Methods for reducing liver triglycerides

Abstract

A method for reducing hepatic triglyceride levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a proprotein convertase 7 (PC7) inhibitor. Also provided herein are compositions and kits comprising the inhibitor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on February 7, 2023, and published in English under PCT Article 21(2), under PCT Application Serial No. PCT / CA2023 / *, which itself claims the benefit of U.S. Provisional Application Serial No. 63 / 267,671, filed on February 8, 2022. All of the above documents are incorporated herein by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT N / A

[0003] The present disclosure relates to methods of reducing hepatic triglycerides, and more particularly, to methods of reducing hepatic triglycerides using proprotein convertase 7 (PC7) inhibitors.

[0004] Sequence Listing Reference In accordance with 37 C.FR1.821(c), the Sequence Listing is hereby submitted as an ASCII-compliant text file entitled Sequence Listing_G12810-00836, created on February 7, 2023, and having a size of 128 kilobytes. The contents of the above file entitled Sequence Listing_G12810-00836 are incorporated herein by reference in their entirety. [Background technology]

[0005] Nonalcoholic fatty liver disease (NAFLD) is a general term that includes several liver diseases that affect people who drink little or no alcohol. NAFLD is characterized by excess fat stored in liver cells. They are becoming increasingly common around the world, especially in Western countries. For example, about 20% of the Canadian population is affected by it, and there is currently no established treatment. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD characterized by inflammation of the liver and damage caused by the accumulation of fat in the liver (see Figure 1, which shows the progression from a healthy liver to a fatty liver and a NASH liver). NASH is associated with high triglyceride levels.

[0006] For example, in NAFLD, alternative methods for reducing hepatic triglyceride levels are needed.

[0007] This description makes reference to several documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0008] More specifically, the present disclosure provides a method for reducing hepatic triglyceride levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a proprotein convertase 7 (PC7) inhibitor. In another specific embodiment, the present disclosure provides a method for treating NAFLD or a symptom thereof.

[0009] More specifically, the present disclosure provides the following: Item 1. A method for reducing hepatic triglyceride levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a proprotein convertase 7 (PC7) inhibitor. Item 2. The method of item 1, wherein the inhibitor is an anti-PC7 antisense oligonucleotide (ASO), an anti-PC7 microRNA (miRNA), an anti-PC7 small interfering RNA (siRNA), or a CRISPR base editor or prime editor. Item 3. The method of item 2, wherein the inhibitor is an ASO. Item 4. The method of item 3, wherein the ASO targets a human PC7 RNA exon or transcript. Item 5. The method of item 2, wherein the inhibitor is a siRNA. Item 6. The method of item 2, wherein the inhibitor is a miRNA. Item 7. The method of item 6, wherein the miRNA targets one or more of human PCSK7 3'-UTR, PCSK7 exon 14, or PCSK7 exon 15. Item 8. The method according to any one of items 1 to 7, wherein the subject has nonalcoholic fatty liver disease (NAFLD) or is a potential candidate for NAFLD. Item 9. The method of item 8, wherein the subject has nonalcoholic steatohepatitis (NASH) or is a potential candidate for NASH. Item 10. The method according to any one of items 1 to 9, wherein the subject is a human. Item 11. A kit for reducing liver triglyceride levels in a subject, comprising: (A) Proprotein convertase 7 (PC7) inhibitors, and (B)(i) another drug for the prevention or treatment of nonalcoholic fatty liver disease (NAFLD) or a symptom thereof; Item 12.(ii) a pharma- ceutically acceptable carrier; Item 13.(iii) A kit comprising instructions for using the kit for reducing liver triglyceride levels, or Item 14.(iv) a combination of at least two of (i) to (iii). Item 15. The kit according to item 11, wherein the PC7 inhibitor is (a) an anti-PC7 antisense oligonucleotide (ASO), (b) an anti-PC7 microRNA (miRNA) against PC7, or (c) an anti-PC7 small interfering RNA (siRNA). Item 16. The kit according to Item 12, wherein the ASO, miRNA, or siRNA is specific to human PC7. Item 17. A composition comprising: (A) a proprotein convertase 7 (PC7) inhibitor; and (B) (i) another agent for the prevention or treatment of nonalcoholic fatty liver disease (NAFLD) or a symptom thereof; (ii) a pharma- ceutically acceptable carrier; or (iv) a combination of at least two of (i) to (iii). Item 18. The composition according to item 14, wherein the PC7 inhibitor is (a) an anti-PC7 antisense oligonucleotide (ASO) specific for PC7, (b) an anti-PC7 microRNA (miRNA), or (c) an anti-PC7 small interfering RNA (siRNA). Item 19. The composition described in Item 15, wherein the ASO, miRNA, or siRNA is specific to human PC7. Item 20. The composition described in Item 16, wherein the ASO targets a human PC7 RNA transcript. Item 21. The composition of item 16, wherein the inhibitor is a siRNA. Item 22. The method of item 16, wherein the inhibitor is miRNA. Item 23. The method of item 19, wherein the miRNA targets one or more of human PCSK7 3'-UTR, PCSK7 exon 14, or PCSK7 exon 15. In another embodiment, a PC7 inhibitor as described herein is provided, including but not limited to anti-PC7 siRNA, for example, as shown in Figure 6A, anti-PC7 ASO, for example, as shown in Figure 33B or 35B, and is optionally further modified to target hepatocytes. For example, the ASO is conjugated at its 5' and / or 3' end with a GalNAc moiety, as described herein. In another embodiment, a composition is provided that includes a PC7 inhibitor and a pharma- ceutically acceptable excipient.

[0010] definition The terms and symbols of genetics, molecular biology, biochemistry, and nucleic acid used herein are in accordance with standard treatises and texts in the field, such as Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984), etc. All terms should be understood in the typical sense established in the relevant art.

[0011] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (particularly in the context of the claims which follow) should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0012] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0013] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein. Every subset of values ​​within a range is also incorporated herein as if it were individually recited herein.

[0014] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0015] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better clarify the disclosure and does not limit the scope of the disclosure, unless otherwise specified.

[0016] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0017] As used herein, the term "about" has its ordinary meaning. In embodiments, "about" may mean plus or minus 10% of the qualified numerical value.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0019] PC7 inhibitors As used herein, the term "PC7" (EC 3.4.21.B27), also referred to as "proprotein convertase 7" or "proprotein convertase subtilisin / kexin type 7 (PCSK7)", belongs to the subtilisin-like proprotein convertase family, which are proprotein convertases that process latent precursor proteins into their biologically active products. It is a calcium-dependent serine endoprotease and is structurally related to its family members furin, PC5 and PACE4. It has a transmembrane segment (residues 668-688) close to its C-terminus and is not secreted, so is concentrated within the membrane-associated transmembrane Golgi network. Without being so limited, human PC7 amino acid and nucleotide sequences are shown in Figures 41A-B (SEQ ID NOs: 1-2) and 42A-D (SEQ ID NOs: 3-4), rat PC7 amino acid and nucleotide sequences are shown in Figures 43 and 44 (SEQ ID NOs: 5-6), and mouse PC7 amino acid and nucleotide sequences are shown in Figures 45 and 46A-G (SEQ ID NOs: 7-11).

[0020] As used herein, the term "PC7 inhibitor" refers to an agent that can reduce the expression (e.g., protein levels) and / or activity of PC7. Without being so limited, PC7 inhibitors include nucleic acid PC7 inhibitors. In some embodiments, the inhibition targets PC7 expression.

[0021] In a specific embodiment, the above-mentioned PC7 inhibitors are targeted to hepatocytes (targeting other tissues and / or reducing the dose) to avoid side effects.

[0022] Nucleic Acid PC7 Inhibitors In specific embodiments, the nucleic acid PC7 inhibitor is a single-stranded antisense oligonucleotide (ASO) specific for PC7 mRNA, a microRNA (miRNA) (non-coding RNA), or a dsRNA (e.g., RNAi, siRNA, miRNA (e.g., a precursor or mimic, which is ultimately processed into a single-stranded miRNA)), or a CRISPR-Cas base editor (cytosine, adenine or prime editor) or a CRISPR-Cas prime editor with a guide RNA (gRNA) or prime editing guide RNA (pegRNA) that targets a specific region of the PCSK7 DNA locus.

[0023] This disclosure is not limited by any particular mechanism of action, but in some embodiments the nucleic acid enters the cell and causes degradation, blocks translation, blocks interaction with other factors, or affects splicing of RNA of complementary or identical sequence, including endogenous RNA (mRNA or non-coding).

[0024] Single-stranded antisense oligonucleotides (ASOs) In specific embodiments, the PC7 inhibitor is an antisense oligonucleotide (ASO) specific for PC7 RNA (e.g., human). ASOs are synthetic single-stranded strings of 8-50 nucleotides in length, preferably 10-35, 15-25, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleic acids (natural or modified (e.g., locked nucleic acid, phosphorothioate, 2'O-methyl, 2'O-methoxy, phosphoramidite, etc.)) in length. In some embodiments, the ASO is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 single-stranded nucleotides in length.

