biopsy, an imaging technique such as ultrasound, a liver function test, a fibrosis test, or any other techniques described in Yki-Jarvinen, H. Diabetologia (2016) 59: 1104; Madrazo Gastroenterol Hepatol (N Y). 2017 Jun; 13(6): 378-380, which are hereby incorporated by reference in their entirety.

[00209] In some embodiments, the diagnostic testing may be performed by others, such as a medical laboratory or clinical test provider.

[00210] In some embodiments, the methods may further include verifying the validity of the genotype and/or protein abnormality in silico.

[00211] In some embodiments, a PCSK9 or ANGPTL3 targeted therapy is any therapy that directly or indirectly impacts PCSK9 or ANGPTL3 activity or expression. PCSK9 or ANGPTL3 gene expression can be measured via any known RNA, mRNA, or protein quantitative assay, including, but not limited to, as RNA-seq, quantitative reverse transcription PCR (qRT-PCR), RNA microarrays, fluorescent in situ hybridization (FISH), antibody binding, Western blotting, ELISA, or any other assay known in the art.

[00212] Non-human animal data, such as mouse in vivo data, showing the impact of small molecule inhibitors or RNAi knockdown of members of the multiple pathways that regulate PCSK9 or ANGPTL3 expression can be used as evidence that the therapy, when administered to a human, modulates expression of the PCSK9 or ANGPTL3 genes. In addition, data obtained in human hepatocytes, including hepatocytes from humans who harbor the PNPLA3 G allele at SNP rs738409, can be used to identify a therapy that modulates expression of the PCSK9 or ANGPTL3 genes

Compounds and compositions

Small molecules

[00213] In some embodiments, compounds used to modulate PCSK9 or ANGPTL3 gene expression can include small molecules. As used herein, the term“small molecule” refers to any molecule having a molecular weight of 5000 Daltons or less. In certain embodiments, at least one small molecule compounds described herein is applied to a genomic system to alter the boundaries of an insulated neighborhood and/or disrupt signaling centers, thereby modulating the expression of PCSK9 or ANGPTL3.

[00214] A small molecule screen may be performed to identify small molecules that act through signaling centers of an insulated neighborhood to alter gene signaling networks which may modulate expression of a select group of disease genes. For example, known signaling agonists/antagonists may be administered. Credible hits are identified and validated by the small molecules that are known to work through a signaling center and modulate expression of the target gene PCSK9 or ANGPTF3.

[00215] In some embodiments, small molecule compounds capable of modulating PCSK9 or ANGPTF3 expression include, but are not limited to, those shown in Table 2. Any one or more of such compounds may be administered to a subject to treat a PCSK9- or ANGPFT3 -associated dyslipidemia disorder. Such dyslipidemia disorders include NAFFD, NASH, and/or AFD.

QSI-027

[00216] In some embodiments, compounds capable of modulating the expression of the PCSK9 or ANGPTF3 gene include OSI-027, or a derivative or an analog thereof. OSI-027, also known as ASP4786, is a selective and potent dual inhibitor of mTORCl and mTORC2. It has a CAS number of 936890-98-1 and PubChem Compound ID of 72698550. The structure of OSI- 027 is shown below:

[00217] OSI-027 inhibits mTORCl and mTORC2 with IC50 values of 22 nM and 65 nM, respectively. OSI-027 also inhibits mTOR signaling of phospho-4E-BPl with an IC50 of 1 mM and 4E-BP1, Akt, and S6 phosphorylation in vivo. OSI-027 shows anti-proliferative activity against a variety of tumor xenografts, including leukemia cell lines U937, KG-l, KBM-3B, ML- 1, HL-60, and MEG-01, and breast cancer cells in vitro.

PF-04691502

[00218] In some embodiments, compounds capable of modulating the expression of the PCSK9 or ANGPTL3 gene include PF-04691502, or a derivative or an analog thereof. PF- 04691502 is a RI3K(a/b/d/g) and mTOR dual inhibitor. It has a CAS number of 1013101-36-4 and PubChem Compound ID of 25033539. The structure of PF-04691502 is shown below:

[00219] PF-04691502 inhibits mTORCl with an IC50 value of 32 nM and inhibits the activation of downstream mTOR and PI3K effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. PF-04691502 shows anti-proliferative activity against a variety of non-small cell lung carcinoma xenografts.

LY 2157299

[00220] In some embodiments, compounds capable of modulating the expression of the PCSK9 or ANGPTL3 gene include LY2157299, or a derivative or an analog thereof.

LY2157299, also known as Galunisertib, is a Transforming Growth Factor (TGF) b receptor I (TGF RI) inhibitor. It has a CAS number of 700874-72-2 and PubChem Compound ID of 10090485. The structure of PF-04691502 is shown below:

[00221] LY2157299 inhibits TGF RI with IC50 value of 56 nM and inhibits TGF RI-induced

Smad2 phosphorylation. LY2157299 stimulates hematopoiesis and angiogenesis in vitro and in vivo. LY2157299 shows anti-proliferative activity against Calu6 and MX1 xenografts in mice. JR-AB2-011

[00222] In some embodiments, compounds capable of modulating the expression of the PCSK9 or ANGPTL3 gene include JR-AB2-011, or a derivative or an analog thereof. JR-AB2- 011 is an mTORC2 inhibitor that blocks the interaction of mTOR and RICTOR. It has a CAS number of 329182-61-8. The structure of JR-AB2-011 is shown below:

Other comyounds

[00223] Any appropriate compound that modulates a PCSK9 or ANGPTL3 transcription factor or pathway to alter PCSK9 or ANGPTL3 gene expression may be used in the present invention. Exemplary compounds are show in Table 2 and Table 7. CAS Number or CAS Registry Number refers to the unique numerical identifier assigned by the Chemical Abstracts Service (CAS) to every chemical substance described in the scientific literature.

Table 2. Compounds for modulating PCSK9 or ANGPTL3 gene expression

Screening Methods

[00224] In another aspect, provided herein are methods to identify candidate compounds based on biochemical activity or activities as described elsewhere in the specification. In an embodiment, a candidate compound with mTOR inhibitory activity inhibits both the mTORC 1 and mTORC2 complexes. In an embodiment, a candidate compound with mTORC2 inhibitory activity inhibits mTORC2 but not mTORC 1. As shown in Example 9, inhibition of mTORC 1 alone via rapamycin treatment is insufficient to decrease PCSK9 expression, while

mTORC l/mTORC2 inhibitors such as OSI-027 decreased PCSK9 expression. Thus, inhibition of mTORC2, but not mTORC 1, is necessary to decrease PCSK9 expression. In an embodiment, a candidate compound selected for further study may thus inhibit either mTORC2 alone, or mTORC 1 and mTORC2.

[00225] In an embodiment, a candidate compound lacks PI3K inhibitory activity. As shown in Example 12, compounds that inhibit mTOR and PI3K also induced higher insulin and serum glucose levels in mice. Thus, inhibition of PI3K to decrease PCSK9 or ANGPTL3 expression also resulted in adverse effects. In an embodiment, a candidate compound selected for further study may thus lack PI3K or RI3Kb inhibitory activity.

[00226] In an embodiment, the activity is mTORC2 inhibitory activity. In an embodiment, the activity is lack of PI3K inhibitory activity. In an embodiment, the activity is lack of RI3Kb inhibitory activity. In an embodiment, the activity is lack of DNA-PK inhibitory activity. In an embodiment, the activity is lack of PIP4K2C inhibitory activity. In an embodiment, the activity is lack of ability to induce hyperinsulinemia. In an embodiment, the activity is lack of ability to induce hyperglycemia. In an embodiment, the activity is PCSK9 or ANGPTL3 gene expression inhibitory activity.

[00227] In some embodiments, the activity is mTOR inhibitory activity. In some

embodiments, the activity is mTORC2 inhibitory activity. In some embodiments, the activity is PCSK9 or ANGPTL3 gene expression inhibitory activity.

[00228] In some embodiments, the activity is lack of PI3K inhibitory activity. In some embodiments, the activity is lack of RI3Kb inhibitory activity. In some embodiments, the activity is lack of DNA-PK inhibitory activity. In some embodiments, the activity is lack of PIP4K2C inhibitory activity. In some embodiments, the activity is lack of the ability to induce hyperinsulinemia. In some embodiments, the activity is lack of the ability to induce

hyperglycemia. [00229] In an embodiment, the activity is any two of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any three of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any four of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce

hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any five of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any six of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any seven of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any eight of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce

hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity. In an embodiment, the activity is any nine of mTOR inhibitory activity, mTORC2 inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of PIP4K2C inhibitory activity, lack of the ability to induce hyperinsulinemia, lack of the ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity.

Assays

[00230] Inhibitory activity of the candidate compound can be determined via an appropriate method known in the art. Inhibition assays include enzymatic assay that measure changes in phosphorylation of kinase target proteins, or binding assays that measure binding of a candidate compound to the kinase target protein. In some embodiments, the assay is a biochemical assay. In some embodiments, the assay is in a cell. In some embodiments, the assay is in a cell lysate.

[00231] For enzymatic assays, any appropriate assay may be used, such as antibody assays including Western blots or ELISAs; or biochemical assays that measure incorporation of radioactive or fluorescent ATP into kinase substrates (Ma et al, Expert Opin Drug Discov, 2008 3(6):607-62l which is hereby incorporated by reference in its entirety).

[00232] Radiometric assays include biochemical assays using purified kinase proteins and substrates. The kinase reaction is performed in solution in the presence of ³²R-g-ATR, ³³R-g- ATP, or ³⁵S-thio-labeled ATP and the candidate inhibitory compound. The radioisotope labeled substrate products are column purified and/or bound to filters or membranes and the free ATP is washed away, allowing for quantification of only the phosphorylated substrate. The radioisotope labeled protein can be measured via autoradiography or phosphorimager techniques known in the art.

[00233] An alternative to columns or membranes is to use a scintillation proximity assay, in which the radiolabeled proteins of interest are bound to beads that contain a scintillant that can emit light after stimulation by beta particles or auger elements. The stimulation of the scintillant occurs only when radiolabeled molecules are bound to the beads. The emission of light can be measured via a scintillation analyzer or flow scintillation analyzer. Commercial radioisotope and scintillation kits are available from multiple vendors, including PerkinElmer and Reaction Biology.

[00234] Fluorescent and luminescent assays include biochemical assays using purified kinase proteins and substrates. Any appropriate fluorescent or luminescent assay, including but not limited to, fluorescence or luminescent intensity, fluorescence polarization, fluorescence resonance energy transfer (FRET), or time resolved fluorescence resonance energy transfer (TRF-FRET).

[00235] Luminescent assays measure the amount of ADP in a sample after a kinase has phosphorylated a substrate using ATP. The remaining ATP after the kinase reaction is depleted and removed, leaving only the newly made ADP in the solution. A detection reagent is added that simultaneously converts the ADP to ATP and the new ATP to light using a

luciferase/luciferin reaction. Commercial luminescent kits are available from Promega (ADP- Glo) and kits specific to PI3 kinases are available as well (ADP-Glo Lipid Kinase Kit).

[00236] Fluorescence intensity assays measure the amount of ADP in a sample after a kinase has phosphorylated a substrate using ATP. The newly made ADP is converted to ADHP (10- Acetyl-3,7-dihydroxyphenoxazine) and linked to hydrogen peroxide, resulting in the synthesis of fluorescent Resorufm. The signal produced by the Resorufm is proportional to the amount of the ADP in the sample, and therefore the activity of the kinase. Compounds that inhibit kinase activity result in less fluorescence signal. Commercial FI kits are available from DiscovRx (ADP Hunter Kit).

[00237] FRET analysis is based on donor and acceptor fluorophores in proximity to each other. An excited donor fluorophore transfers non-radiative energy to a proximal acceptor fluorophore, resulting in excitation and photon emittance of the acceptor fluorophore. Various methods of utilizing FRET for kinase assays are known in the art. In one method, a kinase is mixed with a acceptor fluorophore-tagged substrate and ATP, and the kinase phosphorylates the labeled substrate. Next, a terbium-labeled antibody specific for the phosphorylated substrate is added. The terbium molecule acts a donor fluorophore and transfers energy to the acceptor fluorophore, which is then quantified. The amount of FRET signal is proportional to the amount of phosphorylated substrate and thus the activity of the kinase. Commercial FRET assays for Class I and Class II PI3 kinases are available, including the HTS Kit and HTRF Enzyme Assay Kits from Millipore Sigma. Additional FRET kinase kits are the LANCE Ultra or Classic kits from PerkinElmer, and the LanthaScreen and Z’-LYTE kinase assay kit from ThermoFisher Scientific.

[00238] Detection of phosphorylated substrates can also be accomplished via antibody binding assays, such as ELIS As or Western blots. These assays can be done on both biochemical samples and cell based samples. In the case of a biochemical assay, the substrate is incubated with a kinase, ATP, and optionally a candidate compound. In a cell based assay, the cell is incubated with a candidate compound and then lysed for protein analysis. Once the biochemical kinase reaction is complete or the cell is lysed, the substrate protein or lysate is capture to a membrane by filtration or gel electrophoresis and membrane blotting. An antibody specific to the phosphorylated substrate is added and detected via binding of a fluorescent or enzyme-linked secondary antibody. Total protein can also be measured via antibody detection of total protein, phosphorylated and unphosphorylated via use of a second antibody that is not specific to the phosphorylated substrate. ELISA kits for phosphorylated mTOR and PI3K substrates, including AKT, S6, NDRG1, SGK1, PKC, PIP3, p53 and CHK2 are available from a variety of manufacturers, including Millipore Sigma, Cell Signaling, and Abeam. Antibodies for phosphorylated mTOR, PI3K, DNA-Pk, and PIP4K2C substrates, including AKT, S6, NDRG1, SGK1, PKC, PIP3, p53 and CHK2 are available from a variety of manufacturers, including Cell Signaling, Abeam, and Santa Cruz Biotech.

[00239] For binding assays, any appropriate binding assay known in the art may be used, including but not limited to differential scanning fluorimetry, also known as thermostability shift assay; surface plasmon resonance; or any other appropriate method known in the art. In a differential scanning fluorimetry assay, a target protein is incubated with and without a candidate compound and a fluorescent dye such as SyproOrange. The mixture is heated over a temperature gradient and the thermal unfolding of the protein is assessed via the dye, which is fluorescent in a nonpolar environment and quenched in an aqueous environment. Thus, as the protein unfolds, dye binds to the exposed core of the protein, resulting in a quantifiable increase in the fluorescent intensity of the mixture. Binding of a compound to the target protein stabilizes the protein and shifts the melting temperature (Tm) of the protein. Kinase inhibitor screening using differential scanning fluorimetry is described in Rudolf AF et al, PLoS ONE June 2014, https://doi.org/l0. l37l/joumal.pone.0098800, hereby incorporated by reference in its entirety. Kits for differential scanning fluorimetry or thermoshift assays are available from various vendors, including ThermoFisher Scientific (Protein Thermal Shift Starter Kit) and Biotium (GloMelt).

[00240] Surface plasmon resonance assays may also be used to assess candidate compound binding to kinases. Surface plasmon resonance is a commonly used technique in the protein and molecule binding field to measure the binding of molecules with high sensitivity. SPR has been used to measure binding of small molecules to various protein factors (see e.g, Kennedy AE et al, J. Bio Screen, 2016: 21(1) 96-100 doiTO. l 177/1087057/15607814, hereby incorporated by reference in its entirety). SPR systems and reagents are commercially available from GE Healthcare under the BIAcore brand.

Thresholds

[00241] Inhibitory activity of the candidate compound includes quantifying the IC50 or EC50 of the compound to provide an inhibitory threshold. IC50 or EC50 levels can be the compound enzymatic inhibition level or the compound binding level. An inhibitory threshold to identify a candidate compound can be selected to identify a possible lead compound that is later refined via structure refinement and design informed by structure-activity studies, medicinal chemistry- based studies, or other studies know in the art. An inhibitory threshold can be at least about 100 mM, 95 mM, 90 pM, 85 pM, 80 pM, 75 pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. An inhibitory threshold can be a range of at least 1-100 nM, 1-10 nM, 1-5 nM, 5-10 nM, 10-15 nM, 15-20 nM, 20-25 nM, 25-30 nM, 30-35 nM, 35-40 nM, 40-45 nM, 45-50 nM, 50-55 nM, 55-60 nM, 60-65 nM, 65-70 nM, 70-75 nM, 75-80 nM, 80-85 nM, 85-90 nM, 90-95 nM, 95-100 nM, 1-100 pM, 1-10 pM, 1-5 pM, 5-10 pM, 10-15 pM, 15-20 pM, 20-25 pM, 25-30 pM, 30-35 pM, 35-40 pM, 40-45 pM, 45-50 pM, 50-55 pM, 55-60 pM, 60-65 pM, 65-70 pM, 70-75 pM, 75-80 pM, 80-85 pM, 85-90 pM, 90-95 pM, or 95-100 pM.

Compound Library

[00242] Candidate compounds can be selected from any available library or commercial vendor. Candidate compounds can also by synthesized by the applicant or a third party company using chemistry methods generally known in the art. Libraries of candidate PBK/mTOR/Akt small molecule inhibitors are available from various commercial vendors, including the 223 compound library PBK Akt/mTOR Compound Library from MedChemExpress, catalogue no. HY-L015 and the 145 compound DiscoveryProbe™ PBK/Akt/MTOR Compound Library from ApexBio, catalogue no. L1034. General small molecule libraries are also available from commercial vendors, including the 1496 compound DiscoveryProbe™ FDA-Approved Drug Library from ApexBio, catalogue no. L 1021; the 493 compound DiscoveryProbe™ Kinase Inhibitor Library from ApexBio, catalogue no. L1024, the 1983 compound DiscoveryProbe™ Inhibitor Library from ApexBio, catalogue no. L1048; and the 7853 compound Bioactive Compound Library Plus from MedChemExpress, catalogue no. HY-L001P.

Pharmaceutical compositions

[00243] Methods for treatment of high low density lipoprotein (LDL) cholesterol diseases, liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and/or alcoholic liver disease (ALD) are also encompassed by the present invention. Said methods of the invention include administering a therapeutically effective amount of PCSK9 or ANGPTL3 transcription factor or signaling pathway inhibitor. The PCSK9 or ANGPTL3 transcription factor or signaling pathway inhibitors of the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the PCSK9 or ANGPTL3 transcription factor or signaling pathway inhibitors, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[00244] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[00245] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.

[00246] Whether it is a polypeptide, antibody, nucleic acid, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a“therapeutically effective amount” or

“prophylactically effective amount”(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, l6th edition, Osol, A. (ed), 1980. [00247] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

EXAMPLES

[00248] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[00249] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et ah, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's

Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company,

1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) Vols A and B(l992).

Example 1. Experimental procedures

Hepatocyte cell culture

[00250] Cryopreserved hepatocytes were cultured in plating media for 16 hours, transferred to maintenance media for 4 hours. Cultured on serum-free media for 2 hours, then a compound was added. The hepatocytes were maintained on the serum-free media for 16 hours prior to gene expression analysis. Primary Human Hepatocytes were stored in the vapor phase of a liquid nitrogen freezer (about -l30°C).

[00251] To seed the primary human hepatocytes, vials of cells were retrieved from the LN2 freezer, thawed in a 37°C water bath, and swirled gently until only a sliver of ice remained.

Using a lOml serological pipet, cells were gently pipetted out of the vial and gently pipetted down the side of 50mL conical tube containing 20mL cold thaw medium. The vial was rinsed with about lmL of thaw medium, and the rinse was added to the conical tube. Up to 2 vials may be added to one tube of 20mL thaw medium. [00252] The conical tube(s) were gently inverted 2-3 times and centrifuged at 100 g for 10 minutes at 4°C with reduced braking (e.g. 4 out of 9). The thaw medium slowly was slowly aspirated to avoid the pellet. 4mL cold plating medium was added slowly down the side (8mL if combined 2 vials to 1 tube), and the vial was inverted gently several times to resuspend cells.

[00253] Cells were kept on ice until IOOmI of well-mixed cells were added to 400pl diluted Trypan blue and mixed by gentle inversion. They were counted using a hemocytometer (or Cellometer), and viability and viable cells/mL were noted. Cells were diluted to a desired concentration and seeded on collagen I-coated plates. Cells were pipetted slowly and gently onto plate, only 1-2 wells at a time. The remaining cells were mixed in the tubes frequently by gentle inversion. Cells were seeded at about 8.5xl0⁶ cells per plate in 6mL cold plating medium (lOcm). Alternatively, l.5xl0⁶ cells per well for a 6-well plate (lmL medium/well); 7xl0⁵ cells per well for l2-well plate (0.5mL/well); or 3.75xl0⁵ cells per well for a 24-well plate

(0.5mL/well)

[00254] After all cells and medium were added to the plate, the plate was transferred to an incubator (37°C, 5% CO2, about 90% humidity) and rocked forwards and backwards, then side to side several times each to distribute cells evenly across the plate or wells. The plate(s) were rocked again every 15 minutes for the first hour post-plating. About 4 hours post-plating (or first thing the morning if cells were plated in the evening), cells were washed once with PBS and complete maintenance medium was added. The primary human hepatocytes were maintained in the maintenance medium and transferred to fresh medium daily.

Starvation and compound treatment ofheOatocvtes

[00255] Two to three hours before treatment, cells cultured as described above were washed with PBS and the medium was changed to either: fresh maintenance medium (complete) or modified maintenance medium 4b.

[00256] Compound stocks were prepared at lOOOx final concentration and added in a 2-step dilution to the medium to reduce risk of a compound precipitating out of solution when added to the cells, and to ensure reasonable pipetting volumes. One at a time, each compound was first diluted lO-fold in warm (about 37°C) modified maintenance medium (initial dilution = ID), mixed by vortexing, and the ID was diluted lOO-fold into the cell culture (e.g. 5. Imΐ into 1 well of a 24-well plate containing 0.5mL medium). The plate was mixed by carefully swirling and after all wells were treated and returned to the incubator overnight. If desired, separate plates/wells were treated with vehicle-only controls and/or positive controls. If using multi-well plates, controls were included on each plate. After about 18 hours, cells were harvested for further analysis, e.g., ChIP-seq, RNA-seq, ATAC-seq, etc.

Media composition

[00257] The thaw medium contained 6mL isotonic percoll and l4mL high glucose DMEM (Invitrogen #11965 or similar). The plating medium contained lOOmL Williams E medium (Invitrogen #A 1217601, without phenol red) and the supplement pack #CM3000 from

ThermoFisher Plating medium containing 5mL FBS, 10m1 dexamethasone, and 3.6mL plating/maintenance cocktail. Stock trypan blue (0.4%, Invitrogen #15250) was diluted 1:5 in PBS.

[00258] The ThermoFisher complete maintenance medium contained supplement pack #CM4000 (lpl dexamethasone and 4mF maintenance cocktail) and lOOmF Williams E

(Invitrogen #A1217601, without phenol red).

[00259] The modified maintenance media had no stimulating factors (dexamethasone, insulin, etc.), and contained lOOmF Williams E (Invitrogen #A1217601, without phenol red), lmL L- Glutamine (Sigma #G7513) to 2mM, l.5mL HEPES (VWR #J848) to l5mM, and 0.5mL penicillin/streptomycin (Invitrogen #15140) to a final concentration of 50U/mL each.

DNA purification

[00260] DNA purification was conducted as described in Ji et ah, PNAS 112(12):3841-3846 (2015) Supporting Information, which is hereby incorporated by reference in its entirety. One milliliter of 2.5 M glycine was added to each plate of fixed cells and incubated for 5 minutes to quench the formaldehyde. The cells were washed twice with PBS. The cells were pelleted at 1,300 g for 5 minutes at 4°C. Then, 4 ^c 10⁷ cells were collected in each tube. The cells were lysed gently with 1 mL of ice-cold Nonidet P-40 lysis buffer containing protease inhibitor on ice for 5 minutes (buffer recipes are provided below). The cell lysate was layered on top of 2.5 volumes of sucrose cushion made up of 24% (wt/vol) sucrose in Nonidet P-40 lysis buffer. This sample was centrifuged at 18,000 g for 10 minutes at 4°C to isolate the nuclei pellet (the supernatant represented the cytoplasmic fraction). The nuclei pellet was washed once with PBS/l mM EDTA. The nuclei pellet was resuspended gently with 0.5mL glycerol buffer followed by incubation for 2 minutes on ice with an equal volume of nuclei lysis buffer. The sample was centrifuged at 16,000 g for 2 minutes at 4°C to isolate the chromatin pellet (the supernatant represented the nuclear soluble fraction). The chromatin pellet was washed twice with PBS/l mM EDTA. The chromatin pellet was stored at - 80°C. [00261] The Nonidet P-40 lysis buffer contained 10 mM Tris HCl (pH 7.5), 150 mM NaCl, and 0.05% Nonidet P-40. The glycerol buffer contained 20 mM Tris HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, and 50% (vol/vol) glycerol. The nuclei lysis buffer contained 10 mM Hepes (pH 7.6), 1 mM DTT, 7.5 mM MgCh, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, and 1% Nonidet P-40.

Chromatin immunoOrecwitation sequencing (ChIP-sea)

[00262] ChIP-seq was performed using the following protocol for primary hepatocytes and HepG2 cells to determine the composition and confirm the location of signaling centers.

i. Cell cross-linking

[00263] 2 x 10⁷ cells were used for each run of ChIP-seq. Two ml of fresh 11% formaldehyde

(FA) solution was added to 20ml media on l5cm plates to reach a 1.1% final concentration.

Plates were swirled briefly and incubated at room temperature (RT) for 15 minutes. At the end of incubation, the FA was quenched by adding lml of 2.5M Glycine to plates and incubating for 5 minutes at RT. The media was discarded to a 1L beaker, and cells were washed twice with 20ml ice-cold PBS. PBS (lOml) was added to plates, and cells were scraped off the plate. The cells were transferred to l5ml conical tubes, and the tubes were placed on ice. Plates were washed with an additional 4ml of PBS and combined with cells in l5ml tubes. Tubes were centrifuged for 5 minutes at 1,500 rpm at 4°C in a tabletop centrifuge. PBS was aspirated, and the cells were flash frozen in liquid nitrogen. Pellets were stored at -80°C until ready to use.

ii. Pre-block magnetic beads

[00264] Thirty pi Protein G beads (per reaction) were added to a 1.5ml Protein LoBind Eppendorf tube. The beads were collected by magnet separation at RT for 30 seconds. Beads were washed 3 times with lml of blocking solution by incubating beads on a rotator at 4°C for 10 minutes and collecting the beads with the magnet. Five pg of an antibody was added to the 250pl of beads in block solution. The mix was transferred to a clean tube, and rotated overnight at 4°C. On the next day, buffer containing antibodies was removed, and beads were washed 3 times with l.lml blocking solution by incubating beads on a rotator at 4°C for 10 minutes and collecting the beads with the magnet. Beads were resuspended in 50m1 of block solution and kept on ice until ready to use.

iii. Cell lvsis. genomic fragmentation and chromatin immunonrecinitation

[00265] COMPLETE® protease inhibitor cocktail was added to lysis buffer 1 (LB1) before use. One tablet was dissolved in lml of H2O for a 50x solution. The cocktail was stored in aliquots at -20°C. Cells were resuspended in each tube in 8ml of LB1 and incubated on a rotator at 4°C for 10 minutes. Nuclei were spun down at 1,350 g for 5 minutes at 4°C. LB1 was aspirated, and cells were resuspended in each tube in 8ml of LB2 and incubated on a rotator at 4°C for 10 minutes.

[00266] A COVARIS^® E220EVOLUTION¹'¹ ultrasonicator was programmed per the manufacturer’s recommendations for high cell numbers. HepG2 cells were sonicated for 12 minutes, and primary hepatocyte samples were sonicated for 10 minutes. Lysates were transferred to clean l.5ml Eppendorf tubes, and the tubes were centrifuged at 20,000 g for 10 minutes at 4°C to pellet debris. The supernatant was transferred to a 2ml Protein LoBind Eppendorf tube containing pre-blocked Protein G beads with pre-bound antibodies. Fifty pl of the supernatant was saved as input. Input material was kept at -80°C until ready to use. Tubes were rotated with beads overnight at 4°C.

iv. Wash, elution, and cross-link reversal

[00267] All washing steps were performed by rotating tubes for 5 minutes at 4°C. The beads were transferred to clean Protein LoBind Eppendorf tubes with every washing step. Beads were collected in 1 5ml Eppendorf tube using a magnet. Beads were washed twice with 1. lml of sonication buffer. The magnetic stand was used to collect magnetic beads. Beads were washed twice with 1. lml of wash buffer 2, and the magnetic stand was used again to collect magnetic beads. Beads were washed twice with l.lml of wash buffer 3. All residual Wash buffer 3 was removed, and beads were washed once with l.lml TE + 0.2% Triton X-100 buffer. Residual TE + 0.2% Triton X-100 buffer was removed, and beads were washed twice with TE buffer for 30 seconds each time. Residual TE buffer was removed, and beads were resuspended in 300m1 of ChIP elution buffer. Two hundred fifty mΐ of ChIP elution buffer was added to 50m1 of input, and the tubes were rotated with beads 1 hour at 65°C. Input sample was incubated overnight at 65°C oven without rotation. Tubes with beads were placed on a magnet, and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65°C oven without rotation

v. Chromatin extraction and precipitation

[00268] Input and immunoprecipitant (IP) samples were transferred to fresh tubes, and 300m1 of TE buffer was added to IP and Input samples to dilute SDS. RNase A (20mg/ml) was added to the tubes, and the tubes were incubated at 37°C for 30 minutes. Following incubation, 3m1 of 1M CaCh and 7m1 of 20mg/ml Proteinase K were added, and incubated 1.5 hours at 55°C. MaXtract High Density 2ml gel tubes (Qiagen) were prepared by centrifugation at full speed for 30 seconds at RT. Six hundred mΐ of phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction and transferred in about l.2ml mixtures to the MaXtract tubes. Tubes were spun at 16,000 g for 5 minutes at RT. The aqueous phase was transferred to two clean DNA LoBind tubes (300m1 in each tube), and 1.5m1 glycogen, 30m1 of 3M sodium acetate, and 900m1 ethanol were added. The mixture was precipitated overnight at -20°C or for 1 hour at -80°C, and spun down at maximum speed for 20 minutes at 4°C. The ethanol was removed, and pellets were washed with lml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4°C. Remnants of ethanol were removed, and pellets were dried for 5 min at RT. Twenty-five mΐ of FLO was added to each immunoprecipitant (IP) and input pellet, left standing for 5 minutes, and vortexed briefly. DNA from both tubes was combined to obtain 50m1 of IP and 50m1 of input DNA for each sample. One mΐ of this DNA was used to measure the amount of pulled down DNA using Qubit dsDNA HS assay (ThermoFisher, #Q32854). The total amount of

immunoprecipitated material ranged from several ng (for TFs) to several hundred ng (for chromatin modifications). Six mΐ of DNA was analyzed using qRT-PCRto determine enrichment. The DNA was diluted if necessary. If enrichment was satisfactory, the rest was used for library preparation for DNA sequencing.

vi. Library preparation for DNA sequencing

[00269] Libraries were prepared using NEBNext Ultra II DNA library prep kit for Illumina (NEB, #E7645) using NEBNext Multiplex Oligos for Illumina (NEB, #6609S) according to manufacturer’s instructions with the following modifications. The remaining ChIP sample (about 43 mΐ) and lpg of input samples for library preparations were brought up the volume of 50m1 before the End Repair portion of the protocol. End Repair reactions were run in a PCR machine with a heated lid in a 96-well semi-skirted PCR plate (ThermoFisher, #AB 1400) sealed with adhesive plate seals (ThermoFisher, #AB0558) leaving at least one empty well in-between different samples. Undiluted adapters were used for input samples, 1: 10 diluted adapters for 5- lOOng of ChIP material, and 1:25 diluted adapters for less than 5ng of ChIP material. Ligation reactions were run in a PCR machine with the heated lid off. Adapter ligated DNA was transferred to clean DNA LoBind Eppendorf tubes, and the volume was brought to 96.5m1 using FLO.

[00270] 200-600bp ChIP fragments were selected using SPRIselect magnetic beads (Beckman

Coulter, #B23317). Thirty mΐ of the beads were added to 96.5m1 of ChIP sample to bind fragments that are longer than 600 bp. The shorter fragments were transferred to a fresh DNA LoBind Eppendorf tube. Fifteen mΐ of beads were added to bind the DNA longer than 200bp, and beads were washed with DNA twice using freshly prepared 75% ethanol. DNA was eluted using 17m1 of 0. IX TE buffer. About 15m1 was collected. [00271] Three mΐ of size-selected Input sample and all (15m1) of the ChIP sample was used for PCR. The amount of size-selected DNA was measured using a Qubit dsDNA HS assay. PCR was run for 7 cycles of for Input and ChIP samples with about 5-l0ng of size-selected DNA, and 12 cycles with less than 5 ng of size-selected DNA. One-half of the PCR product (25m1) was purified with 22.5m1 of AMPure XP beads (Beckman Coulter, #A63880) according to the manufacturer’s instructions. PCR product was eluted with 17m1 of 0. IX TE buffer, and the amount of PCT product was measured using Qubit dsDNA HS assay. An additional 4 cycles of PCR were run for the second half of samples with less than 5ng of PCR product, DNA was purified using 22.5m1 of AMPure XP beads. The concentration was measured to determine whether there was an increased yield. Both halves were combined, and the volume was brought up to 50m1 using H2O.

[00272] A second round of purifications of DNA was run using 45 mΐ of AMPure XP beads in 17m1 of 0. IX TE, and the final yield was measured using Qubit dsDNA HS assay. This protocol produces from 20ng to lmg of PCR product. The quality of the libraries was verified by diluting Imΐ of each sample with H2O if necessary using the High Sensitivity BioAnalyzer DNA kit (Agilent, #5067-4626) based on manufacturer’s recommendations

vii. Reagents

[00273] 11% Formaldehyde Solution (50mL) contained 14.9ml of 37% formaldehyde (final cone. 11%), 1 ml of 5M NaCl (final cone. 0.1 M), IOOmI of 0.5M EDTA (pH 8) (final cone. lmM), 50m1 of 0.5M EGTA (pH 8) (final cone. 0.5mM), and 2.5 ml 1M Hepes (pH 7.5) (final cone. 50 mM).

[00274] Block Solution contained 0.5% BSA (w/v) in PBS and 500mg BSA in lOOml PBS. Block solution may be prepared up to about 4 days prior to use.

[00275] Lysis buffer 1 (LB1) (500ml) contained 25ml of 1 M Hepes-KOH, pH 7.5; l4ml of 5M NaCl; 1 ml of 0.5M EDTA, pH 8.0; 50ml of 100% Glycerol solution; 25ml of 10% NP-40; and 12.5ml of 10% Triton X-100. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00276] Lysis buffer 2 (LB2) (1000ml) contained lOml of 1 M Tris-HCL, pH 8.0; 40ml of 5 M NaCl; 2ml of 0.5M EDTA, pH 8.0; and 2ml of 0.5M EGTA, pH 8.0. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00277] Sonication buffer (500ml) contained 25ml of 1M Hepes-KOH, pH 7.5; l4ml of 5M NaCl; lml of 0.5M EDTA, pH 8.0; 50ml of 10% Triton X-100; lOml of 5% Na-deoxycholate; and 5ml of 10% SDS. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4 °C. The pH was re -checked immediately prior to use.

[00278] Proteinase inhibitors were included in the LB1, LB2, and Sonication buffer.

[00279] Wash Buffer 2 (500ml) contained 25ml of 1M Hepes-KOH, pH 7.5; 35 ml of 5M NaCl; lml of 0.5M EDTA, pH 8.0; 50ml of 10% Triton X-100; lOml of 5% Na-deoxycholate; and 5ml of 10% SDS. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4 °C. The pH was re -checked immediately prior to use.

[00280] Wash Buffer 3 (500ml) contained lOml of 1M Tris-HCL, pH 8.0; lml of 0.5M EDTA, pH 8.0; l25ml of 1M LiCl solution; 25ml of 10% NP-40; and 50ml of 5% Na- deoxycholate. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00281] ChIP elution Buffer (500ml) contained 25ml of 1 M Tris-HCL, pH 8.0; lOml of 0.5M EDTA, pH 8.0; 50ml of 10% SDS; and 4l5ml of ddHiO. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

Analysis of ChIP-seq results

[00282] All pass filter reads from each sample were trimmed of sequencing adapters using trim_galore 0.4.4 with default options. Trimmed reads were mapped against the human genome (assembly GRCh38/GCA_00000l405.15“no alt” analysis set merged with

hs38dl/GCA_000786075.2) using bwa version 0.7.15 (Li (2013) arXiv: l303.3997vl) with default parameters. Aligned read duplicates were assessed using picard 2.9.0

(http://broadinstitute.hithub.io/picard) and reads with a MAPQ<20 or those matching standard SAM flags 0x1804 were discarded. Standard QC were applied (read integrity, mapping statistics, library complexity, fragment bias) to remove unsatisfactory samples. Enriched ChIP-seq peaks were identified by comparing samples against whole cell extract controls using MACS2 version 2.1.0 (Zhang et ah, Genome Biol. (2008) 9(9):Rl37), with significant peaks selected as those with an adjusted p-value < 0.01. Peaks overlapping known repetitive“blacklist” regions (ENCODE Project Consortium, Nature (2012) 489(7414:57-74) were discarded. ChIP-seq signals were also normalized by read depth and visualized using the UCSC browser.