[0025] They bind to RNA via standard Watson-Crick base pairing. ASOs encompassed by the present invention interfere with PC7 RNA, resulting in its degradation (Figure 47A-E) (e.g., gapmers), prevent PC7 translation, affect splicing of PC7 mRNA in a manner that affects the resulting protein, resulting in loss of function (e.g., ASO targeting PC7 splice sites), or inhibit binding of proteins important for mRNA processing (e.g., by targeting binding partner sites on PC7). According to the ENCODE database on RNA-binding protein (RBP) profiling based on enhanced cross-linking and immunoprecipitation assays, a non-exhaustive list of RNA-binding proteins (RBPs) that bind to PCSK7 mRNA includes AQR, BCLAF1.BUD13.DDX3X, EIF3H, IGF2BP1, LSM11, PABPN1, U2AF1, and SRSF1.

[0026] ASOs may target exons or introns. In specific embodiments, the ASO comprises a subsequence of a polynucleotide of the present disclosure (e.g., a subsequence of the sequence of Figures 42A-D (SEQ ID NO: 2-4), Figure 44 (SEQ ID NO: 6), or Figures 46A-G (SEQ ID NO: 8-11)). In other specific embodiments, the inhibitor is a gapmer, i.e., an ASO that contains a central block of deoxynucleotide monomers of sufficient length to induce ribonuclease cleavage. In specific embodiments, the ASO of the present disclosure targets an exon. In specific embodiments, the ASO of the present disclosure results in PC7 (e.g., >50% knockdown of PC7) RNA degradation. Examples of ASOs that are specific for and result in degradation of human PC7 and target exons 3, 6, 8, 10, 12, or 14 are shown in Figure 33B, and ASOs that are specific for mouse and target exons 2, 4, or 5 are shown in Figure 35B. In a more specific embodiment, the ASO targets exon 3, exon 12, or exon 14 of human PC7.

[0027] In a specific embodiment, the PC7 ASO described above is modified to target hepatocytes. For example, the ASO is conjugated at its 5' and / or 3' end with a GalNAc moiety. GalNAc moieties include, but are not limited to, triantennary GalNAc, trivalent GalNAc (such as those produced by Trilink), trishexylamino GalNAc, and the like. Examples of such GalNAc-conjugated ASOs are outlined in FIG. 48. In other specific embodiments, the GalNAc is a triantennary N-acetylgalactosamine as described in Prakash et al. Nucleic Acid Research, 2014 42(13)8796-8807. In a specific embodiment, the GalNAc moiety can alternatively or additionally be bound to the membrane of a lipid vesicle (such as a liposome) that encapsulates the ASO.

[0028] In other embodiments, the PC7 inhibitors described above (e.g., PC7 ASOs or miRNA mimics) are chemically modified to increase their stability and / or help them avoid immune responses (e.g., phosphorothioate (PS) (e.g., to increase stability), 2'O-methyl (2'OMe) (e.g., to increase stability and decrease immune response), 2'O-methoxy (2'MOE) (e.g., to increase stability and decrease immune response), phosphoramidite (NP) (e.g., to increase stability), locked nucleic acid or phosphoramidate morpholino (PMO), and peptide nucleic acid (PNA) groups. Such modifications may also aid in loading into the RISC complex and exclusion of the passenger strand.

[0029] double-stranded RNA (dsRNA) molecule In specific embodiments, the PC7 inhibitor is a double-stranded RNA (dsRNA) molecule (or a molecule that includes a double-stranded region). In some embodiments, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more double-stranded nucleotides in length. In some embodiments, the dsRNA is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA) (miRNA mimic).

[0030] Without being so limited, siRNAs for use in the methods disclosed herein include antisense molecules such as those prepared by Ionis pharmaceuticals and those available from Santa Cruz (e.g., sc-40889). Without being so limited, anti-PC7 RNAi inhibitors for use in the methods disclosed herein include antisense molecules such as those prepared by Alnylam. Also provided herein is a double-stranded RNA (dsRNA) molecule comprising a portion of the mature polypeptide coding sequence of any one of the coding sequences of the polypeptides disclosed herein that inhibits expression of that polypeptide in a cell. In a more specific embodiment, the inhibitor is an siRNA, for example, an siRNA of any one of SEQ ID NOs: 12-15 (FIG. 6A).

[0031] When cells are exposed to dsRNA, RNA that contains complementary sequences is selectively degraded by a process called RNA interference (RNAi). In some embodiments, the dsRNA provided herein is used in gene silencing methods. In one aspect, a method is provided for selectively degrading RNA using the dsRNA disclosed herein. In some embodiments, the PC7 inhibitor is an shRNA expressed by a DNA vector transfected or transduced into a target cell. In some embodiments, the PC7 inhibitor is a virus that encodes the shRNA. In some embodiments, the PC7 inhibitor is a vector that encodes the shRNA. This process is alternatively performed in vitro, ex vivo, or in vivo. In one aspect, the dsRNA molecule is used to generate loss-of-function mutations in cells, organs, or organisms. Methods for making and using dsRNA molecules to selectively degrade RNA have been described in the art, see, e.g., U.S. Pat. No. 6,506,559, U.S. Pat. No. 6,511,824, U.S. Pat. No. 6,515,109, and U.S. Pat. No. 6,489,127.

[0032] In some embodiments, the nucleic acid PC7 inhibitors (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) comprise a circular nucleic acid molecule, wherein the nucleic acid PC7 inhibitors (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and the circular oligonucleotide forms a dumbbell-shaped structure having about 19 base pairs and two loops.

[0033] In some embodiments, the circular nucleic acid PC7 inhibitor (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analog thereof) contains two loop motifs, and one or both loop portions of the dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analog thereof) are biodegradable. In some embodiments, the degradation of the loop portion of the circular nucleic acid PC7 inhibitor (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analog thereof) produces a double-stranded nucleic acid PC7 inhibitor (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analog thereof) having a 3'-end overhang, such as a 3'-end overhang comprising about 2 nucleotides. The sense strand of the double-stranded dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analog thereof) can have a terminal cap moiety, such as an inverted deoxybasic moiety, at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand.

[0034] In some embodiments, the 3'-terminal nucleotide overhang of the anti-PC7 dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analogs thereof) comprises ribonucleotides or deoxyribonucleotides chemically modified at the nucleic acid sugar, base, or backbone. In some embodiments, the 3'-terminal nucleotide overhang comprises one or more universal base ribonucleotides. In some embodiments, the 3'-terminal nucleotide overhang comprises one or more acyclic nucleotides.

[0035] In other specific embodiments, the PC7 inhibitor is an oligonucleotide having a nucleic acid sequence of a miRNA (regulatory) specific for PC7 RNA expression (e.g., human) (miRNA mimic). miRNA is a double-stranded RNA (e.g., 18-22 nucleotides long) that post-transcriptionally regulates gene expression by base pairing with the 3' untranslated region of target messenger RNAs and inhibiting their expression. miRNA mimic technology (miR-mimic) is an innovative gene silencing approach to generate non-natural double-stranded miRNA-like RNA fragments. Such RNA fragments are designed to have their 5' ends with motifs that are partially complementary to selected sequences within the 3' UTR unique to the target gene. When introduced into a cell, this RNA fragment, mimicking the endogenous miRNA, can specifically bind to its target gene and cause post-transcriptional repression, more specifically translational inhibition, of the gene. Unlike endogenous miRNA, miR-mimic acts in a gene-specific manner (Xiao, et al., 2007).

[0036] In the present disclosure, miRNAs (or miRNA mimics) are natural or modified (e.g., locked nucleic acid, phosphorothioate, 2'O-methyl, 2'O-methoxy, phosphoramidite, etc.) double-stranded strings of nucleic acids 8-50 nucleotides in length, preferably 10-35, 15-25, 18-22 or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length, that target PCSK7 transcripts. In some embodiments, miRNAs are about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length that target one or more regions of PCSK7 mRNA. In specific embodiments, the oligonucleotides comprise or consist of nucleic acids of miRNAs (miRNA mimics) that target one or more (or two or more, or all three) of exon 14, exon 15, and 3'UTR of PCSK7. In some embodiments, the miRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more single-stranded nucleotides in length. In more specific embodiments, the oligonucleotide comprises or consists of a nucleic acid of miR-125a-5p, miR-143-3p, or miR-409-3p, and any of their mimics (i.e., miRNA mimics of endogenous miRNAs). In more specific embodiments, the oligonucleotide comprises or consists of a nucleic acid of miR-125a-5p.

[0037] Base Editor In a specific embodiment, the PC7 inhibitor is a CRISPR-Cas base editor (CRISPR cytosine base editor, adenine base editor, prime editor) used to induce loss-of-function or gain-of-function mutants of PC7 (e.g., including but not limited to, mutations in the prodomain, catalytic domain, or cytoplasmic tail, such as heterozygous or homozygous G65E, A102T, D187A, H228A, S505E, S505D or R504H, P777L mutants).

[0038] CRISPR adenine base editors can induce targeted A→G edits in DNA (T→C on the opposing strand), whereas CRISPR cytosine base editors can induce targeted C→T edits in DNA (G→A on the opposing strand). These base editors use nickase Cas proteins fused to evolved deoxyadenosine deaminase domains (adenine base editors) or cytidine deaminase domains (cytosine base editors). Used with guide RNAs (gRNAs) to engage double-stranded protospacer DNA sequences adjacent to a protospacer adjacent motif (PAM) sequence at their 3' ends, these base editors chemically modify adenosine (adenine base editors) or cytosine (cytosine base editors) nucleosides on one DNA strand, which in combination with nicking on the other strand allows for A→G or C→T transition mutations, respectively, at the target site. The use of any of the base editors can inactivate genes by disrupting splice donors (canonical GT sequences on the sense strand) or splice acceptors (canonical AG sequences on the sense strand) at exon-intron boundaries, can alter binding sites for transcription factors in regulatory elements that control expression of a target gene, or can cause a loss or gain of function mutant version of a target gene.