RNA-sea

[00283] This protocol is a modified version of the following protocols: MagMAX m rVana Total RNA Isolation Kit User Guide (Applied Biosystems #MAN00l 1131 Rev B.0), NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490), and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420) (New England Biosystems #E7490l).

[00284] The MagMAX mirW ana kit instructions (the section titled“Isolate RNA from cells” on pages 14-17) were used for isolation of total RNA from cells in culture. Two hundred pl of Lysis Binding Mix was used per well of the multiwell plate containing adherent cells (usually a 24-well plate).

[00285] For mRNA isolation and library prep, the NEBNext Poly(A) mRNA Magnetic Isolation Module and Directional Prep kit was used. RNA isolated from cells above was quantified, and prepared in 500pg of each sample in 50pl of nuclease-free water. This protocol may be run in microfiige tubes or in a 96-well plate.

[00286] The 80% ethanol was prepared fresh, and all elutions are done in 0.1X TE Buffer. For steps requiring Ampure XP beads, beads were at room temperature before use. Sample volumes were measured first and beads were pipetted. Section 1.9B (not 1.9A) was used for NEBNext Multiplex Oligos for Illumina (#E6609). Before starting the PCR enrichment, cDNA was quantified using the Qubit (DNA High Sensitivity Kit, ThermoFisher #Q32854). The PCR reaction was run for 12 cycles.

[00287] After purification of the PCR Reaction (Step 1.10), the libraries were quantified using the Qubit DNA High Sensitivity Kit. Imΐ of each sample were diluted to l-2ng/pl to run on the Bioanalyzer (DNA High Sensitivity Kit, Agilent # 5067-4626). If Bioanalyzer peaks were not clean (one narrow peak around 300bp), the AMPure XP bead cleanup step was repeated using a 0.9X or 1.0X beads:sample ratio. Then, the samples were quantified again with the Qubit, and run again on the Bioanalyzer (l-2ng/pl).

[00288] Nuclear RNA from INTACT-purified nuclei or whole neocortical nuclei was converted to cDNA and amplified with the Nugen Ovation RNA-seq System V2. Libraries were sequenced using the Illumina HiSeq 2500.

RNA-seq data analysis

[00289] All pass filter reads from each sample were mapped against the human genome (assembly GRCh38/GCA_00000l405.15“no alt” analysis set merged with

hs38dl/GCA_000786075.2) using two pass mapping via STAR version 2.5.3a (alignment parameters alignIntronMin=20; alignIntronMax= 1000000; outFilterMismatchNmax=999;

outFilterMismatchNoverLmax=0.05; outFilterType=BySJout; outFilterMultimapNmax=20; alignS JoverhangMin=8; alignSJDBoverhangMin=l; alignMatesGapMax= 1000000) (Dobin et ah, Bioinformatics (2012) 29(1): 15-21). Genomic alignments were converted to transcriptome alignments based on reference transcript annotations from The Human GENCODE Gene Set release 24 (Harrow et al, Genome Res. (2012) 22(9): 1760-1774). Using unique and multimapped transcriptomic alignments, gene-level abundance estimates were computed using RSEM version 1.3.0 (Li and Dewey, BMC Bioinformatics (2011) 12:323) in a strand-aware manner, and including confidence interval sampling calculations, to arrive at posterior mean estimates (PME) of abundances (counts and normalized FPKM - fragments per kilobase of exon per million mapped fragments) from the underlying Bayesian model. Standard QC were applied (read integrity, mapping statistics, library complexity, fragment bias) to remove unsatisfactory samples. Differential gene expression was computed using the negative binomial model implemented by DESeq2 version 1.16.1 (Love et al., Genome Biol. (2014) 15(12): 550). Log2 fold change and significance values were computed using PME count data (with replicates explicitly modeled versus pan-experiment controls), median ratio normalized, using maximum likelihood estimation rather than maximum a posteriori, and disabling the use of Cook’s distance cutoff when determining acceptable adjusted p-values. Significantly differential genes were assigned as those with an adjusted p-value < 0.01, a log2 fold change of >=l or <=-l, and at least one replicate with PME FPKM >=l. RNA-seq signals were also normalized by read depth and visualized using the UCSC browser.

ATAC-seq

[00290] Hepatocytes were seeded overnight, then the serum and other factors were removed. After 2-3 hours, the cells were treated with the compound and incubated overnight. The cells were harvested and the nuclei were prepared for the transposition reaction. 50,000 bead bound nuclei were transposed using Tn5 transposase (Illumina FC-121-1030) as described in Mo et al., 2015, Neuron 86, 1369-1384, which is hereby incorporated by reference in its entirety. After 9- 12 cycles of PCR amplification, libraries were sequenced on an Illumina HiSeq 2000. PCR was performed using barcoded primers with extension at 72°C for 5 minutes, PCR, then the final PCR product was sequenced.

[00291] All obtained reads from each sample were trimmed using trim galorc 0.4.1 requiring Phred score > 20 and read length > 30 for data analysis. The trimmed reads were mapped against the human genome (hgl9 build) using Bowtie2 (version 2.2.9) with the parameters: -t -q -N 1 -L 25 -X 2000 no-mixed no-discordant. All unmapped reads, non-uniquely mapped reads and PCR duplicates were removed. All the ATAC-seq peaks were called using MACS2 with the parameters—nolambda -nomodel -q 0.01— SPMR. The ATAC-seq signal was visualized in the UCSC genome browser. ATAC-seq peaks that were at least 2 kb away from annotated promoters (RefSeq, Ensemble and UCSC Known Gene databases combined) were selected as distal ATAC- seq peaks.

qRT-PCR

qRT-PCR was performed as described in North et al, PNAS, 107(40) 17315-17320 (2010), which is hereby incorporated by reference in its entirety. qRT-PCR was performed with cDNA using the iQ5 Multicolor rtPCR Detection system from BioRad with 60°C annealing.

[00292] Analysis of the fold changes in expression as measured by qRT-PCR were performed using the technique below. The control was DMSO, and the treatment was the selected compound (CPD). The internal control was GAPDH or B-Actin, and the gene of interest is the target. First, the averages of the 4 conditions were calculated for normalization:

DMSO: GAPDH, DMSO:Target, CPD: GAPDH, and CPD:Target. Next, the ACT of both control and treatment were calculated to normalize to internal control (GAPDH) using (DMSO: Target) - (DMSO:GAPDH) = ACT control and (CPD:Target) - (CPD: GAPDH) = ACT experimental. Then, the AACT was calculated by ACT experimental - ACT control. The Expression Fold Change was calculated by 2-( AACT) (2 -fold expression change was shown by RNA-Seq results provided herein).

Chromatin Interaction Analysis by Paired-End Tag Sequencing ( ChlA-PET )

[00293] ChlA-PET was performed as previously described in Chepelev et al. (2012) Cell Res. 22, 490-503; Fullwood et al. (2009) Nature 462, 58-64; Goh et al. (2012) J Vis. Exp., http://dx.doi.org/l0.379l/3770; Fi et al. (2012) Cell 148, 84-98; and Dowen et al. (2014) Cell 159, 374-387, which are each hereby incorporated by reference in their entireties. Briefly, embryonic stem (ES) cells (up to lxlO⁸ cells) were treated with 1% formaldehyde at room temperature for 20 minutes and then neutralized using 0.2M glycine. The crosslinked chromatin was fragmented by sonication to size lengths of 300-700 bp. The anti-SMCl antibody (Bethyl, A300-055A) was used to enrich SMCl-bound chromatin fragments. A portion of ChIP DNA was eluted from antibody-coated beads for concentration quantification and for enrichment analysis using quantitative PCR. For ChlA-PET library construction ChIP DNA fragments were end-repaired using T4 DNA polymerase (NEB). ChIP DNA fragments were divided into two aliquots and either linker A or linker B was ligated to the fragment ends. The two linkers differ by two nucleotides which were used as a nucleotide barcode (Finker A with CG; Finker B with AT). After linker ligation, the two samples were combined and prepared for proximity ligation by diluting in a 20ml volume to minimize ligations between different DNA-protein complexes. The proximity ligation reaction was performed with T4 DNA ligase (Fermentas) and incubated without rocking at 22°C for 20 hours. During the proximity ligation DNA fragments with the same linker sequence were ligated within the same chromatin complex, which generated the ligation products with homodimeric linker composition. However, chimeric ligations between DNA fragments from different chromatin complexes could also occur, thus producing ligation products with heterodimeric linker composition. These heterodimeric linker products were used to assess the frequency of nonspecific ligations and were then removed.

i DAY 1

[00294] The cells were crosslinked as described for ChIP. Frozen cell pellets were stored in the -80°C freezer until ready to use. This protocol requires at least 3xl0⁸ cells frozen in six l5ml Falcon tubes (50 million cells per tube). Six IOOmI Protein G Dynabeads (for each ChlA-PET sample) was added to six 1.5ml Eppendorf tubes on ice. Beads were washed three times with 1.5 ml Block solution, and incubated end over end at 4°C for 10 minutes between each washing step to allow for efficient blocking. Protein G Dynabeads were resuspended in 250pl of Block solution in each of six tubes and lOpg of SMC1 antibody (Bethyl A300-055A) was added to each tube. The bead-antibody mixes were incubated at 4°C end-over-end overnight.

ii DAY 2

[00295] Beads were washed three times with l.5ml Block solution to remove unbound IgG and incubated end-over-end at 4°C for 10 minutes each time. Smcl-bound beads were resuspended in IOOmI of Block solution and stored at 4 °C. Final lysis buffer 1 (8ml per sample) was prepared by adding 5 Ox Protease inhibitor cocktail solution to Lysis buffer 1 (LB1) (1:50). Eight ml of Final lysis buffer 1 was added to each frozen cell pellet (8ml per sample x 6). The cells were thoroughly resuspended and thawed on ice by pipetting up and down. The cell suspension was incubated again end-over-end for 10 minutes at 4 °C. The suspension was centrifuged at 1,350 x g for 5 minutes at 4 °C. Concurrently, Final lysis buffer 2 (8ml per sample) was prepared by adding 5 Ox Protease inhibitor cocktail solution to lysis buffer 2 (LB2) (1:50)

[00296] After centrifugation, the supernatant was discarded, and the nuclei were thoroughly resuspended in 8ml Final lysis buffer 2 by pipetting up and down. The cell suspension was incubated end-over-end for 10 minutes at 4°C. The suspension was centrifuged at 1,350 x g for 5 minutes at 4°C. During incubation and centrifugation, the Final sonication buffer (l5ml per sample) was prepared by adding 5 Ox Protease inhibitor cocktail solution to sonication buffer (1:50). The supernatant was discarded, and the nuclei were fully resuspended in l5ml Final sonication buffer by pipetting up and down. The nuclear extract was extracted to fifteen lml Covaris Evolution E220 sonication tubes on ice. An aliquot of 10m1 was used to check the size of unsonicated chromatin on a gel.

[00297] A Covaris sonicator was programmed according to manufacturer’s instructions (12 minutes per 20 million cells = 12x15= 3 hours). The samples were sequentially sequenced as described above. The goal is to break chromatin DNA to 200-600 bp. If sonication fragments are too big, false positives become more frequent. The sonicated nuclear extract was dispensed into l .5ml Eppendorf tubes. l .5ml samples are centrifuged at full speed at 4°C for 10 minutes. Supernatant (SNE) was pooled into a new pre-cooled 50ml Falcon tube, and brought to a volume of l8ml with sonication buffer. Two tubes of 50m1 were taken as input and to check the size of fragments. 250m1 of ChIP elution buffer was added and reverse crosslinking occurred at 65°C overnight in the oven After reversal of crosslinking, the size of sonication fragments was determined on a gel.

[00298] Three ml of sonicated extract was added to 100 mΐ Protein G beads with SMC1 antibodies in each of six clean l5ml Falcon tubes. The tubes containing SNE-bead mix were incubated end-over-end at 4°C overnight (14 to 18 hours).

iii DAY 3

[00299] Half the volume (l .5ml) of the SNE-bead mix was added to each of six pre-chilled tubes and SNE was removed using a magnet. The tubes were sequentially washed as follows: 1) l.5ml of Sonication buffer was added, the beads were resuspended and rotated for 5 minutes at 4°C for binding, then the liquid was removed (step performed twice); 2) l .5ml of high-salt sonication buffer was added, and the beads were resuspended and rotated for 5 minutes at 4°C for binding, then the liquid was removed (step performed twice); 3) l .5ml of high-salt sonication buffer was added, and the beads were resuspended and rotated for 5 minutes at 4°C for binding, then the liquid was removed (step performed twice); 4) l .5ml of LiCl buffer was added, and the cells were resuspended and incubated end-over-end for 5 minutes for binding, then the liquid was removed (step performed twice); 5) l .5ml of IX TE + 0.2% Triton X-100 was used to wash the cells for 5 minutes for binding, then the liquid was removed; and l .5ml of ice-cold TE Buffer was used to wash the cells for 30 seconds for binding, then the liquid was removed (step performed twice). Beads from all six tubes were sequentially resuspended in beads in one l,000ul tube of IX ice-cold TE buffer.

[00300] ChIP-DNA was quantified using the following protocol. Ten percent of beads (by volume), or IOOmI, were transferred into a new l .5ml tube, using a magnet. Beads were resuspended in 300m1 of ChIP elution buffer and the tube was rotated with beads for 1 hour at 65°C. The tube with beads was placed on a magnet and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65°C oven without rotating. Immuno-precipitated samples were transferred to fresh tubes, and 300pl of TE buffer was added to the immuno-precipitants and Input samples to dilute. Five pl of RNase A

(20mg/ml) was added, and the tube was incubated at 37°C for 30 minutes.

[00301] Following incubation, 3m1 of 1M CaCh and 7m1 of 20 mg/ml Proteinase K was added to the tube and incubated 1.5 hours at 55°C. MaXtract High Density 2ml gel tubes (Qiagen) were prepared by centrifuging them at full speed for 30 seconds at RT. 600m1 of

phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction. About l.2ml of the mixtures was transferred to the MaXtract tubes. Tubes were spun at 16,000 g for 5 minutes at RT. The aqueous phase was transferred to two clean DNA LoBind tubes (300m1 in each tube), and Imΐ glycogen, 30 mΐ of 3M sodium acetate, and 900m1 ethanol was added. The mixture was allowed to precipitate overnight at -20°C or for 1 hour at -80°C.

[00302] The mixture was spun down at maximum speed for 20 minutes at 4°C, ethanol was removed, and the pellets were washed with lml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4°C. All remnants of ethanol were removed, and pellets were dried for 5 minutes at RT. H2O was added to each tube. Each tube was allowed to stand for 5 minutes, and vortexed briefly. DNA from both tubes was combined to obtain 50m1 of IP and IOOmI of Input DNA.

[00303] The amount of DNA collected was quantitated by ChIP using Qubit (Invitrogen #Q32856). One mΐ intercalating dye was combined with each measure Imΐ of sample. Two standards that come with the kit were used. DNA from only 10% of the beads was measured. About 400ng of chromatin in 900m1 of bead suspension was obtained with a good enrichment at enhancers and promoters as measured by qPCR.

iv. DAY 3 or 4

[00304] End-blunting of ChIP -DNA was performed on the beads using the following protocol. The remaining chromatin/beads were split by pipetting, and 450m1 of bead suspension was aliquoted into 2 tubes. Beads were collected on a magnet. Supernatant was removed, and then the beads were resuspended in the following reaction mix: 70m1 10X NEB buffer 2.1 (NEB, M0203L), 7m1 lOmM dNTPs, 615.8m1 dFTO. and 7.2m1 of 3U/pl T4 DNA Polymerase (NEB, M0203L). The beads were incubated at 37°C with rotation for 40 minutes. Beads were collected with a magnet, then the beads were washed 3 times with lml ice-cold ChIA-RET Wash Buffer (30 seconds per each wash). [00305] On -Bead A-tailing was performed by preparing Klenow (3 'to 5'exo-) master mix as stated below: 70pl 10X NEB buffer 2, 7m1 lOmM dATP, 616m1 dH20, and 7m1 of 3u/m1 Klenow (3'to 5'exo-) (NEB, M0212L). The mixture was incubated at 37°C with rotation for 50 minutes. Beads were collected with a magnet, then beads were washed 3 times with lml of ice-cold ChIA-RET Wash Buffer (30 seconds per each wash).

[00306] Linkers were thawed gently on ice. Linkers were mixed well with water gently by pipetting, then with PEG buffer, then gently vortexed. Then, 1394m1 of master mix and 6m1 of ligase was added per tube and mixed by inversion. Parafilm was put on the tube, and the tube was incubated at l6°C with rotation overnight (at least 16 hours). The biotinylated linker was ligated to ChIP-DNA on beads by setting up the following reaction mix and adding reagents in order: 1110m1 dEEO, 4m1 200ng/pl biotinylated bridge linker, 280m1 5X T4 DNA ligase buffer with PEG (Invitrogen), and 6m1 30 U/pl T4 DNA ligase (Fermentas).

v DAY 5

[00307] Exonuclease lambda/Exonuclease I On-Bead digestion was performed using the following protocol. Beads were collected with a magnet and washed 3 times with lml of ice-cold ChIA-RET Wash Buffer (30 seconds per each wash). The Wash buffer was removed from beads, then resuspended in the following reaction mix: 70m1 10X lambda nuclease buffer (NEB, M0262L), 618m1 nuclease-free dH20, 6m1 5 U/pl Lambda Exonuclease (NEB, M0262L), and 6pl Exonuclease I (NEB, M0293L). The reaction was incubated at 37°C with rotation for 1 hour. Beads were collected with a magnet, and beads were washed 3 times with lml ice-cold ChlA- PET Wash Buffer (30 seconds per each wash).

[00308] Chromatin complexes were eluted off the beads by removing all residual buffer and resuspending the beads in 300pl of ChIP elution buffer. The tube with beads was rotated 1 hour at 65°C. The tube was placed on a magnet and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65°C in an oven without rotating.

vi DAY 6

[00309] The eluted sample was transferred to a fresh tube and 300pl of TE buffer was added to dilute the SDS. Three mΐ of RNase A (30mg/ml) was added to the tube, and the mixture was incubated at 37°C for 30 minutes. Following incubation, 3pl of 1M CaCh and 7pl of 20 mg/ml Proteinase K was added, and the tube was incubated again for 1.5 hours at 55°C. MaXtract High Density 2ml gel tubes (Qiagen) were precipitated by centrifuging them at full speed for 30 seconds at RT. Six hundred mΐ of phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction, and about l.2ml of the mixture was transferred to the MaXtract tubes. Tubes were spun at 16,000 x g for 5 minutes at RT. [00310] The aqueous phase was transferred to two clean DNA LoBind tubes (300m1 in each tube), and lpl glycogen, 30m1 of 3M sodium acetate, and 900m1 ethanol is added. The mixture was precipitated for 1 hour at -80°C. The tubes were spun down at maximum speed for 30 minutes at 4°C, and the ethanol was removed. The pellets were washed with lml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4°C. Remnants of ethanol were removed, and the pellets were dried for 5 minutes at RT. Thirty mΐ of ThO was added to the pellet and allowed to stand for 5 minutes. The pellet mixture was vortexed briefly, and spun down to collect the DNA.

[00311] Qubit and DNA High Sensitivity ChIP were performed to quantify and assess the quality of proximity ligated DNA products. About 120 ng of the product was obtained.

vii DAY 7

[00312] Components for Nextera tagmentation were then prepared. One hundred ng of DNA was divided into four 25m1 reactions containing 12.5m1 2X Tagmentation buffer (Nextera), Imΐ nuclease-free dfhO. 2.5m1 Tn5 enzyme (Nextera), and 9m1 DNA (25ng). Fragments of each of the reactions were analyzed on a Bioanalyzer for quality control.

[00313] The reactions were incubated at 55°C for 5 minutes, then at l0°C for 10 minutes. Twenty-five mΐ of H2O was added, and tagmented DNA was purified using Zymo columns.

Three hundred fifty mΐ of Binding Buffer was added to the sample, and the mixture was loaded into a column and spun at 13,000 rpm for 30 seconds. The flow through was re-applied and the columns were spun again. The columns are washed twice with 200m1 of wash buffer and spun for 1 minute to dry the membrane. The column was transferred to a clean Eppendorf tube and 25 mΐ of Elution buffer was added. The tube was spun down for 1 minute. This step was repeated with another 25 mΐ of elution buffer. All tagmented DNA was combined into one tube.

[00314] ChlA-PETs was immobilized on Streptavidin beads using the following steps. 2X B&W Buffer (40ml) was prepared as follows for coupling of nucleic acids: 400m1 1M Tris-HCl pH 8.0 (lOmM final), 80m1 1M EDTA (lmM final), l6ml 5M NaCl (2M final), and 23.52ml dHiO. IX B&W Buffer (40ml total) was prepared by adding 20ml dfhO to 20ml of the 2X B&W Buffer.

[00315] MyOne Streptavidin Dynabeads M-280 were allowed to come to room temperature for 30 minutes, and 30m1 of beads were transferred to a new l .5ml tube. Beads were washed with 150m1 of 2X B&W Buffer twice. Beads were resuspended in IOOmI of iBlock buffer (Applied Biosystems), and mixed. The mixture was incubated at RT for 45 minutes on a rotator. [00316] I-BLOCK Reagent was prepared to contain: 0.2% I-Block reagent (0.2 g), IX PBS or IX TBS (10 ml 10X PBS or 10X TBS), 0.05% Tween-20 (50 pl), and HiO to lOOml. 10X PBS and I-BLOCK reagent was added to ThO, and the mixture was microwaved for 40 seconds (not allowed to boil), then stirred. Tween-20 was added after the solution is cooled. The solution remained opaque, but particles dissolved. The solution was cooled to RT for use.

[00317] During incubation of beads, 500ng of sheared genomic DNA was added to 50m1 of ThO and 50m1 of 2X B&W Buffer. When the beads finished incubating with the iBLOCK buffer, they were washed twice with 200m1 of IX B&W buffer. The wash buffer was discarded, and IOOmI of the sheared genomic DNA was added. The mixture was incubated with rotation for 30 minutes at RT. The beads were washed twice with 200m1 of IX B&W buffer. Tagmented DNA was added to the beads with an equal volume of 2X B&W buffer and incubated for 45 minutes at RT with rotation. The beads were washed 5 times with 500m1 of 2xSSC/0.5% SDS buffer (30 seconds each time) followed by 2 washes with 500ml of IX B&W Buffer and incubated each after wash for 5 minutes at RT with rotation. The beads were washed once with IOOmI elution buffer (EB) from a Qiagen Kit by resuspending beads gently and putting the tube on a magnet. The supernatant was removed from the beads, and they were resuspended in 30m1 of EB.

[00318] A paired end sequencing library was constructed on beads using the following protocol. Ten mΐ of beads are tested by PCR with 10 cycles of amplification. The 50m1 of the PCR mixture contains: 10m1 of bead DNA, 15 mΐ NPM mix (from Illumina Nextera kit), 5m1 of PPC PCR primer, 5m1 of Index Primer 1 (i7), 5m1 of Index Primer 2 (i5), and 10m1 ofEhO. PCR was performed using the following cycle conditions: denaturing the DNA at 72°C for 3 minutes, then 10-12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 50 seconds, and a final extension of 72°C for 5 minutes. The number of cycles was adjusted to obtain about 300ng of DNA total with four 25 mΐ reactions. The PCR product may be held at 4°C for an indefinite amount of time.

[00319] The PCR product was cleaned-up using AMPure beads. Beads were allowed to come to RT for 30 minutes before using. Fifty mΐ of the PCR reaction was transferred to a new Low- Bind Tube and (l.8x volume) 90m1 of AMPure beads was added. The mixture was pipetted well and incubated at RT for 5 minutes. A magnet was used for 3 minutes to collect beads and remove the supernatant. Three hundred mΐ of freshly prepared 80% ethanol was added to the beads on the magnet, and the ethanol was carefully discarded. The wash was repeated, and then all ethanol was removed. The beads were dried on the magnet rack for 10 minutes. Ten mΐ EB was added to the beads, mixed well, and incubated for 5 minutes at RT. The eluate was collected, and 1 mΐ of eluate was used for Qubit and Bioanalyzer.

[00320] The library was cloned to verify complexity using the following protocol. One mΐ of the library was diluted at 1: 10. A PCR reaction was performed as described below. Primers that anneal to Illumina adapters were chosen (Tm=52.2°C). The PCR reaction mixture (total volume: 50m1) contained the following: 10m1 of 5X GoTaq buffer, Imΐ of 10 mM dNTP, 5m1 of 10mM primer mix, 0.25m1 of GoTaq polymerase, Imΐ of diluted template DNA, and 32.75m1 of ThO. PCR was performed using the following cycle conditions: denaturing the DNA at 95°C for 2 minutes and 20 cycles at the following conditions: 95°C for 60 seconds, 50°C for 60 seconds, and 72°C for 30 seconds with a final extension at 72°C for 5 minutes. The PCR product may be held at 4°C for an indefinite amount of time.

[00321] The PCR product was ligated with the pGEM® T-Easy vector (Promega) protocol. Five mΐ of 2X T4 Quick ligase buffer, Imΐ of pGEM® T-Easy vector, Imΐ of T4 ligase, Imΐ of PCR product, and 2m1 of EhO were combined to a total volume of 10m1. The product was incubated for 1 hour at RT and 2m1 was used to transform Stellar competent cells. Two hundred mΐ of 500m1 of cells were plated in SOC media. The next day, 20 colonies were selected for Sanger sequencing using a T7 promoter primer. 60% clones had a full adapter, and 15% had a partial adapter.

viii. Reagents

[00322] Protein G Dynabeads for 10 samples were purchased from Invitrogen Dynal, Cat# 10003D. Block solution (50ml) contained 0.25g BSA dissolved in 50ml of ddH20 (0.5% BSA, w/v), and was stored at 4°C for 2 days before use.

[00323] Lysis buffer 1 (LB1) (500ml) contained 25ml of 1M Hepes-KOH, pH 7.5; l4ml of 5M NaCl; lml of 0.5 M EDTA, pH 8.0; 50ml of 100% Glycerol solution; 25ml of 10% NP-40; and 12.5ml of 10% Triton X-100. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4°C. The pH was re-checked immediately prior to use. Lysis buffer 2 (LB2)

(1000ml) contained lOml of 1M Tris-HCL, pH 8.0; 40ml of 5 M NaCl; 2ml of 0.5 M EDTA, pH 8.0; and 2ml of 0.5 M EGTA, pH 8.0. The pH was adjusted to 8.0. The buffer was sterile- filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00324] Sonication buffer (500ml) contained 25ml of 1M Hepes-KOH, pH 7.5; l4ml of 5M NaCl; lml of 0.5 M EDTA, pH 8.0; 50ml of 10% Triton X-100; lOml of 5% Na-deoxycholate; and 5ml of 10% SDS. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use. High-salt sonication buffer (500ml) contained 25ml of 1M Hepes- KOH, pH 7.5; 35ml of 5M NaCl; lml of 0.5 M EDTA, pH 8.0; 50ml of 10% Triton X-100; lOml of 5% Na-deoxycholate; and 5ml of 10% SDS. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00325] LiCl wash buffer (500 ml) contained lOml of 1M Tris-HCL, pH 8.0; lml of 0.5M EDTA, pH 8.0; l25ml of 1M LiCl solution; 25ml of 10% NP-40; and 50ml of 5% Na- deoxycholate. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4 °C. The pH was re-checked immediately prior to use.

[00326] Elution buffer (500ml) used to quantify the amount of ChIP DNA contained 25ml of 1M Tris-HCL, pH 8.0; lOml of 0.5M EDTA, pH 8.0; 50ml of 10% SDS; and 4l5ml of ddHiO. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4 °C. The pH was re checked immediately prior to use.

[00327] ChIA-RET Wash Buffer (50ml) contains 500pl of 1M Tris-HCl, pH 8.0 (final lOmM); IOOmI of 0.5M EDTA, pH 8.0 (final lmM); 5ml of 5M NaCl (final 500mM); and 44.4ml of dH₂0.

HiChIP

[00328] Alternatively to ChIA-RET, HiChIP was used to analyze chromatin interactions and conformation. HiChIP requires fewer cells than ChlA-PET.

i. Cell crosslinking

[00329] Cells were cross-linked as described in the ChIP protocol above. Crosslinked cells were either stored as pellets at -80°C or used for HiChIP immediately after flash-freezing the cells.

ii. Lvsis and restriction

[00330] Fifteen million cross-linked cells were resuspended in 500pL of ice-cold Hi-C Lysis Buffer and rotated at 4°C for 30 minutes. For cell amounts greater than 15 million, the pellet was split in half for contact generation and then recombined for sonication. Cells were spun down at 2500g for 5 minutes, and the supernatant was discarded. The pelleted nuclei were washed once with 500pL of ice-cold Hi-C Lysis Buffer. The supernatant was removed, and the pellet was resuspended in lOOpL of 0.5% SDS. The resuspension was incubated at 62°C for 10 minutes, and then 285 pL of H2O and 50pL of 10% Triton X-100 were added to quench the SDS. The resuspension was mixed well, and incubated at 37°C for 15 minutes. Fifty pL of 10X NEB Buffer 2 and 375 U of Mbol restriction enzyme (NEB, R0147) was added to the mixture to digest chromatin for 2 hours at 37°C with rotation. For lower starting material, less restriction enzyme was used: l5gL was used for 10-15 million cells, 8gL for 5 million cells, and 4gL for 1 million cells. Heat (62°C for 20 minutes) was used to inactivate Mbol.

iii. Biotin Incorporation and Proximity Ligation

[00331] To fill in the restriction fragment overhangs and mark the DNA ends with biotin,

52gL of fill-in master mix was reacted by combining 37.5gL of 0.4mM biotin-dATP (Thermo 19524016); l.5gL of lOmM dCTP, dGTP, and dTTP; and l OgL of 5U/gL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210). The mixture was incubated at 37°C for 1 hour with rotation.

948 gL of ligation master mix was added. Ligation Master Mix contained 150gL of 10X NEB T4 DNA ligase buffer with lOmM ATP (NEB, B0202); 125 gL of 10% Triton X-100; 3gL of 50mg/mL BSA; lOgL of 400 U/gL T4 DNA Ligase (NEB, M0202); and 660gL of water. The mixture was incubated at room temperature for 4 hours with rotation. The nuclei were pelleted at 2500g for 5 minutes, and the supernatant was removed.

iv. Sonication

[00332] For sonication, the pellet was brought up to lOOOgL in Nuclear Lysis Buffer. The sample was transferred to a Covaris millitube, and the DNA was sheared using a Covaris^® E220Evolution^™ with the manufacturer recommended parameters. Each tube (15 million cells) was sonicated for 4 minutes under the following conditions: Fill Level 5; Duty Cycle 5%; PIP 140; and Cycles/Burst 200.

v. Preclearing. Immunoprecipitation. IP Bead Capture and Washes

[00333] The sample was clarified for 15 minutes at l6,l00g at 4°C. The sample was split into

2 tubes of about 400gL each and 750gL of ChIP Dilution Buffer was added. For the Smcla antibody (Bethyl A300-055A), the sample was diluted 1 :2 in ChIP Dilution Buffer to achieve an SDS concentration of 0.33%. 60gL of Protein G beads were washed for every 10 million cells in ChIP Dilution Buffer. Amounts of beads (for preclearing and capture) and antibodies were adjusted linearly for different amounts of cell starting material. Protein G beads were resuspended in 50gL of Dilution Buffer per tube (lOOgL per HiChIP). The sample was rotated at 4°C for 1 hour. The samples were put on a magnet, and the supernatant was transferred into new tubes. 7.5gg of antibody was added for every 10 million cells, and the mixture was incubated at 4°C overnight with rotation. Another 60gL of Protein G beads for every 10 million cells in ChIP Dilution Buffer was added. Protein G beads were resuspended in 50gL of Dilution Buffer (100 gL per HiChIP), added to the sample, and rotated at 4°C for 2 hours. The beads were washed three times each with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffer. Washing was performed at room temperature on a magnet by adding 500gL of a wash buffer, swishing the beads back and forth twice by moving the sample relative to the magnet, and then removing the supernatant

vi. ChIP DNA Elution

[00334] ChIP sample beads were resuspended in lOOpL of fresh DNA Elution Buffer. The sample beads were incubated at RT for 10 minutes with rotation, followed by 3 minutes at 37°C with shaking. ChIP samples were placed on a magnet, and the supernatant was removed to a fresh tube. Another lOOpL of DNA Elution Buffer was added to ChIP samples and incubations were repeated. ChIP sample supernatants were removed again and transferred to a new tube. There was about 200pL of ChIP sample. Ten pL of Proteinase K (20mg/ml) was added to each sample and incubated at 55°C for 45 minutes with shaking. The temperature was increased to 67°C, and the samples were incubated for at least 1.5 hours with shaking. The DNA was Zymo- purified (Zymo Research, #D40l4) and eluted into lOpL of water. Post-ChIP DNA was quantified to estimate the amount of Tn5 needed to generate libraries at the correct size distribution. This assumed that contact libraries were generated properly, samples were not over sonicated, and that material was robustly captured on streptavidin beads. SMC1 Hi ChIP with 10 million cells had an expected yield of post-ChIP DNA from l5ng-50ng. For libraries with greater than l50ng of post-ChIP DNA, materials were set aside and a maximum of l50ng was taken into the biotin capture step

vii. Biotin Pull-Down and Preparation for Illumina Sequencing

[00335] To prepare for biotin pull-down, 5pL of Streptavidin C-l beads were washed with

Tween Wash Buffer. The beads were resuspended in lOpL of 2X Biotin Binding Buffer and added to the samples. The beads were incubated at RT for 15 minutes with rotation. The beads were separated on a magnet, and the supernatant was discarded. The beads were washed twice by adding 500pL of Tween Wash Buffer and incubated at 55°C for 2 minutes while shaking. The beads were washed in lOOpL of IX (diluted from 2X) TD Buffer. The beads were resuspended in 25 pL of 2X TD Buffer, 2.5pL of Tn5 for each 50ng of post-ChIP DNA, and water to a volume of 50pL.

[00336] The Tn5 had a maximum amount of 4 pL. For example, for 25ng of DNA transpose, l.25pL of Tn5 was added, while for l25ng of DNA transpose, 4pL of Tn5 was used. Using the correct amount of Tn5 resulted in proper size distribution. An over-transposed sample had shorter fragments and exhibited lower alignment rates (when the junction was close to a fragment end). An undertransposed sample has fragments that are too large to cluster properly on an Illumina sequencer. The library was amplified in 5 cycles and had enough complexity to be sequenced deeply and achieve proper size distribution regardless of the level of transposition of the library.

[00337] The beads were incubated at 55°C with interval shaking for 10 minutes. Samples were placed on a magnet, and the supernatant was removed. Fifty mM EDTA was added to samples and incubated at 50°C for 30 minutes. The samples were then quickly placed on a magnet, and the supernatant was removed. The samples were washed twice with 50mM EDTA at 50°C for 3 minutes, then were removed quickly from the magnet. Samples were washed twice in Tween Wash Buffer for 2 minutes at 55°C, then were removed quickly from the magnet. The samples were washed with lOmM Tris-HCl, pH8.0.

viii. PCR and Post-PCR Size Selection

[00338] The beads were resuspended in 50pL of PCR master mix (use Nextera XT DNA library preparation kit from Illumina, #15028212 with dual-index adapters # 15055289). PCR was performed using the following program. The cycle number was estimated using one of two methods: (1) A first run of 5 cycles (72°C for 5 minutes, 98°C for 1 minute, 98°C for 15 seconds, 63°C for 30 seconds, 72°C for 1 minute) was performed on a regular PCR and then the product was removed from the beads. Then, 0.25X SYBR green was added, and the sample was run on a qPCR. Samples were pulled out at the beginning of exponential amplification; or (2) Reactions were run on a PCR and the cycle number was estimated based on the amount of material from the post-ChIP Qubit (greater than 50ng was run in 5 cycles, while approximately 50ng was run in 6 cycles, 25ng was run in 7 cycles, l2.5ng was run in 8 cycles, etc.).

[00339] Libraries were placed on a magnet and eluted into new tubes. The libraries were purified using a kit form Zymo Research and eluted into lOpL of water. A two-sided size selection was performed with AMPure XP beads. After PCR, the libraries were placed on a magnet and eluted into new tubes. Then, 25 pL of AMPure XP beads were added, and the supernatant was kept to capture fragments less than 700 bp. The supernatant was transferred to a new tube, and 15pL of fresh beads were added to capture fragments greater than 300 bp. A final elution was performed from the Ampure XP beads into lOpL of water. The library quality was verified using a Bioanalyzer.

ix. Buffers

[00340] Hi-C Lysis Buffer (lOmL) contained lOOpL of 1M Tris-HCl pH 8.0; 20 pL of 5M NaCl; 200pL of 10% NP-40; 200pL of 50X protease inhibitors; and 9.68mL of water. Nuclear Lysis Buffer (lOmL) contained 500pL of 1M Tris-HCl pH 7.5; 200pL of 0.5M EDTA; lmL of 10% SDS; 200pL of 50X Protease Inhibitor; and 8.3mL of water. ChIP Dilution Buffer (lOmL) contained lOpL of 10% SDS; l . lmL of 10% Triton X-100; 24pL of 500mM EDTA; !67pL of 1M Tris pH 7.5; 334pL of 5M NaCl; and 8.365mL of water. Low Salt Wash Buffer (lOmL) contained lOOpL of 10% SDS; lmL of 10% Triton X-100; 40pL of 0.5M EDTA; 200pL of 1M Tris-HCl pH 7.5; 300pL of 5M NaCl; and 8.36mL of water. High Salt Wash Buffer (lOmL) contained lOOpL of 10% SDS; lmL of 10% Triton X-100; 40pL of 0.5M EDTA; 200pL of 1M Tris-HCl pH 7.5; lmL of 5M NaCl; and 7.66mL of water. LiCl Wash Buffer (lOmL) contained lOOpL of 1M Tris pH 7.5; 500pL of 5M LiCl; lmL of 10% NR-40; lmL of 10% Na- deoxycholate; 20pL of 0.5M EDTA; and 7.38mL of water.