[0039] Prime editing uses a catalytically impaired Cas nuclease (nickase) fused to an engineered reverse transcriptase in combination with a prime editing guide RNA (pegRNA) consisting of a spacer sequence that hybridizes to the target DNA site and an extended template sequence for the reverse transcriptase that corresponds to the target site with the desired modification. Prime editors can be designed to induce all different base transversions, as well as insertions or deletions of sequences, making it possible to cause gain or loss of function mutations in the PCSK7 gene.

[0040] For delivery to human hepatocytes, adenine base editor, cytosine base editor or prime editor mRNA is packaged into lipid nanoparticles (which may contain a GalNAc moiety bound to the membrane of the lipid vesicle) along with the relevant PCSK7-specific single guide RNA or pegylated RNA and injected intravenously.

[0041] Generation of nucleic acid PC7 inhibitors In some embodiments, nucleic acid PC7 inhibitor molecules are generated that have a sequence complementary to a target RNA.

[0042] Methods for synthesizing ASOs include the phosphoramidite method and solid-phase or enzymatic synthesis using phosphoramidite building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g., LNA or BNA. ASOs can be purified, for example, by gel electrophoresis or high performance liquid chromatography (HPLC).

[0043] The method of synthesis of synthetic dsRNA molecules includes (a) synthesis of two complementary strands of a dsRNA molecule, and (b) annealing the two complementary strands together under suitable conditions to obtain a double-stranded RNA molecule. In another embodiment, the synthesis of the two complementary strands of the RNA molecule is by solid-phase or enzymatic oligonucleotide synthesis. In yet another embodiment, the synthesis of the two complementary strands of the RNA molecule is by solid-phase tandem oligonucleotide synthesis. In some embodiments, the nucleic acid molecules described herein are synthesized separately and linked together post-synthetically, for example, by ligation or hybridization after synthesis and / or deprotection. Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using any suitable method. The dsRNA constructs can be purified by gel electrophoresis or can be purified by HPLC. In some embodiments, anti-PC7 dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and their analogs) is assembled from two separate oligonucleotides, one strand is sense strand, the other strand is antisense strand, and antisense strand and sense strand are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to the nucleotide sequence of the other strand; e.g., antisense strand and sense strand form a double-stranded or double-stranded structure, e.g., the double-stranded region is about 19 base pairs).In some embodiments, antisense strand of anti-PC7 dsRNA molecule (e.g., siRNA molecule, miRNA molecule, and their analogs) comprises nucleotide sequence that is complementary to the nucleotide sequence of target nucleic acid molecule or a part thereof, and sense strand comprises nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof. In some embodiments, the anti-PC7 dsRNA molecules (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are linked by a nucleic acid-based or non-nucleic acid-based linker(s).In some embodiments, the anti-PC7 dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analogs thereof) comprises a single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (e.g., such dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analogs thereof) does not require that the nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof is present in the dsRNA molecule (e.g., RNAi molecule, siRNA molecule, miRNA molecule, and analogs thereof)), and the single-stranded polynucleotide further comprises a terminal phosphate group such as 5'-phosphate or 5',3'-diphosphate. The terminal structure of the dsRNA molecule described herein is either blunt or sticky (overhanging). In some embodiments, the sticky (overhanging) end structure is a 3' overhang or a 5' overhang. In some embodiments, the number of overhanging nucleotides is any length as long as the overhang does not impair the gene silencing activity. In some embodiments, the overhang sequence is not complementary (antisense) or identical (sense) to the PC7 sequence. In some embodiments, the overhang sequence comprises a low molecular weight structure (e.g., a natural RNA molecule such as tRNA, rRNA, or tumor or CTC RNA, or an artificial RNA molecule).

[0044] The total length of a dsRNA molecule having a linked end structure is expressed as the sum of the length of the paired double-stranded portion and the length of the pair including the overhanging single strand at both ends. For example, in the exemplary case of a 19 bp double-stranded RNA with four nucleotide overhangs at both ends, the total length is expressed as 23 bp. In some embodiments, the end structure of an anti-PC7 dsRNA molecule (e.g., an RNAi molecule, an siRNA molecule, an miRNA molecule, and analogs thereof) has a stem-loop structure in which the ends of one side of the double-stranded nucleic acid are connected by a linker nucleic acid, e.g., a linker RNA. In some embodiments, the length of the double-stranded region (stem-loop portion) is 15 to 49 bp, often 15 to 35 bp, and more commonly about 21 to 30 bp.

[0045] In some embodiments, the anti-PC7 dsRNA molecule is a polynucleotide having a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure and has self-complementary sense and antisense regions, where the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence of a separate target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.

[0046] Selection of Nucleic Acid PC7 Inhibitors In some embodiments, the nucleic acid PC7 inhibitors disclosed herein (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are capable of specifically binding to a desired PC7 variant, while not being able to specifically bind to an undesired PC7 variant.

[0047] In some embodiments, nucleic acid PC7 inhibitors (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are selected for use in the methods disclosed herein based on prediction of the stability of the molecule. In some embodiments, the prediction of stability is achieved by adopting theoretical melting curves where a higher theoretical melting curve indicates increased stability of the molecule and a concomitant decrease in cytotoxic effects. In some embodiments, the stability of nucleic acid PC7 inhibitors (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) is empirically determined, for example, by measuring the hybridization of a single modified RNA strand containing one or more universal binding nucleotide(s) to a complementary PC7 sequence in a polynucleotide array. In some embodiments, the melting temperature (i.e., Tm value) of each modified RNA and complementary RNA immobilized on the array is determined, and the relative stability of the modified RNA paired with the complementary RNA molecule is determined from the Tm value.

[0048] In some embodiments, nucleic acid PC7 inhibitors (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are selected for use in the methods disclosed herein based on "off-target" profiling, in which one or more nucleic acid molecules are administered to a cell(s) either in vivo or in vitro, total RNA is collected, and used to probe a microarray containing oligonucleotides having one or more nucleotide sequences from a panel of known genes, including non-target genes. The "off-target" profile of the modified RNA molecule (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) is quantified by determining the number of non-target genes that have reduced expression levels in the presence of the nucleic acid molecule. The presence of "off-target" binding indicates that the nucleic acid PC7 inhibitor (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) can specifically bind to one or more non-target genes. Ideally, nucleic acid PC7 inhibitors applicable for therapeutic use (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and their analogs) would exhibit high Tm values ​​while showing little or no "off-target" binding.

[0049] In some embodiments, nucleic acid PC7 inhibitors (e.g., ASOs, RNAi molecules, siRNA molecules, miRNA molecules, and analogs thereof) are selected for use in the methods disclosed herein by the use of a report gene assay. In some embodiments, the reporter gene construct comprises a constitutive promoter, e.g., a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of regulating the expression of, one or more reporter genes, such as, for example, a luciferase gene, a chloramphenicol (CAT) gene, and / or a β-galactosidase gene, which is in turn operably fused in frame with an oligonucleotide (typically about 15 base pairs to about 40 base pairs, more typically about 19 base pairs to about 30 base pairs, most typically 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 base pairs) comprising the target sequence of one or more nucleic acid PC7 inhibitors. In some embodiments, individual reporter gene expression constructs are co-transfected with one or more nucleic acid PC7 inhibitors. In some embodiments, the ability of a given nucleic acid PC7 inhibitor (e.g., ASO, RNAi molecule, siRNA molecule, miRNA molecule, and analogs thereof) to reduce the expression level of each of the contemplated gene variants is determined by comparing the measured reporter gene activity from cells transfected with the modified nucleic acid molecule to untransfected cells.

[0050] In some embodiments, nucleic acid inhibitors (e.g., ASOs, siRNA molecules, miRNA molecules, and their analogs) are selected for use in the methods disclosed herein by assaying their ability to specifically bind to RNA, such as RNA expressed by liver cells (e.g., PCSK7 transcripts).

[0051] Functional properties of inhibitors As used herein, the term "PC7 activity" refers to direct or indirect PC7 activity independent of its enzymatic activity (proteolysis independent). More specifically, but not so limited to, PC7's elevation of hepatic triglyceride levels, positive regulation of apoB levels in hepatocytes, positive regulation of apoB secretion from hepatocytes (Example 2, Figures 6C, 7F-G), protection of apoB from ER autophagy and / or lysosomal degradation (Example 2, Figures 6B-C, 7F), binding of PC7 to apoB (Example 2, Figures 7H-I), as well as the specific membrane-bound substrate transferrin receptor 1 (hTfR1) (Example 8, Figures 23A-B), and the cancer-associated protein CASC4 (PMID: 32820145).