[00341] DNA Elution Buffer (5mL) contained 250pL of fresh 1M NaHCCh; 500pL of 10% SDS; and 4.25mL of water. Tween Wash Buffer (50mL) contained 250pL of 1M Tris-HCl pH 7.5; 50pL of 0.5M EDTA; lOmL of 5M NaCl; 250pL of 10% Tween-20; and 39.45mL of water. 2X Biotin Binding Buffer (lOmL) contained lOOpL 1M Tris-HCl pH 7.5; 20pL of 0.5M; 4mL of 5M NaCl; and 5.88mL of water. 2X TD Buffer (lmL) contains 20pL of 1M Tris-HCl pH 7.5; lOpL of 1M MgCh; 200pL of 100% Dimethylformamide; and 770pL of water.

Drug dilutions for administration to hepatocytes

[00342] Prior to compound treatment of hepatocytes, lOOmM stock drugs in DMSO were diluted to lOmM by mixing 0. lmM of the stock drug in DMSO with 0.9ml of DMSO to a final volume of l.Oml. Five pl of the diluted drug was added to each well, and 0.5ml of media was added per well of drug. Each drug was analyzed in triplicate. Dilution to lOOOx was performed by adding 5pl of drug into 45m1 of media, and the 50m1 being added to 450m1 of media on cells.

[00343] Bioactive compounds were also administered to hepatocytes. To obtain lOOOx stock of the bioactive compounds in lml DMSO, 0.1 ml of IO,OOOC stock was combined with 0.9ml DMSO.

siRNA knockdown

[00344] Primary human hepatocytes were reverse transfected with siRNA with 6 pmol siRNA using RNAiMAX Reagent (ThermoFisher Cat#l3778030) in 24 well format, 1 mΐ per well. The following morning, the medium was removed and replaced with modified maintenance medium for an additional 24 hours. The entire treatment lasted 48 hours, at which point the medium was removed and replaced with RLT Buffer for RNA extraction (Qiagen RNeasy 96 QIAcube HT Kit Cat#74l7l). Cells were processed for qRT-PCR analysis and then levels of target mRNA were measured.

[00345] The siRNAs were obtained from Dharmacon and were a pool of four siRNA duplex all designed to target distinct sites within the specific gene of interest (“SMARTpool”). Example 2. ChIP-seq study in hepatocvtes

[00346] The ChIP-seq method described in Example 1 was used to identify chromatin binding proteins that bind the PCSK9 and ANGPTL3 insulated neighborhoods in primary human hepatocytes.

[00347] The transcription factors or signaling pathways listed in Table 1A were identified to bind the PCSK9 insulated neighborhood. The transcription factors or signaling pathways listed in Table 1B were identified to bind the ANGPTL3 insulated neighborhood.

Example 3. siRNA knockdown of identified transcription factors and signaling pathways

[00348] To further interrogate the pathways that control PCSK9 expression, hepatocytes were treated with siRNAs against specific members of various pathways and transcription factors. Cells were treated with siRNA and mRNA harvested as previously described in Example 1. siRNA for were purchased from Dharmacon. Dharmacon siRNA catalogue numbers were: M- 008822-01-0005 for siATF5, M-003401-04-0005 for siESRl, M-003265-01-0005 for siFOS, M- 010319-01-0005 for siFOXAl, M-003896-00-0005 for siGFIl, M-008215-01-0005 for siHNFlA, M-003406-02-0005 for siHNF4A, M-003413-01-0005 for siNRlFB, M-019872-01- 0005 for siOneCut2, M-003436-02-0005 for siPPARG, M-003443-02-0005 for siRXRA, M- 020067-00-0005 for siSMAD3, M-015791-00-0005 for siSMAD5, M-003544-02-0005 for siSTAT3, M-005169-02-0005 for siSTAT5A, M-012611-00-0005 for siTEAD2, M-016083-00- 0005 for siWWTRl, M-012200-00-0005 for siYAPl, M-011796-02-0005 for siYYl, M-019064- 01-0005 for siZGPAT.

[00349] The siRNA results are shown in Table 3A. PCSK9 expression after each siRNA knockdown is shown relative to the housekeeping gene GUSB.

[00350] Table 3A. SD stands for standard deviation.

[00351] To further interrogate the pathways that control ANGPTL3 expression, hepatocytes were treated with siRNAs against specific members of various pathways and transcription factors. Cells were treated with siRNA and mRNA harvested as previously described in Example 1. siRNA for were purchased from Dharmacon. Dharmacon siRNA catalogue numbers were: M- 003900-05 for siJUND, and M-020067-00 for siSMAD3.

[00352] The siRNA results are shown in Table 3B. ANGPTL3 expression after each siRNA knockdown is shown relative to the housekeeping gene GUSB.

[00353] Table 3B. SD stands for standard deviation.

Example 4: In vitro hepatocvte assays with mTOR inhibitors

[00354] Cells from three different primary human hepatocyte donors were treated with 1 mM or 10 pM of the mTOR inhibitors OSI-027 or PF04691502 for 16 hours. Human hepatocyte samples were also treated with DMSO as a control. After treatment, the cells were collected and total RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 3 shows the relative PCSK9 mRNA levels in each sample after treatment normalized to the control sample. The experiment was repeated in triplicate, error bars indicate the standard deviation. The mTOR inhibitors resulted in a decrease in the level of PCSK9 mRNA as compared to control samples.

[00355] Mouse hepatocytes were also treated with 1 pM or 10 pM of the mTOR inhibitors OSI-027 or PF04691502 for 16 hours. Mouse hepatocyte samples were also treated with DMSO as a control. After treatment, the cells were collected and total RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 4 shows the relative PCSK9 mRNA levels in each sample after treatment normalized to the control sample. The experiment was repeated in triplicate, error bars indicate the standard deviation. The mTOR inhibitors resulted in a decrease in the level of PCSK9 mRNA as compared to control samples. Quantification of the relative levels of PCSK9 mRNA is shown in Table 4.

Table 4. Quantification of PCSK9 mRNA

Examnle 5: In vivo assay with mTOR inhibitors

[00356] C57/B16 male mice were fed a chow diet. Mice were given an oral gavage of 50 mg/kg O SI-027 and sacrificed 3 hours post treatment. Control mice were administered vehicle without the drug as a control. The livers were extracted, flash frozen, and pulverized. RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 5 shows the relative mRNA levels in the control and treated mice after treatment, normalized to a housekeeping gene. Each dot represents an individual mouse, error bars indicate the standard deviation p value was determined by ANOVA. The relative PCSK9 levels of the control mice were 1.019±0.238, while treatment with OSI-027 resulted in decreased PCSK9 mRNA level of 0.663±0.06l.

[00357] Next, mice on a high sucrose diet were treated with mTOR inhibitors. C57/B16 mice were fed a high sucrose diet during the dark cycle on days 1-3. Mice were administered the mTOR inhibitors OSI-027 (lOmg/kg) or PF04691502 (50mg/kg) by oral gavage at the beginning of the dark cycle on days 1, 2, 3, and 4. Control mice were administered vehicle without the drug as a control. The mice were starved on day 4, and provided food for 6 hours on day 5. Another dose of the indicated mTOR drug was administer on day 5 with the food. After the 6 hours on day 5, animals were sacrificed, and the livers extracted, flash frozen, and pulverized. RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 6 shows the relative mRNA levels in the control and treated mice after treatment, normalized to a housekeeping gene. Each dot represents an individual mouse, error bars indicate the standard deviation p values were determined by ANOVA. The relative PCSK9 levels of the control mice treated with vehicle were 1.11±0.50, while treatment with PF04691502 and OSI-027 resulted in decreased PCSK9 mRNA levels of 0. l5±0.07 for PF04691502 and 0.07±0.02 for OSI-027.

[00358] As a comparison, mice were treated with simvastatin, a drug used to treat high cholesterol and triglyceride levels. C57/B16 mice were fed a chow diet during the dark cycle. Mice were given an oral gavage of 100 mg/kg simvastatin at the beginning of the dark cycle for two consecutive days. Control mice were administered vehicle (DMSO) without the drug as a control. Mice were sacrificed 12 hours after the second drug dose, and the livers extracted, flash frozen, and pulverized. RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 7 shows the relative mRNA levels in the control and treated mice after treatment, normalized to a housekeeping gene. Each dot represents an individual mouse, error bars indicate the standard deviation p values were determined by ANOVA. Simvastatin treatment resulted in an increase in PCSK9 mRNA as compared to control mice. The relative PCSK9 levels of the control mice treated with vehicle were 1.008±0.141, while treatment with simvastatin resulted in increased PCSK9 mRNA levels of l .869±0.55 l.

Example 6: In vivo dose response studies

[00359] Next, an in vivo dose titration study was completed for OSI-027, PF-04691502, and LY2157299 (Galunisertib). C57BL/6J mice were divided into 14 groups of 6 mice each. Each group had 6 male mice. All mice were given an HS diet for 6 days. On Day 7, mice were administered decreasing amounts of a candidate compound four times QD daily via oral gavage for four consecutive days. Table 5 shows the treatment and dose for each animal group. The animals received no food at night on Day 10. Animals were sacrificed 6 hours post-last dose on Day 11. Organs including liver, spleen, kidney, adipose, plasma, and muscle were collected.

[0100] Mouse liver tissues were pulverized in liquid nitrogen and aliquoted into small microtubes. TRIzol (Invitrogen Cat# 15596026) was added to the tubes to facilitate cell lysis from tissue samples. The TRIzol solution containing the disrupted tissue was then centrifuged and the supernatant phase was collected. Total RNA was extracted from the supernatant using Qiagen RNA Extraction Kit (Qiagen Cat#74l82) and the target mRNA levels were analyzed using qRT-PCR. mRNA levels for PCSK9 and ANGPTL3 were assessed. ACTB, GUSB, PPIA, B2M, HPRT, and GAPDH values were used to calculate a GeoMean for mRNA normalization. Table 5. Mouse Groups and Compound Doses

[00360] Relative PCSK9 mRNA for each treatment group is shown in FIG. 8A. Individual mRNA levels for each animal in a specific treatment group are shown in FIGS. 8B-G.

[00361] Mice in groups 2-6 treated with O SI-027 had a dose dependent decrease in PCSK9 at 6 hours post dose (FIG. 8A and 8B). OSI-027 treatment reduced ANGPTL3 mRNA at 6 hours post dose at each concentration tested (FIG. 8A and 8E).

[00362] All concentrations of PF-04691502 tested resulted in a decrease in PCSK9 and ANGPTL3 mRNA at 6 hours post dose in mice in groups 7-10 (FIG. 8C and 8F, respectively).

[00363] All concentrations of LY 2157299 tested resulted in a decrease in ANGPTL3 mRNA at 6 hours post dose (FIG. 8G), and the three lower doses (50 mg/kg, 25 mg/kg, and 10 mg/kg) resulted in a decrease in PCSK9 mRNA at 6 hours post dose (FIG.8D).

Example 7: In vivo assay with additional mTOR and PI3K inhibitors

[00364] The in vivo dose titration study was repeated with the mTOR inhibitors OSI-027 and PF-04691502, the PI3K inhibitor CH5132799 and the dual mTOR/PI3K inhibitor VS5584, to assess the relative effect of PI3K or mTOR pathway inhibition on PCSK9 and ANGPTL3 gene expression. In addition, food was provided the final night of the experiment and the time between the last dose and sacrifice was decreased to 4 hours.

[00365] C57BL/6J mice were divided into 11 groups of 8 mice each. All mice were given an HS diet for 6 days. On Day 6, mice were administered decreasing amounts OSI-027 and PF- 04691502, or a single concentration of CH5132799 and VS5584, for a total of 5 doses four times QD daily via oral gavage for four consecutive days. Dosing started in the evening of Day 6. Table 6 shows the treatment and dose for each animal group. Food was left in the cage until the final day, Day 10. The fifth inhibitor dose was administered in the morning of Day 10. Animals were sacrificed 4 hours post-last dose on Day 10. Organs including liver, spleen, kidney, adipose, plasma, and muscle were collected. Liver and plasma samples were processed for mRNA extraction and analysis.

[00366] Mouse liver tissues were processed for mRNA extraction and qRT-PCR as previously described. mRNA levels for PCSK9 was assessed. ACTB, GUSB, B2M, HPRT, and GAPDH mRNA values were used to calculate a GeoMean for PCSK9 and ANGPLT3 mRNA normalization.

[00367] Individual PCSK9 or ANGPLT3 mRNA levels for each animal in a treatment group are shown in FIGS. 9A-E.

[00368] Mice in groups 2-5 treated with O SI-027 had a dose dependent decrease in PCSK9 at 4 hours post dose (FIG. 9A). OSI-027 treatment reduced ANGPTL3 mRNA at the lower concentrations tested, 10 mg/kg, 5 mg/kg, and 2 mg/kg, but had inconsistent inhibition of ANGPLT3 at the highest dose tested, 25 mg/kg (FIG. 9D).

[00369] Mice in groups 6-9 treated with PF-04691502 had a dose dependent decrease in PCSK9 (FIG. 9B) and ANGPLT3 (FIG. 9E).

[00370] The dual PI3K/mTOR inhibitor VS-5584 also significantly reduced PCSK9 expression in 5 of the 6 mice (FIG. 9C). In contrast, the PI3K inhibitor CH5132799 did not significantly reduce PCSK9 mRNA expression (FIG. 9C). CH5132799 is selective for class I PI3Ks. Taken together, this data suggests that inhibition of the mTOR pathway is more critical for inhibition of PCSK9 expression than inhibition of the PI3K pathway. Example 8: Additional inhibitor compound testing for in vitro protein

phophorylation assay

[00371] Additional compounds to reduce PCSK9 expression and reduce activation of the mTOR and/or PI3K pathways were tested in human and mouse primary as previously described. Additional compounds tested are shown in Table 7.

[00372] Parallel samples of human hepatocytes were treated with 3 mM each of the additional inhibitors for 16 hours. One set of samples were harvested for Western Blots using Laemmli buffer (2% SDS, 10% glycerol, 75mM Tris-Cl, pH 6.8, 5% beta-mercaptoethanol, bromphenol blue). The other set was harvest for mRNA processing as previously described. Hepatocyte cell lysates were loaded onto 4-12% Bis-Tris gels with 35,000 cells/ l5uL per lane. Blots were incubated with primary antibodies overnight in Odyssey blocking buffer. Antibodies used were pAKT (Ser473) Rabbit mAb 4060 (Cell Signaling (1: 1000)), pS6 Ser235/236 Rabbit mAb 4858 (Cell Signaling (1: 1000)), pNDRGl T346 Rabbit mAb 5482 (Cell Signaling (1: 1000)), p4EBPlc (Thr37/46) Rabbit mAb 2855 (Cell Signaling (1: 1000)), AKT (pan) Mouse mAb 2920 (Cell Signaling (1: 1000)), Ribosomal Protein S6 (C-8) sc-74459 Mouse mAb (Santa Cruz Biotech (1:2000)), NDRG1 A-5 sc-398823 Mouse mAb (Santa Cruz Biotech (1:200)) and 4EBP1 (53H11) Rabbit mAb 9644 (Cell Signaling (1: 1000)). Blots were incubated with secondary antibodies IRDye® 800CW Donkey anti-Rabbit IgG (H + L) 926-32213 or Donkey Anti-Mouse IgG Polyclonal Antibody (IRDye® 680LT) 926-68022 at 1: 10,000 in Odyssey blocking buffer for lhour, and imaged using Odyssey Licor Scanner. Image Studio software was used to quantify phosphorylated protein abundance to total protein abundance, relative to DMSO control from each timepoint.

[00373] Levels of phosphorylated 4-EBP1, S6, AKT, and NDRG1 proteins were determined as compared to total S6, AKT, and NDRG1 protein. PCSK9 mRNA expression was quantified and normalized to housekeeping gene GUSB.

[00374] Table 7: Additional compounds tested

[00375] Provided in Table 8 are fold changes in PCSK9 mRNA expression in primary hepatocytes (donor 4178) relative to GUSB. All compounds were tested at 3mM concentrations. Also shown are changes in phosphorylated 4-EBP1, S6, AKT, and NDRG1 protein levels in hepatocytes after treatment with the compounds. P/T indicates the ratio of the phosphorylated protein to total protein measured. PCSK9 is shown as fold change (FC) as normalized to GUSB.

[00376] Table 8

Example 9: In vitro hepatocvte assay with additional mTOR inhibitors and inhibition time course

[00377] Primary human hepatocytes were treated with four concentrations of various inhibitors or activators for 16 hours. Inhibitors were used at 3 mM, 1 pM, 0.3 pM, or 0.1 pM starting four hours after cells were plated. Human hepatocyte samples were also treated with DMSO as a control. After treatment, the cells were collected and total RNA was extracted and processed for cDNA synthesis and q-PCR. FIG. 10, 11, 12, and 13 shows the relative PCSK9 mRNA levels in each sample after treatment normalized to the control sample using the housekeeping gene GUSB. The experiment was repeated in triplicate, error bars indicate the standard deviation. The inhibitors used are shown in Table 9, as well as the fold change in PCSK9 mRNA normalized to GUSB. FC stands for fold change, SD stands for standard deviation.

[00378] Table 9. Fold change in relative PCSK9 mRNA

[00379] Treatment of hepatocytes with mTOR, PI3K, or mTOR/PI3K inhibitors resulted in a decrease in the level of PCSK9 mRNA as compared to control samples. FIG. 10 provides the relative PCSK9 mRNA expressed in hepatocytes after treatment with 3 mM of the indicated compound. FIG. 11 provides the relative PCSK9 mRNA expressed in hepatocytes after treatment with 1 pM of the indicated compound. FIG. 12 provides the relative PCSK9 mRNA expressed in hepatocytes after treatment with 0.3 pM of the indicated compound. FIG. 13 provides the relative PCSK9 mRNA expressed in hepatocytes after treatment with 0.1 pM of the indicated compound.

[00380] A select number of the mTOR inhibitors used in the previous assay were then tested in a time course assay in Hu4l75 primary hepatocytes to assess PCSK9 gene expression inhibition. Hepatocytes were incubated with 3 pM of PF-04691502, OSI-027, CH5132799, rapamycin, or Alpelisb, for 35min, 1 hr, 2 hr, 3 hr, 4.5 hr, or 20 hrs. Cells were collected for mRNA quantification as described above. PCSK9 gene expression was determined via q-RT- PCR and normalized to housekeeping gene GUSB. The fold change in the relative PCSK9 mRNA after treatment with each inhibitor is show in FIG. 14. The inhibitors that affected both mTORCl and mTORC2, OSI-027 and PF-04691502, resulted in the best inhibition of PCSK9 expression over the course of the time course. The most PCSK9 gene inhibition in samples treated with OSI-027 and PF-04691502 was between 3 hr and 20 hr post-treatment. In contrast, rapamycin, which is an mTORC 1 specific inhibitor, did not result in PCSK9 inhibition until the 20 hr time point, and the overall PCSK9 gene expression reduction was not significant, suggesting that inhibition of mTORC2 is necessary for inhibition of PCSK9 gene expression.

[00381] The RI3Ka/b inhibitor CH5132799 also resulted in inhibition of PCSK9 gene inhibition in a time-dependent manner. In contrast, treatment with the PI3Ka inhibitor Alpelisib did not result in any significant PCSK9 gene inhibition, indicating that inhibition of the RI3Kb kinase, and not the PI3Ka kinase, is necessary for inhibition of PCSK9 gene expression. Example 10: In vitro hepatocvte assay in PNPLA3 mutant cell line

[00382] Primary human hepatocytes homozygous for the I148M mutation in the Patatin-like phospholipase domain-containing protein 3 (PNPLA3) protein mutation (Y ecuris cell line, PNPLA3 1148 M/M homozygous) were treated with various concentrations of the indicated inhibitors or activators for 16 hours. Inhibitors were used at 3.3 mM, 1.1 pM, 0.37 pM, 0.12 pM, or 0.04 pM starting four hours after cells were plated. Hepatocyte samples were also treated with DMSO as a control. After treatment, the cells were collected and total RNA was extracted and processed for cDNA synthesis and q-PCR as previously described. FIG. 14 shows the relative PCSK9 mRNA levels in each sample after treatment normalized to the control sample using the housekeeping gene GUSB. The experiment was repeated in triplicate, error bars indicate the standard deviation. The inhibitors used are shown in Table 10, as well as the fold change in PCSK9 mRNA normalized to GUSB. FC stands for fold change, SD stands for standard deviation.

[00383] Table 10. Fold change in relative PCSK9 mRNA

[00384] As shown in FIG. 15 and Table 10, treatment of the PNPLA3 I148M hepatocytes with OSI-027, PF-04691502, and Mubritinib resulted in a dose dependent decrease in PCSK9 inhibition. Mubritinib is a HER2 inhibitor that works upstream of mTOR. Thus, mubritinib inhibition of PCSK9 may also be via the mTOR pathway.

[00385] Treatment with the RI3Ka/b inhibitor WYE-125132 resulted in PCSK9 gene inhibition at all concentrations tested, with the exception of the lowest concentration.

Example 11: In vivo inhibitor assay in PNPLA3 mutant mice

[00386] Next, an in vivo study was completed for OSI-027 or PF-04691502, in the Yecuris mouse model. Yecuris mice are C57/BL6 humanized mice that are knockouts for the Fah, Rag, and Ilrg genes. Knock out of the Fah gene results in liver damage. The mouse liver can then be repopulated via engraftment with human or mouse hepatocytes of a desired genotype. This model is commonly used in liver disease and damage studies. For this assay, the human hepatocytes used for repopulation and engraftment were mutant hepatocytes homozygous for the PNPFA3 I148M mutation.

[00387] Mice were divided into 3 groups of 6 mice each. All mice were given a high fat diet for 8 weeks before treatment with the inhibitors was initiated. Mice were administered 5 mg/kg OSI-027 or 2.5 mg/kg PF-04691502 once daily via oral gavage for four weeks (weeks 9-12). Organs including liver, spleen, kidney, adipose, plasma, and muscle were collected. Fivers were divided into three areas (left, F; medial, M; and right, R) to account for differences in human hepatocyte engraftment.

[00388] Mouse liver tissues were processed for mRNA extraction and qRT-PCR as previously described. mRNA levels for PCSK9 was assessed. ACTB, GUSB, PPIA, B2M, HPRT, and GAPDH values were used to calculate a GeoMean for mRNA normalization.

[00389] FIG. 16 shows the relative PCSK9 mRNA levels after treatment with OSI-027 or PF- 04691502 in the left (F), medial (M), or right (R) kidney sections. OSI-027 treatment resulted in significant reduction in PCSK9 mRNA expression. PF-04691502 resulted in moderate reduction in PCSK9 mRNA expression. Thus, treatment of mice with homozygous PNPFA3 I148M mutant hepatocytes with the mTORCl/C2 inhibitor OSI-027 resulted in a significant decrease in PCSK9 mRNA expression in hepatocytes.

Example 12: In vivo glucose and insulin quantification after inhibitor treatment

[00390] The effects of mTOR and mTOR/PI3K inhibitors on in vivo insulin and glucose levels were assessed.

In vivo dosing materials and methods

[00391] 7-8 week old C57BF/6J mice were divided into 9 groups. Each group had 8 male mice. All mice were given a high sucrose diet for 10 days (Diet no. 901683; 74% kCal from sucrose, MP Biomedicals, Santa Ana, CA) at the start of the dark cycle, about 7 pm. Food was removed at the start of the light cycle, about 7 am, except on the last day, when food was left in the cage until termination. On day 7-10, mice were administered daily (QD) via oral gavage, candidate compounds at a volume of lOmL/kg with the compound in vehicle solution (0.5% methylcellulose/ 0.2% tween20). Vehicle alone was administered to control group 1. OSI-027 was administered at 25 mg/kg, 10 mg/kg, 5 mg/kg, and 2 mg/kg to groups 2-5. PF-04691502 was administered at lOmg/kg, 5 mg/kg, 2 mg/kg, and 1 mg/kg to groups 6-9. The treatment was administered in the evening on Days 7 to 10 and in the morning on Day 11, starting at 5 am. On Day 11, mice were terminated 4 hours post last dose at 9 am, for a total of 5 doses of each candidate compound. Mice were weighed 2X/week until Day 11. Liver and blood samples were collected after mice were terminated. Liver samples were process for mRNA extraction as previously described. Blood samples were processed for serum collection. The geometric mean for the mRNA analysis was calculated by averaging the PCR CTs from the housekeeping genes ACTB, GAPDH, GUSB, HPRT, and B2M from the same cDNA sample.

Serum glucose quantification

[00392] Serum glucose levels were measured in a single-reagent coupled-enzyme assay, against a glucose standard curve, colorimetrically. The glucose assay reagent was prepared as follows: one capsule of glucose oxidase/peroxidase (Sigma, cat# G3660-1 CAP) was dissolved in 19.6 ml of deionized water. Separately, one vial of O-Dianisidine reagent (Sigma, cat# D2679) was dissolved in 0.5 ml of deionized water. 0.4ml of the O-Dianisidine reagent was added into the enzyme mix to make 20 ml of 2X Glucose assay reagent. The glucose assay reagent was made fresh prior to running the assay.

[00393] A glucose standard curve was prepared by serially diluting D-glucose two-fold from 200ug/ml to l2.5ug/ml in IX PBS. A no glucose control was included as a reagent blank.

[00394] Mouse serum samples were diluted 30-fold in IX PBS. 50 pl of the sample (or standard) was combined with 50 mΐ of the glucose assay reagent in a 96-well microplate. The reaction was incubated at 37 °C for 30 min. IOOmI of 2N sulfuric acid was then added to quench the reaction. The color developed was read spectrophotometrically at 540nm. The amount of glucose in the samples were determined based on the parameters of the linear fit obtained from the glucose standard curve.

Serum insulin quantification

[00395] Serum insulin levels in mouse samples were quantified using an ELISA kit purchased from Crystal Chem (Catalog# 90080), per the manufacturer’s instructions. Results

[00396] Treatment of mice with OSI-027 and PF-04691502 resulted in different serum glucose and serum insulin outcomes. Only the highest dose of OSI-027 treatment, 25 mg/kg, resulted in significant increased serum glucose (FIG. 17A) and serum insulin (FIG. 17B), while the three lower doses, 10 mg/kg, 5 mg/kg, and 2 mg/kg, resulted in no significant changes in serum glucose or serum insulin levels in the mice. In contrast, PF-04691502 treatment resulted in statistically significant increases in the insulin and glucose levels at the 10 mg/kg, 5 mg/kg, and 2 mg/kg doses. Thus, OSI-027 treatment at three different concentrations did not result in an adverse increase in serum insulin or glucose. Statistical analysis was performed with one way ANOVA, followed by Dunnett test for multiple comparisons, ns = not significant.

[00397] In contrast, mice treated with PF-04691502 experienced significant serum glucose and serum insulin increases at the three highest doses, 10 mg/kg, 5 mg/kg, and 2 mg/kg (FIG. 17A and FIG. 17B), and moderate increases in serum insulin at the lowest dose, 1 mg/kg (FIG. 17B). The lowest dose of PF-0469150 still induced moderate increased insulin and glucose levels in the mice.

[00398] Thus, the dual PBka/b and mTORCl/C2 inhibitor PF-0469150 induced increased levels of serum glucose and insulin, while the mTOR only inhibitor OSI-027 had only minimal adverse side effects. Based on these results, inhibition of the PBka/b pathway leads to adverse in vivo results, e.g. increased serum glucose and insulin levels. Increased levels of serum insulin, or hyperinsulinemia, is associated with pre-diabetes, hypertension, obesity, dyslipidemia, and glucose intolerance. High blood sugar, or hyperglycemia, can lead to nerve damage, blood vessel damage, or organ damage, as well as decreased healing, increased skin and mucosal infections, vision problems, or gastrointestinal issues such as constipation or diarrhea.

[00399] Therefore inhibition of only the mTOR pathway to reduce PCSK9 or ANGPTL3 expression is preferable, due to the adverse effects induced by inhibition of the PI3K pathway.

Example 13: mTOR inhibitory activity

[00400] A candidate compound is tested for mTOR inhibitory activity via an antibody binding assay.

[00401] Human hepatocytes are treated with various concentrations of the candidate compound for 35 min, 1 hr, 2 hrs, 3 hrs, 4.5 hrs, or 20 hrs. Cells are harvested for protein immunoblots using Laemmli buffer (2% SDS, 10% glycerol, 75mM Tris-Cl, pH 6.8, 5% beta- mercaptoethanol, bromphenol blue). Hepatocyte cell lysates are loaded onto 4-12% Bis-Tris gels with 35,000 cells/l5uL per lane. Blots are incubated with primary antibodies overnight in Odyssey blocking buffer. Antibodies used include pAKT (Ser473) Rabbit mAb 4060 (Cell Signaling (1: 1000)), pS6 Ser235/236 Rabbit mAb 4858 (Cell Signaling (1: 1000)), pNDRGl T346 Rabbit mAb 5482 (Cell Signaling (1: 1000)), p4EBPlc (Thr37/46) Rabbit mAb 2855 (Cell Signaling (1: 1000)), AKT (pan) Mouse mAb 2920 (Cell Signaling (1: 1000)), Ribosomal Protein S6 (C-8) sc-74459 Mouse mAb (Santa Cruz Biotech (1:2000)), NDRG1 A-5 sc-398823 Mouse mAb (Santa Cruz Biotech (1:200)) and 4EBP1 (53H11) Rabbit mAb 9644 (Cell Signaling (1: 1000)), pSGKl (Ser78) rabbit mAB 5599 (Cell Signaling), SGK1 rabbit mAb 12103 (Cell Signaling), pPKC (Thr4l0) rabbit mAb 2060 (Cell Signaling), PKC rabbit mAb 9960 (Cell Signaling). Blots are incubated with secondary antibodies IRDye® 800CW Donkey anti-Rabbit IgG (H + L) 926-32213 or Donkey Anti -Mouse IgG Polyclonal Antibody (IRDye® 680LT) 926- 68022 at 1 : 10,000 in Odyssey blocking buffer for 1 hour, and are imaged using Odyssey Licor Scanner. Image Studio software is used to quantify phosphorylated protein abundance to total protein abundance, relative to DMSO control from each timepoint.

[00402] Levels of at least one of phosphorylated S6, AKT, SGK1, PKC, NDRG1, and

4EBPlc proteins are determined as compared to total S6, AKT, SGK1, PKC, NDRG1, and 4EBPlc protein levels.

[00403] Cells treated with candidate compounds that have mTORCl/C2 inhibitory activity show a decrease in the relative amount of phosphorylated S6, AKT, SGK1, PKC, NDRG1, and/or 4EBPlc. mTORC2 specific inhibitors show decreased levels of phosphorylated AKT, SGK1, PKC, and/or NDRG1 but not S6 and/or 4EBPlc. mTORCl specific inhibitors show decreased levels of phosphorylated S6 and/or 4EBPlc but not AKT, SGK1, PKC, and/or NDRG1. mTORCl/C2 inhibitors show decreased levels of both phosphorylated S6 and/or 4EBPlc and AKT, SGK1, PKC, and/or NDRG1.

Examnle 14: PI3K inhibitory activity

[00404] Compounds identified in Example 13 as mTOR inhibitors are assessed for PI3K inhibitory activity in a biochemical assay.

[00405] Purified PI3Ka or RI3Kb is purchased from Promega (catalogue no. V1721 or V 1751 ). An ADP-Glo kit with PIP2 is purchased from Promega (catalogue no . V 1791 ) .

Alternatively, an ADP-Glo kit with PI is purchased from Promega (catalogue no. VI 781).

[00406] A standard curve of the kinase substrate is prepared according to the manufactures instructions. A working solution of the PI3K kinase in reaction buffer with the substrate is prepared. Serial dilutions of the candidate compound are made in buffer. The candidate compound samples are added to the kinase and substrate mixture and incubated to allow binding of the kinase to the substrate. Control sample with no enzyme (background control) or no candidate compound (negative control) are run. A known PI3K inhibitor, such as CH51332799, is used as a positive control. The reaction is started by adding ATP to a final concentration of 25 mM and incubated for 1 hr. The reaction is halted by adding ADP-Glo Reagent. Kinase Detection Reagent is added to the samples to convert the ADP to ATP, and the luciferase and luciferin to detect the new ATP. The luminescence of the samples is quantified with a luminescent plate reader. The IC50 of a candidate compound is determined from the serial dilution curve, as compared to the luminescence of the sample with no candidate compound (100% activity).

[00407] Inhibition of the PI3K kinase reaction results in reduced luminescence of the samples. Thus, samples treated with compounds with PI3K inhibitory activity show decreased luminescence, while samples treated with compounds that do not inhibit PI3K do not show decreased luminescence in the assay.

[00408] Candidate compounds selected for further analysis and development are those that have mTORC 1/2 or mTORC2 inhibitory activity and do not inhibit the activity of PI3K, including RI3Kb.

Examnle 15: DNA-PK inhibitory activity

[00409] Compounds identified in Example 13 as mTOR inhibitors are assessed for DNA-PK inhibitory activity in a biochemical assay.

[00410] Purified DNA-PK and the DNA-PK substrate is purchased from Promega in a kit (catalogue no. V4106). An ADP-Glo kit is purchased from Promega (catalogue no. V9101, or V4107 when purchased with the DNA-PK kit).

[00411] A dose response curve of the DNA-PK kinase substrate is prepared according to the manufactures instructions to determine the optimal kinase and ATP concentration. A working solution of the DNA-PK kinase in reaction buffer with the substrate is prepared. Serial dilutions of the candidate compound are made in buffer. The candidate compound samples are added to the kinase and substrate mixture and incubated to allow binding of the kinase to the substrate. Control sample with no enzyme (background control) or no candidate compound (negative control) are run. A known DNA-PK inhibitor, such as LY3023414 or CC-l 15, is used as a positive control. The reaction is started by adding ATP to a final concentration as previously determined and incubated for 1 hr. The reaction is halted by adding ADP-Glo Reagent. Kinase Detection Reagent is added to the samples to convert the ADP to ATP, and the luciferase and luciferin to detect the new ATP. The luminescence of the samples is quantified with a luminescent plate reader. The IC50 of a candidate compound is determined from the serial dilution curve, as compared to the luminescence of the sample with no candidate compound (100% activity).

[00412] Inhibition of the DNA-PK kinase reaction results in reduced luminescence of the samples. Thus, samples treated with compounds with DNA-PK inhibitory activity show decreased luminescence, while samples treated with compounds that do not inhibit DNA-PK do not show decreased luminescence in the assay.

[00413] Candidate compounds selected for further analysis and development are those that have mTORC 1/2 or mTORC2 inhibitory activity and do not inhibit the activity of DNA-PK.

Example 16: Insulin and glucose assays

[00414] Compounds identified in Example 13 as mTOR inhibitors are assessed for the ability to increase insulin and glucose levels in vivo.

[00415] Mice are dosed with candidate compounds and serum is collected for glucose and insulin quantification as described in Example 12. Increased levels of serum insulin or glucose are observed in mice treated with compounds that increase insulin or glucose.

[00416] Candidate compounds selected for further analysis and development are those that have mTORC 1/2 or mTORC2 inhibitory activity and do not increase insulin or glucose.

Example 17: PCSK9 or ANGPTL3 gene expression assays

[00417] Compounds identified in Example 13 as mTOR inhibitors are assessed for the ability to decrease PCSK9 or ANGPTL3 expression. Hepatocytes are treated with a candidate compound and PCSK9 or ANGPTL3 expression is quantified as described in Examples 4, 9, and 10. Decreased PCSK9 or ANGPTL3 mRNA is observed in cells treated with compounds that reduce PCSK9 or ANGPTL3 gene expression.

[00418] Candidate compounds selected for further analysis and development are those that have mTORC 1/2 or mTORC2 inhibitory activity and decrease PCSK9 or ANGPTL3 gene expression.

Example 18: In vitro hepatocvte assay with YY1 modulators

[00419] Human hepatocyte cells are contacted with effective amounts of the YY 1 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed. Example 19: In vitro hepatocvte assay with HNF4A modulators

[00420] Human hepatocyte cells are contacted with effective amounts of the HNF4A modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 20: In vitro hepatocvte assay with HNF1A modulators

[00421] Human hepatocyte cells are contacted with effective amounts of the HNF1A modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 21: In vitro hepatocvte assay with ONECUT1 modulators

[00422] Human hepatocyte cells are contacted with effective amounts of the ONECUT1 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 22: In vitro hepatocvte assay with MYC modulators

[00423] Human hepatocyte cells are contacted with effective amounts of the MY C modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 23: In vitro hepatocvte assay with NR1H4 modulators

[00424] Human hepatocyte cells are contacted with effective amounts of the NR1H4 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 24: In vitro hepatocvte assay with NR3C1 modulators

[00425] Human hepatocyte cells are contacted with effective amounts of the NR3C1 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 25: In vitro hepatocvte assay with NRTA2 modulators

[00426] Human hepatocyte cells are contacted with effective amounts of the NRTA2 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed. Example 26: In vitro hepatocvte assay with RXRA modulators

[00427] Human hepatocyte cells are contacted with effective amounts of the RXRA modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 27: In vitro hepatocvte assay with VDR modulators

[00428] Human hepatocyte cells are contacted with effective amounts of the VDR modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 28: In vitro hepatocvte assay with CREB1 modulators

[00429] Human hepatocyte cells are contacted with effective amounts of the CREB 1 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 29: In vitro hepatocvte assay with ESR1 modulators

[00430] Human hepatocyte cells are contacted with effective amounts of the ESR1 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 transcript levels is observed.