[0052] The functional properties of PC7 inhibitors provided herein (e.g., anti-PC7 dsRNA, ASO, miRNA) are, in some embodiments, tested in vitro and in vivo. For example, in some embodiments, PC7 inhibitors are tested for their ability to: reduce lipid droplet size in hepatocytes or liver (see, e.g., FIG. 2G, Example 2, FIG. 8; Example 1, FIG. 2A-C; FIG. 37A-B); reduce lipid droplet volume (FIG. 39A, 2H-I, FIG. 8, 14L, FIG. 16D, FIG. 10G); reduce lipid droplet number (FIG. 8, 14L, 16D, 39A); reduce plasma and liver TG levels (see, e.g., FIG. 4A, 2K, 10A, 18L); increase protein levels of carnitine palmitoyltransferase 1a (Cpt1a) (see, e.g., Example 1, FIG. 4B); in hepatocytes relative to controls. 14reduce C-oleic acid (see, e.g., Example 4, FIG. 12A); increase LC3-II levels (Example 4, FIG. 13); increase autophagosome biogenesis (Example 4, FIG. 13); e.g., increase ER-associated autophagy under a normal diet (FIG. 12B); increase lipophagy in hepatocytes (FIG. 10E); e.g., increase TG hydrolysis under a normal diet (FIG. 11); increase β-oxidation in mitochondria (FIG. 4A); increase circulating blood Decreases plasma apoB100 and apo48 levels and decreases hepatic apoB100 and apo48 levels (Example 1, Figures 2C-2D; Example 1, Figures 3A-B; Example 2, Figures 6B, 7A-B, 14B, 14D-E, and 38D); Increases cotranslational degradation of apoB in hepatocytes (Example 1, Figure 5A; Example 2, Figures 7C-7D); Increases polyubiquitination of apoB100 (Figure 7E); Reduces apoB100 secretion (Example 1, Figure 15C); Reduces approximately 5% of apoB in hepatocytes increase the production of the 0 kDa secretory fragment (Example 1, Figure 5D); increase ER-60 protein levels (Figure 5F); increase fatty acid β-oxidation (Figure 21, Figure 10F); increase apoB degradation, for example, via the ERAD pathway (Figures 5E-F); decrease TG-rich very low density lipoprotein (VLDL) particles (Figures 6C, Figures 24A-B); modulate the intracellular trafficking and lysosomal localization of PC7 (Figures 22A-C); and inhibit the expression of a specific membrane-bound substrate, transferrin receptor (Figure 26). reduce the ability of PC7 to scavenge receptor 1 (hTfR1) (Figure 2B, Figure 32A-B); reduce LDL cholesterol (Figure 24A-B); increase weight loss (Figure 17G); reduce steatosis score (Figure 18B-D, 39B); reduce lobular / liver inflammation (Figure 18B-D, 39B); reduce hepatocyte ballooning (Figure 18B-D, 39B); reduce liver fibrosis score (Figure 18F, 18H, 39C); reduce ALT-induced hepatotoxicity (Figure 18J); increase IRE1α mRNA levels (Figure 10H, Figure 19A); increase GRP78 mRNA levels (Figure 19A); increase IRE1α protein levels (Figure 19D); decrease hepatic Pcsk7 total expression (e.g., mRNA) (Figure 38A-B), decrease hepatic / hepatocyte Pcsk7 expression (e.g., mRNA) (Figure 38B, 39D).

[0053] In some embodiments, the above assays are performed with mutant forms of PC7 that further reduce apoa5 intracellular and / or extracellular concentration(s) (e.g., hPC7 S505E, S505D and R504H) or other mutants associated with reduced hepatic TG (e.g., P777L). In some embodiments, PC7 inhibitors are tested for their effect on apoa5 activity (e.g., reduction of blood TG (circulating) and / or LDL levels).

[0054] In some embodiments, transgenic mice are genetically modified to express hPC7. In some embodiments, PC7 inhibitors are tested for their ability to reduce TG blood levels in these models or in non-genetically modified animals.

[0055] In some embodiments, the kinetics of TG clearance from plasma is measured in animals. 125 I]-labeled TG was injected, and blood samples were obtained 0, 5, 10, 15, and 30 min after injection. 125 It is determined by quantifying [I]-TG.

[0056] The increase in TG clearance in animals administered PC7 inhibitors indicates that the drug inhibits PC7 apo a5 activity in vivo.

[0057] In some embodiments, a decrease in blood TG in response to treatment with a PC7 inhibitor indicates therapeutic efficacy of the PC7 inhibitor. In some embodiments, the lipid profile is determined by colorimetric, gas-liquid chromatographic, or enzymatic methods using commercially available kits.

[0058] As used herein, the terms "decrease" or "reduction" (e.g., of PC7 activity or of NAFLD or NASH symptoms) refer to a reduction of at least 10% compared to a control, in an embodiment at least 20% lower, in a further embodiment at least 30% lower, in a further embodiment at least 40% lower, in a further embodiment at least 50% lower, in a further embodiment at least 60% lower, in a further embodiment at least 70% lower, in a further embodiment at least 80% lower, in a further embodiment at least 90% lower, and in a further embodiment a 100% reduction (complete inhibition).

[0059] Similarly, as used herein, the terms "increase" or "increasing" (e.g., of PC7 activity) refer to an increase of at least 10% compared to a control, in embodiments at least 20% higher, in further embodiments at least 30% higher, in further embodiments at least 40% higher, in further embodiments at least 50% higher, in further embodiments at least 60% higher, in further embodiments at least 70% higher, in further embodiments at least 80% higher, in further embodiments at least 90% higher, in further embodiments 100% higher, in further embodiments 200% higher, etc.

[0060] A "control" for use as a reference in the methods disclosed herein is a subject (human or model animal) that is not treated with a cell or inhibitor of the disclosure (e.g., for methods of testing PC7 inhibitors in vitro or in cellulo). For methods of preventing or treating NAFLD (or NASH) or a symptom thereof, it can be, for example, a control subject (or model animal) that has NAFLD (or NASH) and is not treated with an agent of the disclosure.

[0061] Methods / assays for determining PC7 activity are described below.

[0062] In some embodiments, PC7 inhibitors are tested for their ability to reduce PC7 activity in the human hepatic cell lines HepG2 or HuH7. In some embodiments, the assay consists of adding either transfected or purified wild-type (WT), mutant or chimeric PC7 directly to the culture supernatant in the presence or absence of the test compound. Each "dose-response" experiment is performed in triplicate at 4-6 different doses.

[0063] WT PC7. In some embodiments, PC7 inhibitors are tested in assays involving the addition of wild-type (WT) PC7, either as conditioned medium from transfected cells or as purified medium, added to the culture supernatant in the presence or absence of a PC7 inhibitor.

[0064] Mutant PC7 (gain of function). To further characterize whether a PC7 inhibitor inhibits the function of a gain of function mutation, cells are incubated with purified mutant proteins in the presence or absence of different doses of a PC7 inhibitor. In some embodiments, the purified PC7 mutant is PC7D374Y, S505E, S505D, or R504H. In some embodiments, the doses selected for exogenously added PC7 and gain of function mutants such as D374Y are 1 μg / ml and 0.2 μg / ml. In some embodiments, the assay is performed using media harvested from cells transfected with a gain of function of a PC7 mutant.

[0065] Formulations and methods of administration of PC7 nucleic acid inhibitors In some embodiments, compositions provided herein are administered by one or more routes of administration using one or more of a variety of suitable methods.

[0066] As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. The route of administration of PC7 inhibitors (e.g., anti-PC7 siRNA or ASO) for use and methods herein includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, spinal or other parenteral administration routes, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrathoracic injection and infusion. Alternatively, the PC7 inhibitors provided herein are administered by non-parenteral routes, such as topical, epithelial or mucosal administration routes, such as oral, intranasal, vaginal, rectal, sublingual, or topical.

[0067] Method of ASO administration When the PC7 inhibitor of the present disclosure is an ASO, the ASO can be administered to a subject in need thereof parenterally or orally in the form of naked nucleic acid or encapsulated nucleic acid (e.g., lipid-based particles such as liposomes).As noted above, in specific embodiments, the ASO of the present invention is chemically modified to increase nuclease resistance, not induce an immune response, and allow administration as naked nucleic acid.

[0068] Suitable parenteral routes for the ASOs of the disclosure include intravenous, intraperitoneal, or subcutaneous administration.

[0069] Method of administering double-stranded RNA The present disclosure also encompasses vectors (plasmids) that contain the above-mentioned dRNAs (e.g., shRNAs). It is contemplated that the vectors can be of any type suitable for, for example, expression of the polypeptide in a particular organism or propagation of the gene encoding the polypeptide. In some embodiments, the organism is of eukaryotic or prokaryotic origin. The specific choice of vector depends on the host organism and is known to those skilled in the art. In embodiments, the vector comprises a transcriptional regulatory sequence or promoter that is operably linked to a nucleic acid that includes a sequence encoding PC7. A first nucleic acid sequence is "operably linked" to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, in reading frame. However, some polynucleotide elements are operably linked but not contiguous, for example because enhancers generally function when separated by several kilobases from the promoter and intron sequences are often of variable length. "Transcriptional regulatory sequence" or "transcriptional regulatory element" is a general term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, that induce or control the transcription of a protein-coding sequence to which they are operably linked.