Example 30: In vitro hepatocvte assay with SMAD2 modulators

[00431] Human hepatocyte cells are contacted with effective amounts of the SMAD2 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 or ANGPTL3 transcript levels is observed.

Example 31: In vitro hepatocvte assay with SMAD3 modulators

[00432] Human hepatocyte cells are contacted with effective amounts of the SMAD3 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 or ANGPTL3 transcript levels is observed.

Example 32: In vitro hepatocvte assay with STAT3 modulators

[00433] Human hepatocyte cells are contacted with effective amounts of the STAT3 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 or ANGPTL3 transcript levels is observed.

Example 33: In vitro hepatocvte assay with TGFB receptor and SMAD2, SMAD3 or

SMAD4 modulators

[00434] Human hepatocyte cells are contacted with effective amounts of the TGF receptor and SMAD2, SMAD3 or SMAD4 inhibitors shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in PCSK9 or ANGPTL3 transcript levels is observed.

Example 34: In vitro hepatocvte assay with NF-kB modulators

[00435] Human hepatocyte cells are contacted with effective amounts of the NF-KB modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in ANGPTL3 transcript levels is observed.

Example 35: In vitro hepatocvte assay with BRD4 modulators

[00436] Human hepatocyte cells are contacted with effective amounts of the BRD4 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in ANGPTL3 transcript levels is observed.

Example 36: In vitro hepatocvte assay with TP53 modulators

[00437] Human hepatocyte cells are contacted with effective amounts of the TP53 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in ANGPTL3 transcript levels is observed.

Example 37: In vitro hepatocvte assay with TCF7L2 modulators

[00438] Human hepatocyte cells are contacted with effective amounts of the TCF7L2 modulators shown in Table 2. Samples are collected and processed for RNA and q-PCR. A reduction in ANGPTL3 transcript levels is observed.

Example 38: Human treatment using PSCK9 or ANGPTL3 inhibitors

[00439] A human subject is administered an effect amount of any of the compounds in the forgoing examples and Table 2, such as PF04691502, OSI-027, OPB-31121, OPB-51602, STAT3 inhibitor XIII, danvatirsen, MYC-targeting siRNA DCR-MYC, AVI-4126, obeticholic acid apomine, rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate,

fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate, 1 lbeta hydrocortisone acetate, betamethasone, dexamethasone, budesonide, fluticasone propionate, beclomethasone dipropionate, betamethasone acetate/betamethasone phosphate, betamethasone acetate, triamcinolone acetonide, ciprofloxacin/hydrocortisone, ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone dipropionate/calcipotriene, fluticasone furoate,

budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate, beclomethasone 17 -monopropionate, dexamethasone/lenalidomide/sorafenib, docetaxel/prednisone, carmustine/prednisone, cabazitaxel/prednisone,

mitoxantrone/prednisone, docetaxel/hydrocortisone, cytarabine/dexamethasone,

dexamethasone/pomalidomide, bortezomib/dexamethasone,

cyclophosphamide/dexamethasone/thalidomide, bortezomib/dexamethasone/doxorubicin, bortezomib/dexamethasone/lenalidomide, bortezomib/dexamethasone/thalidomide, carfdzomib/dexamethasone/lenalidomide,

cyclophosphamide/mitoxantrone/prednisone/vincristine,

cyclophosphamide/etoposide/prednisone/vincristine, dexamethasone/fludarabine

phosphate/mitoxantrone/rituximab, chlorambucil/mitoxantrone/prednisone,

chlorambucil/mitoxantrone/prednisone/rituximab, clocortolone, alclometasone,

prednisone/somatotropin, carfilzomib/dexamethasone/rituximab,

cyclophosphamide/prednisolone/vincristine,

cyclophosphamide/prednisolone/rituximab/vincristine,

cytarabine/dexamethasone/oxaliplatin/rituximab, everolimus/prednisone,

cyclophosphamide/gemcitabine/prednisolone/rituximab/vincristine,

cabazitaxel/prednisolone, prednisone/vincristine, carfilzomib/dexamethasone,

ciprofloxacin/fluocinolone acetonide, dexamethasone/vincristine,

glycopyrrolate/indacaterol/mometasone furoate, indacaterol/mometasone furoate,

fluticasone/salmeterol, betamethasone/clotrimazole, aprepitant/dexamethasone,

bortezomib/dexamethasone/pomalidomide, dexamethasone/imatinib/vincristine,

acetaminophen/diphenhydramine/prednisolone,

acetaminophen/diphenhydramine/methylprednisolone,

cytarabine/dasatinib/dexamethasone/methotrexate,

cytarabine/dexamethasone/imatinib/methotrexate,

cyclophosphamide/daunorubicin/imatinib/prednisolone/vincristine,

methylprednisolone/mycophenolate mofetil, mycophenolate mofetil/prednisone,

infliximab/methylprednisolone, prednisone/tacrolimus, infliximab/prednisone,

cyclophosphamide/methylprednisolone, methylprednisolone/tacrolimus, methylprednisolone acetate, mometasone furoate, amcinonide, methylprednisolone succinate, betamethasone phosphate, fluocinonide, prednicarbate, hydrocortisone cypionate, hydrocortisone succinate, prednisolone phosphate, betamethasone valerate, betamethasone benzoate, fludrocortisone acetate, prednisolone tebutate, betamethasone dipropionate, hydrocortisone buteprate, alclometasone dipropionate, hydrocortisone butyrate, fluorometholone acetate, hydrocortisone valerate, loteprednol etabonate, hydrocortisone phosphate, methylprednisolone, halobetasol propionate, flunisolide, mifepristone, etretinate, daunorubicin/tretinoin, idarubicin/tretinoin, doxorubicin/tretinoin, bexarotene, acitretin, tretinoin, alitretinoin, of calcipotriene,

ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3- hydroxypropoxy)calcitriol, betamethasone dipropionate/calcipotriene, alfacalcidol, calcium carbonate/cholecalciferol, paricalcitol, doxercalciferol, cholecalciferol, calcitriol, calcifediol, seocalcitol, l7-alpha-ethinylestradiol, fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF-4227, bazedoxifene, estradiol valerate/testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol/ethynodiol diacetate, estradiol acetate, esterified estrogens, estradiol cypionate/medroxyprogesterone acetate, estradiol/norethindrone acetate, estradiol cypionate/testosterone cypionate, synthetic conjugated estrogens, B, etonogestrel, CC8490, MITO-4509, cyproterone acetate/ethinyl estradiol, ethinyl estradiol/etonogestrel, pipendoxifene, chlorotrianisene, icaritin, megestrol acetate/tamoxifen, sulindac/tamoxifen, sulindac/toremifene, raloxifene/sulindac, F18 l6-alpha- fluoroestradiol, ARN-810, Z-endoxifen, goserelin/tamoxifen, raloxifene/teriparatide, AZD9496, elacestrant, SRN-927, fulvestrant/palbociclib, anastrozole/tamoxifen,

fulvestrant/letrozole/tamoxifen, anastrozole/exemestane/fulvestrant,

anastrozole/goserelin/tamoxifen, anastrozole/fulvestrant/tamoxifen, exemestane/fulvestrant, fulvestrant/letrozole, letrozole/tamoxifen, exemestane/tamoxifen,

exemestane/fulvestrant/goserelin/letrozole/tamoxifen, anastrozole/fulvestrant,

fulvestrant/pertuzumab/trastuzumab, pertuzumab/tamoxifen/trastuzumab, LSZ102, selective estrogen receptor modulator, desogestrel/ethinyl estradiol, drospirenone/ethinyl estradiol, ethinyl estradiol/norelgestromin, ethinyl estradiol/norethindrone, ethinyl estradiol/levonorgestrel, ethinyl estradiol/norgestrel, ethinyl estradiol/norgestimate, diethylstilbestrol, ospemifene, toremifene, tamoxifen, raloxifene, everolimus/tamoxifen, H3B-6545, arzoxifene, clomiphene, SAR439859, estramustine phosphate, diethylstilbestrol diphosphate, estradiol/levonorgestrel, tamoxifen/trastuzumab, fulvestrant/trastuzumab, everolimus/fulvestrant, TTC-352,

medroxyprogesterone acetate, desogestrel, danazol, trilostane, fluoxymesterone, norgestimate, progesterone, S-equol, SC75741, BAY 11-7082, JSH-23, and Neferine, FL-411, ZL0420, ZEN- 3411, and PLX51107, PK11007, Serdemetan, RITA, JNJ-26854165, and MI-773, LY2090314,

A 1070722, and AZD2858. A reduction in low density lipoprotein (LDL) cholesterol is observed in the subject after the treatment.

[00440] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[00441] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

1. A method for modulating PCSK9 expression in a cell, comprising: contacting the cell with a compound that modulates a first target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, thereby modulating PCSK9 expression.

2. A method for modulating ANGPTL3 expression in a cell, comprising: contacting the cell with a compound that modulates a second target selected from the group consisting of mTOR, Transforming Growth Factor b receptor (TGF R) I, TGF receptor II, SMAD2, SMAD3, STAT1, NF-KB, BRIM, p53, and TCF7F2 thereby modulating ANGPTF3 expression.

3. The method of any of the above claims, wherein the cell has a PNPFA3 mutation.

4. The method of claim 3, wherein the PNPFA3 mutation is a gain of function mutation.

5. The method of any one of claims 3-4, wherein the mutation is the presence of a G allele at SNP rs738409.

6. The method any one of claims 3-5, wherein the cell is homozygous for the PNPFA3 G allele at SNP rs738409.

7. The method any one of claims 3-5, wherein the cell is heterozygous for the PNPFA3 G allele at SNP rs738409.

8. The method of any one of claims 3-4, wherein the mutation is an I148M mutation in the PNPFA3 protein.

9. The method any one of claims 3-4 or 8, wherein the cell is homozygous for the mutant PNPFA3 protein carrying the I148M mutation.

10. The method any one of claims 3-4 or 8, wherein the cell is heterozygous for the mutant PNPFA3 protein carrying the I148M mutation.

11. The method of any one of claims 3-10, further comprising determining the presence of the PNPFA3 mutation in the cell using a method selected from the group consisting of a mass spectroscopy assay, an oligonucleotide microarray analysis, an allele-specific hybridization assay, an allele-specific PCR assay, and a nucleic acid sequencing assay.

12. The method of any of the above claims, wherein the cell is a hepatocyte.

13. The method of any of the above claims, wherein the contacting is done in vivo or ex- vivo.

14. The method of any of the above claims, wherein the compound is a compound selected from Table 2 or Table 7.

15. The method of any of the above claims, wherein the modulating PCSK9 or ANGPTL3 expression reduces PCSK9 or ANGPTL3 expression.

16. The method of any of the above claims, wherein the target is mTOR and the compound is selected from the group consisting of OSI-027, PF-04691502, WYE-125132, CC-223,

Everolimus, Palomid 529 (P529), GDC-0349, Torin 1, PP242, WAY600, CZ415, INK128, TAK659, AZD-8055, Deforolimus, and JR-AB2-011.

17. The method of any of the above claims, wherein the mTOR inhibitor is OSI-027.

18. The method of any of the above claims, wherein the mTOR inhibitor inhibits mTORC2.

19. The method of claim 18, wherein the mTORC2 inhibitor inhibits RICTOR.

20. The method of claim 19, wherein the mTORC2 inhibitor is JR-AB2-011.

21. The method of any of the above claims, wherein the compound comprises a small interfering RNA (siRNA) directed against the first or the second target.

22. The method of claim 21, wherein the siRNA targets a gene selected from the group consisting of RICTOR, mTOR, Deptor, AKT, mLST8, mSINl, and Protor.

23. The method of any one of claims 2-15, wherein the first and/or second target is TGF RI, TGF RII, SMAD2, or SMAD3 and the compound is selected from the group consisting of LY2157299, LY-364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R-268712, SB-431542, EW-7197, LDN-212854, Halofuginone, ITD-l, LDN- 214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83-01 sodium salt, A 83-01, LDN193189 (Hydrochloride), Oxymatrine, Kartogenin, SRI-011381 (hydrochloride),

Halofuginone, SIS3, LY2157299.

24. The method of claim 23, wherein the first and/or second target is TGF RI, and the compound is LY2157299.

25. The method of any one of claims 2-15, wherein the first and/or second target is NF-KB, and the compound is selected from the group consisting of SC75741, BAY 11-7082, JSH-23, and Neferine.

26. The method of any one of claims 2-15, wherein the first and/or second target is BRIM, and the compound is selected from the group consisting of FL-411 , ZL0420, ZEN-3411 , and PLX51107.

27. The method of any one of claims 2-15, wherein the first and/or second target is TP53, and the compound is selected from the group consisting of PK11007, Serdemetan, RITA, JNJ- 26854165, and MI-773.

28. The method of any one of claims 2-15, wherein the first and/or second target is TCF7L2, and the compound is selected from the group consisting of LY2090314, A 1070722, and AZD2858.

29. The method of any one of claims 1-15, wherein the first and/or second target is STAT1 or STAT3 and the compound is selected from the group consisting of AG 18, Stattic,

Alantolactone, Napabucasin, OPB-31121, OPB-51602, STAT3 inhibitor XIII, danvatirsen, WP1066, Chrysophanol, SMI-l6a, RG13022, TCS-PIM-l-4a, RG14620, Nifuroxazide, Dihydroisotanshinone I, STAT5-IN-1, Hispidulin, Tyrphostin AG 528, AG-1478, Tyrphostin AG 879, AG 555, Niclosamide, PD158780, Piml/AKKl-IN-l, PD153035, NSC 74859, TCS PIM-l 1, AZD1208, CL-387785, EAI045, Artesunate, BIBX 1382, Icotinib, PD153035 (Hydrochloride), AS1517499, HJC0152 hydrochloride, Diosgenin, Fedratinib (SAR302503, TG101348), TP-3654, Morusin, Icotinib (Hydrochloride), PF-06459988, AEE788, AZD3759, CX-6258, Scutellarin, HO-3867, Pelitinib, Mubritinib, CP-724714, Dacomitinib, C188-9, Sapitinib, Irbinitinib, Gefitinib (hydrochloride), AZ-5104, Olmutinib, Poziotinib, WZ4002, AZD-9291, CNX-2006, TAK-285, Lazertinib, CO-1686, Neratinib, Canertinib

(dihydrochloride), BMS-599626 (Hydrochloride), AZD-9291 (mesylate), Avitinib (maleate), SH-4-54, BP-1-102, CO-1686 (hydrobromide), Saikosaponin D.

30. The method of any one of claims 1 or 3-15, wherein the first and/or second target is Myc and the compound is selected from the group consisting of Myc-targeting siRNA DCR-MY C and AVI-4126.

31. The method of any one of claims 1 or 3-15, wherein the first and/or second target is NR3C1 and the compound is selected from the group consisting of rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate, fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate, 1 lbeta hydrocortisone acetate, betamethasone, dexamethasone, budesonide, fluticasone propionate, beclomethasone dipropionate, betamethasone

acetate/betamethasone phosphate, betamethasone acetate, triamcinolone acetonide, ciprofloxacin/hydrocortisone, ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone dipropionate/calcipotriene, fluticasone furoate, budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate, beclomethasone 17-monopropionate, dexamethasone/lenalidomide/sorafenib, docetaxel/prednisone, carmustine/prednisone, cabazitaxel/prednisone, dexamethasone/lenalidomide, hydrocortisone/prednisone,

dexamethasone/thalidomide, cyclophosphamide/prednisone/vincristine,

hydrocortisone/mitoxantrone, mitoxantrone/prednisone, docetaxel/hydrocortisone,

cyclophosphamide/mitoxantrone/prednisone/vincristine,

cyclophosphamide/etoposide/prednisone/vincristine, dexamethasone/fludarabine

phosphate/mitoxantrone/rituximab, chlorambucil/mitoxantrone/prednisone,

chlorambucil/mitoxantrone/prednisone/rituximab, clocortolone, alclometasone,

prednisone/somatotropin, carfilzomib/dexamethasone/rituximab,

cyclophosphamide/prednisolone/vincristine,

cyclophosphamide/prednisolone/rituximab/vincristine,

cytarabine/dexamethasone/oxaliplatin/rituximab, everolimus/prednisone,

cyclophosphamide/gemcitabine/prednisolone/rituximab/vincristine,

cyclophosphamide/epirubicin/prednisone/vincristine, dexamethasone/enzalutamide, abiraterone/prednisolone, dexamethasone/palonosetron, docetaxel/prednisolone, cabazitaxel/prednisolone, prednisone/vincristine, carfilzomib/dexamethasone, ciprofloxacin/fluocinolone acetonide, dexamethasone/vincristine,

glycopyrrolate/indacaterol/mometasone furoate, indacaterol/mometasone furoate,

fluticasone/salmeterol, betamethasone/clotrimazole, aprepitant/dexamethasone,

bortezomib/dexamethasone/pomalidomide, dexamethasone/imatinib/vincristine,

acetaminophen/diphenhydramine/prednisolone,

acetaminophen/diphenhydramine/methylprednisolone,

cytarabine/dasatinib/dexamethasone/methotrexate,

cytarabine/dexamethasone/imatinib/methotrexate,

cyclophosphamide/daunorubicin/imatinib/prednisolone/vincristine,

methylprednisolone/mycophenolate mofetil, mycophenolate mofetil/prednisone,

infliximab/methylprednisolone, prednisone/tacrolimus, infliximab/prednisone,

32. The method of any one of claims 1 or 3-15, wherein the first and/or second target is VDR and the compound is selected from the group consisting of calcipotriene, ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3-hydroxypropoxy)calcitriol, betamethasone dipropionate/calcipotriene, alfacalcidol, calcium carbonate/cholecalciferol, paricalcitol, doxercalciferol, cholecalciferol, calcitriol, calcifediol, and seocalcitol.