[0070] A recombinant expression vector comprising the double-stranded nucleic acid sequence provided herein is introduced into a cell, e.g., a host cell, including a living cell capable of expressing the PC7 inhibitor provided herein encoded by the recombinant expression vector, in some embodiments. Thus, also provided herein are cells, such as host cells, comprising such nucleic acids and / or vectors. Suitable host cells are, for example, any cell of eukaryotic or prokaryotic (bacterial) origin suitable for expression of nucleic acids. In some embodiments, the eukaryotic cell line is of mammalian, yeast, or invertebrate origin. The specific selection of cell line is known to those of skill in the art. The selection of bacterial strains depends on the task at hand and is known to those of skill in the art. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Often, such progeny are not actually identical to the parent cell, as certain modifications often occur later, either by mutation or environmental influence, but still fall within the scope of the term as used herein. The vector is introduced into the cell via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, and viral mediated transfection. Suitable methods for transforming or transfecting a host cell can be found, for example, in Sambrook et al. (supra), Sambrook and Russell (supra), and other laboratory manuals. Methods for introducing nucleic acid into mammalian cells in vivo are also known and are often used to deliver vector DNA of the PC7 inhibitors provided herein to a subject for gene therapy.

[0071] The nucleic acids or vectors described above are in some embodiments delivered to cells in vivo using methods well known in the art, such as direct injection of nucleic acids, receptor-mediated nucleic acid uptake, viral-mediated or non-viral transfection, and lipid-based transfection, all of which often involve the use of gene therapy vectors. Direct injection is used to introduce naked or chemically modified nucleic acids into cells in vivo. In some embodiments, a delivery device (e.g., a "gene gun") is used to inject nucleic acids into cells in vivo. In some embodiments, such devices are commercially available (e.g., from BioRad). In some embodiments, naked or chemically modified nucleic acids are introduced into cells by complexing the nucleic acid to a cation such as polylysine, which is bound to a ligand for a cell surface receptor. In some embodiments, binding of the nucleic acid-ligand complex to the receptor promotes uptake of the nucleic acid by receptor-mediated endocytosis. In some embodiments, degradation of the complex by intracellular lysosomes is avoided by using a nucleic acid-ligand complex bound to an adenovirus capsid that disrupts the endosome, thereby releasing the material into the cytoplasm.

[0072] Defective retroviruses have been well characterized for use as gene therapy vectors (for a review, see Miller, AD, Blood 76:271 (1990)). Protocols for producing recombinant retroviruses and infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, FM et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are well known to those skilled in the art. Examples of suitable packaging virus lines include psiCrip, psiCre, psi2, and psiAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo.

[0073] For use as a gene therapy vector, in some embodiments, the genome of an adenovirus is engineered so that it encodes and expresses a nucleic acid of a PC7 inhibitor provided herein (e.g., a nucleic acid encoding an anti-PC7 adsRNA that targets PC7), but is inactivated with respect to its ability to replicate in a normal lytic virus life cycle. Suitable adenovirus vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and are often used to infect a wide variety of cell types, including airway epithelium, endothelial cells, hepatocytes, and muscle cells.

[0074] In some embodiments, adeno-associated virus (AAV) is used as a gene therapy vector to deliver DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as adenovirus or herpesvirus, as a helper virus for efficient replication and productive life cycle. In some embodiments, AAV is used to integrate DNA into non-dividing cells. In some embodiments, lentiviral gene therapy vectors are adapted for use in the methods provided herein.

[0075] DsRNA (eg, siRNA) of the disclosure can also be administered to a subject in need thereof as a nucleic acid encapsulated in a lipid-based particle, such as a liposome.

[0076] Pharmaceutical Compositions In a specific embodiment, the PC7 inhibitors provided herein (e.g., anti-PC7 siRNA or ASO) are formulated to ensure proper distribution in vivo.

[0077] For example, the therapeutic compounds provided herein are formulated in lipid-based nanoparticles (e.g., liposomes) in some embodiments. In some embodiments, the liposomes contain one or more moieties that selectively transport to specific cells (e.g., hepatocytes), tissues, or organs (e.g., liver), thus enhancing targeted drug delivery (see, e.g., Ranade VV, J. Clin. Pharmacol. 29:685 (1989)). In one embodiment, the PC7 inhibitors provided herein are formulated to be delivered to the liver (i.e., hepatocytes). Without being so limited, the nucleic acid can be encapsulated in lipid-based nanoparticles that can optionally contain GalNac on the surface of the target liver. Biodegradable, biocompatible polymers such as lipids, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid are used in some embodiments. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In some embodiments, therapeutic compositions are administered using medical devices known in the art.

[0078] In another aspect, compositions, e.g., pharmaceutical compositions, are provided that include one or a combination of the PC7 inhibitors provided herein (e.g., anti-PC7 siRNA or ASO) formulated together with a pharma- ceutically acceptable carrier and / or excipient.

[0079] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, etc. that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the PC7 inhibitor is coated with a material to protect the compound from the action of acids and other natural conditions that inactivate the compound in some embodiments.

[0080] In some embodiments, the pharmaceutical compositions provided herein include one or more pharma- ceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM et al., J.Pharm.Sci.66:1-19 (1977)). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0081] In some embodiments, the pharmaceutical compositions provided herein include a pharma- ceutically acceptable antioxidant. Examples of pharma-ceutically acceptable antioxidants include water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc., metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0082] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0083] In some embodiments, the composition also contains adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms is ensured in some embodiments by both sterilization procedures (described above) and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. In some embodiments, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. In addition, in some embodiments, prolonged absorption of the injectable pharmaceutical form is brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0084] Pharmaceutically acceptable carriers or excipients include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharma-ceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the transduced cells, its use in the pharmaceutical compositions provided herein is contemplated. In some embodiments, supplementary active compounds are incorporated into the composition.

[0085] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. In some embodiments, the compositions are formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition.

[0086] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0087] In some embodiments, sterile injectable solution is prepared by incorporating PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) into a suitable solvent with one or a combination of the above-listed ingredients as needed, followed by sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preparation method is high vacuum drying and freeze-drying (lyophilization), which obtains a powder of active ingredient and any additional desired ingredient from its previously sterile-filtered solution.

[0088] The amount of PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) combined with the carrier material to create a single dosage form varies depending on the subject being treated and the particular method of administration. The amount of active ingredient that can be combined with the carrier material to create a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount ranges from about 0.01% to about 99% active ingredient, about 0.1% to about 70%, or about 1% to about 30% active ingredient in combination with a pharma- ceutically acceptable carrier.

[0089] Dosage and Regimen Dosage regimens may be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, in some embodiments, a single bolus is administered. In some embodiments, several divided doses are administered over time. In some embodiments, the dose is proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary doses for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of PC7 inhibitors provided herein are determined by and directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the inherent limitations in the art of synthesizing such active compounds for the treatment of susceptibility in individuals.

[0090] In some embodiments, for administration of a PC7 inhibitor (e.g., anti-PC7 siRNA or ASO), an exemplary dosage range includes, but is not limited to, about 0.0001 to about 100 mg / kg host body weight. In some embodiments, the dosage range includes about 0.01 to about 5 mg / kg host body weight. In some embodiments, the dosage is about 0.3 mg / kg body weight, about 1 mg / kg body weight, about 3 mg / kg body weight, about 5 mg / kg body weight, or about 10 mg / kg body weight, or within the range of about 1 to 10 mg / kg. An exemplary treatment regime involves administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Exemplary dosage regimes provided herein for PC7 inhibitors (e.g., anti-PC7 siRNA or ASO) include, but are not limited to, about 1 mg / kg body weight or about 3 mg / kg body weight by intravenous administration.

[0091] The PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) is usually administered on multiple occasions. The interval between single doses is, for example, weekly, monthly, every three months, or yearly. In some embodiments, the interval is irregular, as indicated by measuring the blood level of the PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) in the patient. In some methods, the dosage is adjusted to achieve a plasma concentration of the PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

[0092] Alternatively, the PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) is administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the inhibitor in the patient. Dosage and frequency vary in some embodiments depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals for a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages may be required at relatively short intervals until the progression of the disease is reduced or terminated, or until the patient shows partial or complete improvement of the symptoms of the disease. Then, in some embodiments, the patient is administered a prophylactic regimen.

[0093] The actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein will vary, in some embodiments, to obtain an amount of the active ingredient that is effective, but not harmful to the individual, to achieve the desired therapeutic response (e.g., reduced hepatic TG levels) for a particular individual, composition, and mode of administration. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition, or ester, salt, or amide thereof, employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0094] combination The pharmaceutical compositions provided herein, in some embodiments, are administered in combination therapy, i.e., in combination with other agents or therapies known to prevent or treat NAFLD or a symptom(s) thereof.

[0095] In a specific embodiment, the PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) is administered in conjunction with another therapy (e.g., a non-pharmacological therapy such as a therapeutic agent or weight loss achieved through a combination of calorie reduction, exercise, and dietary modification) known to prevent or treat NAFLD or at least a symptom thereof or another TG-related disorder.

[0096] In specific embodiments, the PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) is administered in combination with another therapeutic agent known to prevent or treat NAFLD, or at least a symptom thereof, or another TG-related disorder. Such compositions, in some embodiments, comprise one or a combination of (e.g., two or more different) PC7 inhibitors. In some embodiments, the pharmaceutical compositions provided herein comprise one or a combination of ASOs. In some embodiments, the combination therapy comprises a PC7 inhibitor (e.g., ASO or anti-PC7 siRNA) in combination with at least one triglyceride-reducing agent.

[0097] Other agents potentially useful in preventing or treating NAFLD or at least the symptoms thereof include omega-3 fatty acids, anti-cholesterol agents, and the like. In some embodiments, examples of active ingredients, including agents useful for the prevention or treatment of TG-related disorders, that may be administered in combination with a PC7 inhibitor provided herein include: (a) HMG-CoA reductase inhibitors (e.g., statins, including lovastatin, simvastatin, fluvastatin, rosuvastatin, pravastatin, rivastatin, atorvastatin, itavastatin, pitavastatin, cerivastatin, and other statins); (b) cholesterol absorption inhibitors, such as stanol esters, sterol glycosides, such as beta-sitosterol, thixides, and azetidinones, such as ezetimibe; (c) inhibitors of cholesterol ester transfer protein (CETP), currently in clinical trials to increase HDL and reduce LDL cholesterol (e.g., anacetrapib or dalcetrapib); (d) niacin and related compounds, such as nicotinyl alcohol, nicotinamide, and nicotinic acid or their salts; (e) bile acid sequestrants (cholestyramine, nicotinamide, nicotinic acid ... (f) acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, such as avasimibe and melinamide, including selective ACAT-1 and ACAT-2 inhibitors and dual inhibitors; (g) PPARy agonists, such as gemfibrozil and fenofibric acid derivatives (fibrates), including clofibrate, fenofibrate, bezafibrate, ciprofibrate, and etofibrate; (h) microsomal triglyceride transfer protein (MTP) / apo B secretion inhibitors, (i) antioxidant vitamins, such as vitamins C and E and beta-carotene; (k) thyromimetics; (l) LDL receptor derivatives; (m) platelet aggregation inhibitors, such as glycoprotein Ilb / IIIa fibrinogen receptor antagonists and aspirin;(n) Vitamin B12 (also known as cyanocobalamin), (o) folic acid or its pharma- ceutically acceptable salts or esters, such as sodium and methylglucamine salts, (p) FXR and LXR ligands (including both inhibitors and agonists), (q) agents that enhance ABCA1 gene expression, (r) ileal bile acid transporters; and (s) PCSK9 inhibitors, such as anti-PCSK9 monoclonal antibodies (evolocumab and alirocumab), siRNA (inclisiran), vaccines, ASOs, or other compounds that improve a patient's lipid profile, such as small molecules that target PCSK9;

[0098] In some embodiments, the two are alternatively administered sequentially in either order, or simultaneously (in the same composition or different compositions).

[0099] In some embodiments, the combination therapy regimen is additive. In some embodiments, the combination therapy regimen produces synergistic results (e.g., a greater reduction in liver TG levels than would be expected for the combination of two agents). In some embodiments, the combination therapy of a PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) with another agent that prevents or treats NAFLD or a symptom thereof produces synergistic results (e.g., a synergistic reduction in liver TG levels). In some subjects, this allows the dose of other agents for preventing or treating NAFLD and / or agents useful for preventing or treating TG-related disorders ("other active agents") to be reduced to achieve desired TG levels. In some embodiments, the PC7 inhibitor is useful for subjects who are intolerant to therapy with other active agents or for whom therapy with other active agents has produced inadequate results (e.g., subjects who have experienced inadequate TG reduction with statin therapy).

[0100] disease In a specific embodiment, the subject has NAFLD or a symptom thereof. NAFLD is a reversible condition in which large vacuoles of triglyceride fat accumulate in liver cells through the process of steatosis. NASH is a type of NAFLD condition of fatty liver characterized by ballooning, inflammation, and fibrosis. (See FIG. 1, which shows the progression from healthy liver to fatty liver and NASH liver). As used herein, the term "NAFLD condition" includes, but is not limited to, one or more of steatosis, liver / lobular inflammation, hepatocyte ballooning, fibrosis, pancreatitis, and diabetes. In a specific embodiment, the disease is diet-induced NAFLD. In a specific embodiment, the NAFLD condition includes one or more of fibrosis, steatosis, liver / lobular inflammation, and hepatocyte ballooning.

[0101] The term "TG-related disorder" as used herein refers to disorders in which HTG is a factor or symptom, including, but not limited to, cardiovascular disease (CVD), type 2 diabetes (T2D), fibrosis, metabolic syndrome, and acute pancreatitis. Reduction of TG and increase of HDL-c are important components of primary and secondary programs for preventing CVD, NAFLD, atherogenic dyslipidemia, or T2D.

[0102] subject As used herein, the term "subject" is meant to refer to any animal, such as a mammal, including a human, mouse, rat, dog, cat, pig, cow, monkey, horse, etc. In a specific embodiment, the subject refers to a human.

[0103] As used herein, the terms "a subject is a probable candidate for NAFLD" and "a subject is a probable candidate for NASH" are meant to refer to obese subjects, subjects with alcoholism, and subjects with a genetic predisposition to NAFLD or NASH or a triglyceride-related disorder, or subjects with a family history that includes NAFLD or NASH or a triglyceride-related disorder.

[0104] Treatment and Prevention As used herein, the terms "treat / treating / treatment" and "prevent / preventing / prevention" refer to inducing a desired biological response, i.e., a therapeutic effect and a preventive effect, respectively. According to the disclosure herein, the therapeutic effect includes one or more of the following: a reduction / reduction in the severity of human disease (e.g., reduction in liver triglyceride (TG) level), a reduction / reduction in at least one NAFLD symptom (e.g., reduction in the number / amount of lipid droplets in the liver, etc.), and an improvement in at least one NAFLD symptom after administering at least one PC7 inhibitor (e.g., anti-PC7 siRNA or ASO) or a composition comprising a PC7 inhibitor as encompassed herein in combination with another agent for preventing or treating NAFLD or its symptoms. According to the disclosure provided herein, in some embodiments, the preventive effect includes complete or partial avoidance / inhibition of excess hepatic TG or at least one NAFLD symptom following administration of at least one PC7 inhibitor encompassed herein, or a composition comprising a PC7 inhibitor, in combination with another agent for the prevention or treatment of NAFLD or a symptom thereof.

[0105] In some embodiments, a "therapeutically effective amount" or "effective amount" or "therapeutically effective dosage" of a PC7 inhibitor (e.g., an anti-PC7 siRNA or ASO) provided herein results in a reduction in hepatic TG levels in a subject, a decrease in the severity of at least one NAFLD symptom, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease affliction in a subject.

[0106] Methods of detection In one embodiment, anti-PC7 antibodies are used to detect the presence or level of PC7. In some embodiments, this is accomplished by contacting a sample (e.g., a biological sample such as blood, serum, plasma, or a cell sample) with an anti-PC7 antibody under conditions that allow the formation of a complex between the anti-PC7 antibody and PC7. Any complexes formed between the antibody and PC7 are detected and compared in the sample and in a control sample. Standard detection methods well known in the art, such as, for example, ELISA and flow cytometry assays, are contemplated to be performed using the anti-PC7 antibodies disclosed herein.

[0107] Thus, in one aspect, there is provided a method of detecting the presence of PC7 (e.g., hPC7) or measuring the amount of PC7 (e.g., an active form of PC7) in a sample, comprising contacting the sample with an anti-PC7 antibody provided herein under conditions that allow for the formation of a complex between the anti-PC7 antibody and PC7. The formation of the complex is then detected, where a difference in complex formation between the sample compared to a control sample indicates the presence of PC7 in the sample.

[0108] kit Also provided herein is a kit including (A) a PC7 inhibitor provided herein (e.g., an anti-PC7 siRNA or ASO) or a composition provided herein, and (B) (i) another agent for preventing or treating non-alcoholic fatty liver disease (NAFLD) or a symptom thereof, (ii) a pharma- ceutically acceptable carrier and / or excipient, (iii) instructions for use or a combination thereof, or (iv) a combination of at least two of (i)-(iii). In some embodiments, the kit further includes at least one additional reagent, or one or more additional PC7 inhibitors provided herein. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit, or that otherwise accompanies the kit. In some embodiments, the kit further includes one or more containers, reagents, administration devices (e.g., syringes).

[0109] The disclosure of this specification is further illustrated by the following examples and claims, which are illustrative and not intended to be further limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are within the scope of this disclosure and claims. The contents of all references cited throughout this application, including issued patents and published patent applications, are hereby incorporated by reference in their entirety.