33. The method of any one of claims 1 or 3-15, wherein the first and/or second target is ESR1 and the compound is selected from the group consisting of l7-alpha-ethinylestradiol, fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF-4227, bazedoxifene, estradiol valerate/testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol/ethynodiol diacetate, estradiol acetate, esterified estrogens, estradiol cypionate/medroxyprogesterone acetate, estradiol/norethindrone acetate, estradiol cypionate/testosterone cypionate, synthetic conjugated estrogens, B, etonogestrel, CC8490, MITO-4509, cyproterone acetate/ethinyl estradiol, ethinyl estradiol/etonogestrel, pipendoxifene, chlorotrianisene, icaritin, megestrol acetate/tamoxifen, sulindac/tamoxifen, sulindac/toremifene, raloxifene/sulindac, F18 l6-alpha-fluoroestradiol, ARN-810, Z-endoxifen, goserelin/tamoxifen, raloxifene/teriparatide, AZD9496, elacestrant, SRN-927,

fulvestrant/palbociclib, anastrozole/tamoxifen, fulvestrant/letrozole/tamoxifen,

anastrozole/exemestane/fulvestrant, anastrozole/goserelin/tamoxifen,

anastrozole/fulvestrant/tamoxifen, exemestane/fulvestrant, fulvestrant/letrozole,

letrozole/tamoxifen, exemestane/tamoxifen, exemestane/fulvestrant/letrozole/tamoxifen, anastrozole/exemestane/fulvestrant/tamoxifen, anastrozole/fulvestrant/goserelin/tamoxifen, exemestane/fulvestrant/tamoxifen, exemestane/fulvestrant/goserelin/letrozole/tamoxifen, anastrozole/fulvestrant, fulvestrant/pertuzumab/trastuzumab,

pertuzumab/tamoxifen/trastuzumab, FSZ102, selective estrogen receptor modulator, desogestrel/ethinyl estradiol, drospirenone/ethinyl estradiol, ethinyl estradiol/norelgestromin, ethinyl estradiol/norethindrone, ethinyl estradiol/levonorgestrel, ethinyl estradiol/norgestrel, ethinyl estradiol/norgestimate, diethylstilbestrol, ospemifene, toremifene, tamoxifen, raloxifene, everolimus/tamoxifen, H3B-6545, arzoxifene, clomiphene, SAR439859, estramustine phosphate, diethylstilbestrol diphosphate, estradiol/levonorgestrel, tamoxifen/trastuzumab, fulvestrant/trastuzumab, everolimus/fulvestrant, TTC-352, fulvestrant/ribociclib, 4- hydroxytamoxifen, dienestrol, acolbifene, estramustine, medroxyprogesterone acetate, desogestrel, danazol, trilostane, fluoxymesterone, norgestimate, progesterone, and S-equol.

34. The method of any of the above claims, wherein the modulation of the first and/or second target alters binding of the target to a PCSK9 or ANGPTL3 enhancer region.

35. The method of any of the above claims, wherein the alteration of binding reflects binding of the compound to the first and/or second target or binding of the compound to the enhancer and the alteration is selected from group consisting of an alteration in phosphorylation of the first and/or second target, an alteration in localization of the first and/or second target, an alteration in the expression level of the first and/or second target, an alteration in methylation of the first and/or second target, an alteration in acetylation of the first and/or second target, an alteration in ubiquitination of the first and/or second target, an alteration in glycosylation of the first and/or second target, an alteration in sumoylation of the first and/or second target, an alteration in stability of the first and/or second target, and an alteration in degradation of the first and/or second target.

36. The method of any one of the above claims, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced by at least about 30%, 50% or 70%.

37. The method of claim 36, wherein the reduction is determined in a population of cells and the amount of reduction is determined by reference to a matched control cell population.

38. A method for treating a disease comprising: administering to a mammalian subject an effective amount of a compound that modulates a first target selected from the group consisting of mTOR, ONECUT1, Myc, NR3C1, VDR, ESR1, SMAD2, SMAD3 and STAT3, wherein said modulating of the target reduces PCSK9 expression and thereby treats the disease.

39. A method for treating a disease comprising: administering to a mammalian subject an effective amount of a compound that modulates a second target selected from the group consisting of mTOR, Transforming Growth Factor b receptor (TGF R) I, TGF receptor II, SMAD2, SMAD3, STAT1, NF-kB, BRIM, p53, and TCF7F2, wherein said modulating of the target reduces ANGPTF3 expression and thereby treats the disease.

40. The method of claim 38 or 39, wherein the disease is a liver disease or a disease associated with a blood or serum ratio of high density lipoprotein (HDF)-cholesterol/ low density lipoprotein (FDF)-cholesterol of less than or equal to 0.3, optionally wherein the disease is selected from the group consisting of: non-alcoholic fatty liver disease (NAFFD), non alcoholic steatohepatitis (NASH), alcoholic liver disease (AFD), and a high FDF-cholesterol associated disease.

41. The method of claim 40, wherein the high LDL-cholesterol associated disease occurs in a subject having a PCSK9-activating (GOF) mutation, a marked elevation of low density lipoprotein particles in the plasma, primary hypercholesterolemia, or heterozygous Familial Hypercholesterolemia (heFH).

42. The method of any one of claims 38-41, wherein the subject has a PNPLA3 mutation.

43. The method of claim 42, wherein the PNPLA3 mutation is a gain of function mutation.

44. The method of claim 42 or 43, wherein the mutation is the presence of a G allele at SNP rs738409.

45. The method any one of claims 42-44, wherein a cell from the subject is homozygous for the PNPLA3 G allele at SNP rs738409.

46. The method any one of claims 42-44, wherein the cell is heterozygous for the PNPLA3 G allele at SNP rs738409.

47. The method of any one of claims 42-43, wherein the mutation is an I148M mutation in the PNPLA3 protein.

48. The method any one of claims 42-43 or 47, wherein the cell is homozygous for the mutant PNPLA3 protein carrying the I148M mutation.

49. The method any one of claims 42-43 or 47, wherein the cell is heterozygous for the mutant PNPLA3 protein carrying the I148M mutation.

50. The method of any one of claims 42-49, further comprising determining the presence of the PNPLA3 mutation in the cell using a method selected from the group consisting of a mass spectroscopy assay, an oligonucleotide microarray analysis, an allele-specific hybridization assay, an allele-specific PCR assay, and a nucleic acid sequencing assay.

51. The method of any one of claims 38-50, wherein the subject is human.

52. The method of any one of claims 38-51, wherein the compound is a compound selected from Table 2 or Table 7.

53. The method of any one of claims 38-52, wherein the target is mTOR and the compound is selected from the group consisting of OSI-027, PF-04691502, WYE-125132, CC-223, Everolimus, Palomid 529 (P529), GDC-0349, Torin 1, PP242, WAY600, CZ415, INK128, TAK659, AZD-8055, Deforolimus, and JR-AB2-011.

54. The method of claim 53, wherein the mTOR inhibitor is OSI-027.

55. The method of any one of claims 38-54, wherein the mTOR inhibitor inhibits mTORC2.

56. The method of claim 55, wherein the mTORC2 inhibitor inhibits RICTOR.

57. The method of claim 56, wherein the mTORC2 inhibitor is JR-AB2-011.

58. The method any one of claims 38-52, wherein the compound comprises a small interfering RNA (siRNA) directed against the first or the second target.

59. The method of claim 58, wherein the one or more siRNA targets one or more genes selected from the group consisting of RICTOR, mTOR, Deptor, AKT, mLST8, mSINl, and Protor.

60. The method of any one of claims 38-59, wherein the administration of the compound capable of modulating the expression of the PCSK9 or ANGPTL3 gene does not induce hyperinsulinemia in the subject.

61. The method of any one of claims 38-60, wherein the administration of the compound capable of modulating the expression of the PCSK9 or ANGPTL3 gene does not induce hyperglycemia in the subject.

62. The method of any one of claims 38-52, wherein the first and/or second target is TGF RI, TGF RII, SMAD2, or SMAD3 and the compound is selected from the group consisting ofLY2l57299, LY-364947, A 77-01, RepSox, SJ000291942, SB-505124, SB 525334, K02288, ML347, SD-208, R-268712, SB-431542, EW-7197, LDN-212854,

Halofuginone, ITD-l, LDN-214117, GW788388, LY3200882, EW-7197 Hydrochloride, A 83- 01 sodium salt, A 83-01, LDN193189 (Hydrochloride), Oxymatrine, Kartogenin, SRI-011381 (hydrochloride), Halofuginone, SIS3, LY2157299.

63. The method of claim 62, wherein the first and/or second target is TGF RI, and the compound is LY2157299.

64. The method of any one of claims 39-52, wherein the first and/or second target is NF-KB, and the compound is selected from the group consisting of SC75741, BAY 11-7082, JSH-23, and Neferine.

65. The method of any one of claims 39-52, wherein the first and/or second target is is BRIM, and the compound is selected from the group consisting of FL-411, ZL0420, ZEN-3411, and PLX51107

66. The method of any one of claims 39-52, wherein the first and/or second target is TP53, and the compound is selected from the group consisting of PK11007, Serdemetan, RITA, JNJ- 26854165, and MI-773.

67. The method of any one of claims 39-52, wherein the first and/or second target is TCF7L2, and the compound is selected from the group consisting of LY2090314, A 1070722, and AZD2858

68. The method of any one of claims 38-52, wherein the first and/or second target is STAT1 or STAT3 and the compound is selected from the group consisting of AG 18, Stattic,

69. The method of any one of claims 38-52, wherein the first and/or second target is MYC and the compound is selected from the group consisting of MY C-targeting siRNA DCR-MY C and AVI-4126.

70. The method of any one of claims 38-52, wherein the first and/or second target is NR3C1 and the compound is selected from the group consisting of rimexolone, medrysone, clocortolone pivalate, diflorasone diacetate, fluorometholone, dexamethasone phosphate, cortisone acetate, halcinonide, flurandrenolide, desoximetasone, desonide, prednisolone, clobetasol propionate, fluocinolone acetonide, prednisone, hydrocortisone, triamcinolone, dexamethasone 21 -acetate,

ciprofloxacin/dexamethasone, ORG 34517, ciclesonide, betamethasone dipropionate/calcipotriene, fluticasone furoate, budesonide/formoterol, deacylcortivazol, difluprednate, formoterol/mometasone furoate, beclomethasone, fluticasone furoate/vilanterol, azelastine/fluticasone propionate, beclomethasone 17-monopropionate,

dexamethasone/thalidomide, cyclophosphamide/prednisone/vincristine,

hydrocortisone/mitoxantrone, mitoxantrone/prednisone, docetaxel/hydrocortisone,

cyclophosphamide/mitoxantrone/prednisone/vincristine,

cyclophosphamide/etoposide/prednisone/vincristine, dexamethasone/fludarabine

phosphate/mitoxantrone/rituximab, chlorambucil/mitoxantrone/prednisone,

chlorambucil/mitoxantrone/prednisone/rituximab, clocortolone, alclometasone,

prednisone/somatotropin, carfilzomib/dexamethasone/rituximab,

cyclophosphamide/prednisolone/vincristine,

cyclophosphamide/prednisolone/rituximab/vincristine,

cytarabine/dexamethasone/oxaliplatin/rituximab, everolimus/prednisone,

cyclophosphamide/gemcitabine/prednisolone/rituximab/vincristine,

cabazitaxel/prednisolone, prednisone/vincristine, carfilzomib/dexamethasone,

ciprofloxacin/fluocinolone acetonide, dexamethasone/vincristine,

glycopyrrolate/indacaterol/mometasone furoate, indacaterol/mometasone furoate, fluticasone/salmeterol, betamethasone/clotrimazole, aprepitant/dexamethasone,

bortezomib/dexamethasone/pomalidomide, dexamethasone/imatinib/vincristine,

acetaminophen/diphenhydramine/prednisolone,

acetaminophen/diphenhydramine/methylprednisolone,

cytarabine/dasatinib/dexamethasone/methotrexate,

cytarabine/dexamethasone/imatinib/methotrexate,

cyclophosphamide/daunorubicin/imatinib/prednisolone/vincristine,

methylprednisolone/mycophenolate mofetil, mycophenolate mofetil/prednisone,

infliximab/methylprednisolone, prednisone/tacrolimus, infliximab/prednisone,

71. The method of any one of claims 38-52, wherein the first and/or second target is VDR and the compound is selected from the group consisting of calcipotriene, ergocalciferol, inecalcitol, ILX-23-7553, alendronate/cholecalciferol, 2-(3-hydroxypropoxy)calcitriol, betamethasone dipropionate/calcipotriene, alfacalcidol, calcium carbonate/cholecalciferol, paricalcitol, doxercalciferol, cholecalciferol, calcitriol, calcifediol, and seocalcitol.

72. The method of any one of claims 38-52, wherein the first and/or second target is ESR1 and the compound is selected from the group consisting ofl7-alpha-ethinylestradiol, fulvestrant, beta-estradiol, estradiol l7beta-cypionate, estriol, estrone, estradiol valerate, estrone sulfate, mestranol, CHF-4227, bazedoxifene, estradiol valerate/testosterone enanthate, TAS-108, ethynodiol diacetate, ethinyl estradiol/ethynodiol diacetate, estradiol acetate, esterified estrogens, estradiol cypionate/medroxyprogesterone acetate, estradiol/norethindrone acetate, estradiol cypionate/testosterone cypionate, synthetic conjugated estrogens, B, etonogestrel, CC8490, MITO-4509, cyproterone acetate/ethinyl estradiol, ethinyl estradiol/etonogestrel, pipendoxifene, chlorotrianisene, icaritin, megestrol acetate/tamoxifen, sulindac/tamoxifen, sulindac/toremifene, raloxifene/sulindac, F18 l6-alpha-fluoroestradiol, ARN-810, Z-endoxifen, goserelin/tamoxifen, raloxifene/teriparatide, AZD9496, elacestrant, SRN-927,

anastrozole/exemestane/fulvestrant, anastrozole/goserelin/tamoxifen,

73. The method of any one of claims 38-72, wherein the modulating the first and/or second target alters binding of the target to a PCSK9 or ANGPTF3 enhancer region.

74. The method of any one of claims 38-73, wherein the alteration of binding reflects binding of the compound to the first and/or second target or binding of the compound to the enhancer and the alteration is selected from group consisting of an alteration in phosphorylation of the first and/or second target, an alteration in localization of the first and/or second target, an alteration in the expression level of the first and/or second target, an alteration in methylation of the first and/or second target, an alteration in acetylation of the first and/or second target, an alteration in ubiquitination of the first and/or second target, an alteration in glycosylation of the first and/or second target, an alteration in sumoylation of the first and/or second target, an alteration in stability of the first and/or second target, and an alteration in degradation of the first and/or second target.

75. The method of any one of claims 38-74, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced in the liver of the subject.

76. The method of claim 75, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatocytes of the subject.

77. The method of claim 75, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatic stellate cells of the subject.

78. The method of claim 75, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced in the hepatocytes and hepatic stellate cells of the subject.

79. The method of any one of claims 38-78, wherein the method further comprises assessing or having assessed a hepatic triglyceride content in the subject.

80. The method of claim 79, wherein the assessing or having assessed step comprises using a method selected from the group consisting of liver biopsy, liver ultrasonography, computer- aided tomography (CAT) and nuclear magnetic resonance (NMR).

81. The method of claim 80, wherein the assessing or having assessed step comprises proton magnetic resonance spectroscopy (¹H-MRS).

82. The method of claim 79, wherein the subject is eligible for treatment based on a hepatic triglyceride content greater than 5.5% volume/volume.

83. The method of any one of claims 38-82, wherein the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a matched control population.

84. The method of any one of claims 38-82, wherein the reduction is determined in a population of test subjects and the amount of reduction is determined by reference to a pre treatment baseline measurement.

85. A method for identifying a compound that reduces PCSK9 or ANGPTL3 gene expression comprising

a. providing a candidate compound;

b. assaying the candidate compound for at least two of the activities selected from the group consisting of: mTOR inhibitory activity, mTORC2 inhibitory activity, PI3K inhibitory activity, RI3Kb inhibitory activity, DNA-PK inhibitory activity, ability to induce hyperinsulinemia, ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity; and

c. identifying the candidate compound as the compound based on results of the two or more assays that indicate the candidate compound has two or more desirable properties.

86. The method of claim 85, wherein the desirable properties are selected from the group consisting of: mTOR inhibitory activity, lack of PI3K inhibitory activity, lack of RI3Kb inhibitory activity, lack of DNA-PK inhibitory activity, lack of ability to induce

hyperinsulinemia, lack of ability to induce hyperglycemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity.

87. The method of claim 86, wherein mTOR inhibitory activity comprises inhibition of mTORC2 activity.

88. The method of claim 86, wherein the mTOR inhibitory activity is mTORCl and mTORC2 inhibitory activity.

89. The method of claim 86, wherein the PI3K inhibitory activity is RI3Kb inhibitory activity.

90. The method of any of claims 85-89, wherein the assaying step comprises assaying for at least three of the activities.

91. The method of any of claims 85-89, wherein the assaying step comprises assaying for at least four of the activities.

92. The method of any of claims 85-89, wherein the assaying step comprises assaying for at least five of the activities.

93. The method any of claims 85-89, wherein the at least two assays of step (b) comprise assays for mTOR inhibitory activity and PI3K inhibitory activity.

94. The method any of claims 85-89, wherein the at least two assays of step (b) comprise assays for mTORC2 inhibitory activity and RI3Kb inhibitory activity.

95. The method of claim 90, wherein the at least three assays of step (b) comprise assays for mTOR inhibitory activity, PI3K inhibitory activity, and ability to induce hyperinsulinemia.

96. The method of claim 91, wherein the at least four assays of step (b) comprise mTOR inhibitory activity, PI3K inhibitory activity, ability to induce hyperinsulinemia, and PCSK9 or ANGPTL3 gene expression inhibitory activity.

97. The method of any one of claims 85-96, wherein the assay is a biochemical assay.

98. The method of any one of claims 85-96, wherein the assay is a cellular assay.

99. The method of claim 98, wherein the cell is a mammalian cell.

100. The method of claim 99, wherein the cell is a human cell.

101. The method of any one of claims 98-100, wherein the cell is a wild type cell.

102. The method of any one of claims 98-100, wherein the cell comprises the G allele at SNP rs738409 of a PNPLA3 gene or a mutant I148M PNPLA3 protein.

103. The method of claim 102, wherein the cell is homozygous for the PNPLA3 G allele at SNP rs738409.

104. The method of claim 102, wherein the cell is heterozygous for the PNPLA3 G allele at SNP rs738409.

105. The method of claim 102, wherein the cell is homozygous for the mutant PNPLA3 protein carrying the I148M mutation.

106. The method of claim 102, wherein the cell is heterozygous for the mutant PNPLA3 protein carrying the I148M mutation.

107. The method of claim 85, wherein assaying the PCSK9 or ANGPTL3 gene expression comprises a method selected from the group consisting of: mass spectroscopy, oligonucleotide microarray analysis, allele-specific hybridization, allele-specific PCR, and nucleic acid sequencing.

108. The method of claim 107, wherein the expression of the PCSK9 or ANGPTL3 gene is reduced by at least about 30%, 50% or 70%.

109. The method of claim 108, wherein the reduction is determined in a population of cells and the amount of reduction is determined by reference to a matched control cell population.