[0110] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. [Brief description of the drawings]

[0111] In the accompanying drawings: [Figure 1] FIG. 1 is a schematic diagram showing the progression of a healthy liver to a fatty liver, a NASH liver, a cirrhotic liver, and hepatocellular carcinoma. [Figure 2A] Total plasma cholesterol (FIG. 2A) and triglyceride (TG) (FIG. 2B) levels in wild-type (WT) and Pcsk7− / − (KO) mice on a chow diet. [Figure 2B] Total plasma cholesterol (FIG. 2A) and triglyceride (TG) (FIG. 2B) levels in wild-type (WT) and Pcsk7− / − (KO) mice on a chow diet. [Figure 2C] ApoB100 protein levels in plasma (FIG. 2C) and liver (FIG. 2D) and apoB hepatic mRNA levels (FIG. 2D) in wild-type (WT) and Pcsk7− / − (KO) mice fed a chow diet. [Figure 2D] ApoB100 protein levels in plasma (FIG. 2C) and liver (FIG. 2D) and apoB hepatic mRNA levels (FIG. 2D) in wild-type (WT) and Pcsk7− / − (KO) mice fed a chow diet. [Figure 2E] Western blot of apoB100 and MTP protein levels in WT and Pcsk7− / − mice on high-fat diet (HFD). Levels are normalized to tubulin. [Figure 2F] Shown is the hepatic content of lipid droplets (LDs) and triglycerides in WT and Pcsk7− / − mice on HFD. [Figure 2G] Total LD ​​(LD, TG-rich vesicles) accumulation in wild-type (WT) and Pcsk7- / - (KO) mice fed a chow diet (Figure 2G), LD content (total area stained) (Figure 2H), average size (average area) (Figure 2I), total number (average number) of LDs (Figure 2J), and total hepatic TG (Figure 2K). [Figure 2H] Total LD ​​(LD, TG-rich vesicles) accumulation in wild-type (WT) and Pcsk7- / - (KO) mice fed a chow diet (Figure 2G), LD content (total area stained) (Figure 2H), average size (average area) (Figure 2I), total number (average number) of LDs (Figure 2J), and total hepatic TG (Figure 2K). [Figure 2I] Total LD ​​(LD, TG-rich vesicles) accumulation in wild-type (WT) and Pcsk7- / - (KO) mice fed a chow diet (Figure 2G), LD content (total area stained) (Figure 2H), average size (average area) (Figure 2I), total number (average number) of LDs (Figure 2J), and total hepatic TG (Figure 2K). [Figure 2J] Total LD ​​(LD, TG-rich vesicles) accumulation in wild-type (WT) and Pcsk7- / - (KO) mice fed a chow diet (Figure 2G), LD content (total area stained) (Figure 2H), average size (average area) (Figure 2I), total number (average number) of LDs (Figure 2J), and total hepatic TG (Figure 2K). [Figure 2K] Total LD ​​(LD, TG-rich vesicles) accumulation in wild-type (WT) and Pcsk7- / - (KO) mice fed a chow diet (Figure 2G), LD content (total area stained) (Figure 2H), average size (average area) (Figure 2I), total number (average number) of LDs (Figure 2J), and total hepatic TG (Figure 2K). [Figure 3A]Under normal diet (ND), Pcsk7 KO mice exhibit approximately 50% lower circulating apoB100 and apoB48 levels, without changes in hepatic microsomal triglyceride transfer protein (MTP) levels (not shown) or apoB mRNA levels. ApoB protein expression detected by Western blot shows apoB100 (500 kDa) and apoB48 (250 kDa). [Figure 3B] ApoB mRNA levels are shown graphically. [Figure 4A] Hepatic lipids were extracted from mouse livers and TG concentrations were measured (8–11 mice per group). Values ​​are means ± SEM. Statistical comparisons with controls were performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 4B] Western blot quantification of hepatic protein for total acetyl-coenzyme A carboxylase 1, Acc1 (tAcc1), the ratio of inhibitory phosphorylated Acc1 (pAcc1c) to total Acc (tAcc1), and carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for the b-oxidation of FAs. Values ​​are means ± SEM. Statistical comparisons with controls were performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5A] Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Pulse-chase analysis of hepatic apoB from medium and cell lysates of WT and Pcsk7- / - primary hepatocytes (n=2 independent experiments performed in duplicate). Cotranslational degradation of apoB in Pcsk7- / - hepatocytes. Statistical comparison with control was performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5B] Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Pulse-chase analysis of hepatic apoB from medium and cell lysates of WT and Pcsk7- / - primary hepatocytes (n=2 independent experiments performed in duplicate). Cotranslational degradation of apoB in Pcsk7- / - hepatocytes. Statistical comparison with control was performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5C]Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Pulse-chase analysis of hepatic apoB from media and cell lysates of WT and Pcsk7- / - primary hepatocytes (n=2 independent experiments performed in duplicate). ApoB100 secretion is accompanied by increased production of the ∼50 kDa secretory fragment of apoB in Pcsk7- / - hepatocytes over the chase period. Statistical comparisons with controls were performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5D] Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Pulse-chase analysis of hepatic apoB from media and cell lysates of WT and Pcsk7- / - primary hepatocytes (n=2 independent experiments performed in duplicate). ApoB100 secretion is accompanied by increased production of the ∼50 kDa secretory fragment of apoB in Pcsk7- / - hepatocytes over the chase period. Statistical comparisons with controls were performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5E] Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Gel (FIG. 5E) and graph (FIG. 5F) Western blot analysis of ER-60 protein levels in liver extracts of WT and Pcsk7 KO mice. Statistical comparison with controls was performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 5F] Pcsk7 KO mice show enhanced degradation in primary hepatocytes. Gel (FIG. 5E) and graph (FIG. 5F) Western blot analysis of ER-60 protein levels in liver extracts of WT and Pcsk7 KO mice. Statistical comparison with controls was performed by unpaired two-tailed t-test. *p<0.05, ***p<0.001. [Figure 6A] Oligonucleotide sequences of the PCSK7-siRNA pool (siRNA sequences 1, 2, 3 and 4 (SEQ ID NOs: 12 to 15)). Statistical comparison with control was performed by unpaired two-tailed t-test. **p<0.05. [Figure 6B]siRNA-mediated knockdown of PC7 reduces secreted apoB levels from immortalized human hepatocytes (IHH) & HepG2 cells based on WB analysis. Statistical comparison with control was performed by unpaired two-tailed t-test. **p<0.05. [Figure 6C] ApoB-associated TG VLDL secretion (IHH cells). IHH cells were pulse-labeled with 14C-oleate for 2 hours and secreted 14C-labeled VLDL particles were quantified. Statistical comparison with control was performed by unpaired two-tailed t-test. **p<0.05. [Figure 7A] PCSK7 can function as a molecular chaperone-like binding to apoB in the ER and facilitating its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on gel and graph) of apoB100 and PC7 protein levels in WT and PCSK7- / - IHH cells. Levels are normalized to α-tubulin. [Figure 7B] PCSK7 can function as a molecular chaperone-like binding apoB in the ER and facilitating its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on gel and graph) of apoB100 and PC7 protein levels over time (0, 15 and 30 min) in WT and PCSK7- / - IHH cells incubated with cycloheximide (CHX) to block de novo mRNA translation. Levels are normalized to α-tubulin. [Figure 7C]PCSK7 can function as a molecular chaperone-like molecule that binds to apoB in the ER and promotes its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on gels and graphs) of cellular apoB levels in WT and PCSK7- / - IHH cells incubated with brefeldin A (BFA), which blocks exit from the endoplasmic reticulum (ER), the autophagy inhibitor 3-methyladenine (3MA), the proteasome inhibitor MG132, lactacystin, DMSO or a non-treated (NT) control. [Figure 7D] PCSK7 can function as a molecular chaperone-like molecule that binds to apoB in the ER and promotes its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on gels and graphs) of cellular apoB levels in WT and PCSK7- / - IHH cells incubated with brefeldin A (BFA), which blocks exit from the endoplasmic reticulum (ER), the autophagy inhibitor 3-methyladenine (3MA), the proteasome inhibitor MG132, lactacystin, DMSO or a non-treated (NT) control. [Figure 7E] PCSK7 can function as a molecular chaperone-like molecule that binds to apoB in the ER and promotes its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on the gel) of cellular apoB levels and polyubiquitinated apoB100 in WT and PCSK7- / - IHH cells incubated with lactacystin. [Figure 7F]PCSK7 can function as a molecular chaperone-like binding apoB in the ER and facilitating its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Schematic diagram of apoB100 and apoB21 proteins. Western blots of apoB100 and PC7 protein levels in WT and PCSK7- / - naive IHH cells overexpressing PCSK7 (presented on gel and in graph), as well as a western blot of a secreted short N-terminal fragment of apoB (apoB21; aa 1-965aa) with the cell-permeable pan-proprotein convertase inhibitor decanoyl-RVKR-cmk, which blocks PCSK7 enzymatic activity. Levels are normalized to β-actin. [Figure 7G] PCSK7 can function as a molecular chaperone-like binding apoB in the ER and facilitating its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blot (presented on gel) of apoB21 (s-apoB) protein levels in naive WT IHH cells co-expressing rat PCSK7 (rPC7) and its soluble KDEL variant (retained in the ER) (rPC7-KDEL). Levels are normalized to α-tubulin. [Figure 7H] PCSK7 can function as a molecular chaperone-like molecule that binds to apoB in the ER and promotes its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blot (presented on gel) of cellular apoB levels in IHH cells overexpressing PC7 (tagged with V5 at the C-terminus). Cell lysates were immunoprecipitated with mAb-V5, followed by Western blot (WB) with apoB100 antibody. [Figure 7I]PCSK7 can function as a molecular chaperone-like binding apoB in the ER and facilitating its secretion, and its absence leads to rapid degradation of apoB by the proteasome. IHH cells that do not express PCSK7 were generated by CRISPR knockout (PCSK7- / -). Western blots (presented on gel) of endogenous apoB with V5-tagged human PC7 in IHH cells overexpressing PC7-V5 versus cells expressing either 7B2-V5 or SKI-1-V5 as controls. Cell lysates were immunoprecipitated with human apoB antibody, complexes were resolved by SDS-PAGE, and co-immunoprecipitated (co-IP) proteins were revealed by western blot to the V5 tag. PC7 directly binds apoB, as it can be pulled down with endogenous apoB. [Figure 8] CRISPR silencing of PCSK7 in IHH cells reduces the levels and size of intracellular lipid droplets and enhances cell proliferation. Control IHH cells and cells silenced for endogenous PCSK7 by CRISPR were incubated with oleic acid for 72 hours, then the cells were washed and analyzed for TG levels by Oil Red-O labeling. [Figure 9A] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9B] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9C] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9D]qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9E] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9F] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9G] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9H] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9I] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9J] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9K] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9L]qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9M] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9N] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9O] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9P] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9Q] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9R] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9S] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9T]qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9U] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9V] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 9W] qPCR analysis of mRNA levels of 13 representative genes in hepatocyte WT (white bars) and Pcsk7 KO mice (black bars) fed a chow diet or a NASH diet for 12 or 16 weeks. [Figure 10A] Plasma TG levels measured in vivo after administration of poloxamer-407 (P-407), a detergent that blocks TG-lipolysis. Six hours after injection of P-407, plasma TG levels were reduced by approximately 50% in Pcsk7- / - mice, revealing a significant reduction in hepatic secretion of VLDL-TG in Pcsk7- / - mice. [Figure 10B] Primary hepatocytes from WT and Pcsk7- / - mice were incubated with 3H-acetate for 2 h, 3H-TG was extracted, and newly incorporated radioactivity was counted. No significant difference in de novo lipogenesis between WT and Pcsk7- / - could be detected. [Figure 10C] Pyruvate kinase (PKLR), the hepatic receptor for TG-enriched VLDL (LDL receptor-related protein 1, LRP1), and the scavenger receptor for plasma nonesterified free FAs (NEFAs) and lipoproteins (CD36) were unchanged in WT and Pcsk7− / − mice. [Figure 10D]Pyruvate kinase (PKLR), the hepatic receptor for TG-enriched VLDL (LDL receptor-related protein 1, LRP1), and the scavenger receptor for plasma nonesterified free FAs (NEFAs) and lipoproteins (CD36) were unchanged in WT and Pcsk7− / − mice. [Figure 10E] A significant increase in a functional macroautophagy-lysosomal pathway in primary hepatocytes from Pcsk7− / − mice, whereby the levels of LC3-II were high both in the absence and presence of chloroquine, suggesting enhanced functional lipophagy. [Figure 10F] FA oxidation potential compared in human hepatic IHH cells expressing or completely lacking PCSK7 (IHH-CRISPR PCSK7 KO). Cells were incubated with 14C-oleate and released 14CO2 was captured at different time points, revealing enhanced FA β-oxidation (increased 14CO2) at both 8 and 12 hours after the start of incubation. [Figure 10G] IHH and IHH-CRISPR PCSK7 KO cells were incubated overnight with 0.3 mM oleic acid (OA), washed, and incubated in medium lacking OA for 88 h. Cells were then stained with Oil Red O, revealing reduced LD staining in the absence of PCSK7, as observed in Pcsk7- / - mice. [Figure 10H] Comparison of protein levels of ER stress markers in IHH cells versus IHH-CRISPR PCSK7 KO cells. Data show higher levels of phospho-IRE1α and phospho-PERK in cells lacking PCSK7, suggesting activated unfolded protein response (UPR) signaling in the absence of PCSK7. In summary, upregulated UPR signaling leads to the observation of lower apoB, leading to loss of the potential apoB chaperone PCSK7, higher autophagy, and lower lipid accumulation in hepatocytes via enhanced β-oxidation. [Figure 11]FIG. 13 is a schematic diagram of absence of PC7: enhanced autophagy, decreased apoB levels, and steatosis. [Figure 12A] Primary hepatocytes from Pcsk7 KO mice accumulate less fat and show no difference in adipogenesis, but show 6- to 7-fold enhanced autophagy and enhanced fatty acid β-oxidation. Pcsk7 KO hepatocytes appear to be resistant to fat accumulation. Primary hepatocytes from KO and WT mice were incubated with radiolabeled oleic acid [14C] and analyzed for TG content at different time points after lipid extraction. [Figure 12B] Primary hepatocytes from Pcsk7 KO mice accumulate less fat and show no difference in adipogenesis, but show 6- to 7-fold enhanced autophagy and enhanced fatty acid β-oxidation. In the absence of PC7, there is no change in lipogenesis. Primary hepatocytes were incubated with 13C-acetate for 24 h and total cellular radiolabeled lipids were quantified. [Figure 13] Immunoblots of LC3 in primary hepatocytes from WT and Pcsk7- / - mice incubated without or with 100 μM chloroquine for 6 h (n=2 independent experiments performed in triplicate). Values ​​are means ± SEM. Statistical comparisons with controls were performed by unpaired two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 14A] Figure 1 shows the effect of loss of PCSK7 expression on the extent of fat accumulation in the liver after a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. Schematic diagram of the experiment performed. Four groups of 20 WT and 20 Pcsk7- / - mice aged 8 weeks were fed a chow diet or a HFFC diet for 12 weeks. At the end of the 12-week feeding period, half of them (20 weeks old) were sacrificed for evaluation of the development of NAFLD, and the other half were allowed to recover under a chow diet for another 4 weeks (until 24 weeks of age). [Figure 14B]Impact of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. ApoB100 levels were reduced in Pcsk7- / - mice compared to WT at the end of a 16-week (12+4 weeks) period under a chow diet or after 4 weeks of recovery following a 12-week HFFC diet in plasma (Figure 41B-C) and liver (Figure 41D-E). The data show that apoB100 levels were approximately 50-60% lower in Pcsk7- / - mice compared to WT in both plasma and liver, with no significant changes in apoB48. [Figure 14C] Impact of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. ApoB100 levels were reduced in Pcsk7- / - mice compared to WT at the end of a 16-week (12+4 weeks) period under a chow diet or after 4 weeks of recovery following a 12-week HFFC diet in plasma (Figure 41B-C) and liver (Figure 41D-E). The data show that apoB100 levels were approximately 50-60% lower in Pcsk7- / - mice compared to WT in both plasma and liver, with no significant changes in apoB48. [Figure 14D] Impact of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. ApoB100 levels were reduced in Pcsk7- / - mice compared to WT at the end of a 16-week (12+4 weeks) period under a chow diet or after 4 weeks of recovery following a 12-week HFFC diet in plasma (Figure 41B-C) and liver (Figure 41D-E). The data show that apoB100 levels were approximately 50-60% lower in Pcsk7- / - mice compared to WT in both plasma and liver, with no significant changes in apoB48. [Figure 14E]Impact of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. ApoB100 levels were reduced in Pcsk7- / - mice compared to WT at the end of a 16-week (12+4 weeks) period under a chow diet or after 4 weeks of recovery following a 12-week HFFC diet in plasma (Figure 41B-C) and liver (Figure 41D-E). The data show that apoB100 levels were approximately 50-60% lower in Pcsk7- / - mice compared to WT in both plasma and liver, with no significant changes in apoB48. [Figure 14F] Effect of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. Oil Red O (ORO) staining of liver sections revealed robust accumulation of LDs following the HFFC diet in both genotypes, suggesting that lack of PCSK7 does not prevent the development of diet-induced NAFLD. [Figure 14G] Effect of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. Oil Red O (ORO) staining of liver sections revealed robust accumulation of LDs following the HFFC diet in both genotypes, suggesting that lack of PCSK7 does not prevent the development of diet-induced NAFLD. [Figure 14H] Effect of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. In contrast, after a recovery period, the absence of PCSK7 dramatically reduced the extent of LD accumulation, as evidenced by a reduction in the total area stained by ORO, a decrease in the average area, and an increase in the number of LDs. [Figure 14I]Effect of loss of PCSK7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which induces a robust NAFLD-like phenotype in mice. In contrast, after a recovery period, the absence of PCSK7 dramatically reduced the extent of LD accumulation, as evidenced by a reduction in the total area stained by ORO, a decrease in the average area, and an increase in the number of LDs. [Figure 15A] Western blot analysis of apoB levels in (FIG. 15A) plasma and (FIG. 15B) liver in mice fed a chow diet or a Western diet high in fat, fructose, and cholesterol (HFFC) (NASH) for 12 weeks. [Figure 15B] Western blot analysis of apoB levels in (FIG. 15A) plasma and (FIG. 15B) liver in mice fed a chow diet or a Western diet high in fat, fructose, and cholesterol (HFFC) (NASH) for 12 weeks. [Figure 16A] Twelve-week-old mice fed a chow diet showed no significant changes in LD in Pcsk7− / − mice versus WT. [Figure 16B] Twelve-week-old mice fed a chow diet showed no significant changes in LD in Pcsk7− / − mice versus WT. [Figure 16C] In contrast, 16-week-old mice fed a chow diet showed a significant ∼20% reduction in LD in Pcsk7− / − mice versus WT. [Figure 16D] In contrast, 16-week-old mice fed a chow diet showed a significant ∼20% reduction in LD in Pcsk7− / − mice versus WT. [Figure 16E] Twelve week old mice fed the HFFC diet (Figure 16E) or 12 week old mice fed the HFFC diet followed by 4 weeks of recovery under a chow diet (Figure 16F) did not show any differences in total liver cholesterol between both genotypes. [Figure 16F]Twelve week old mice fed the HFFC diet (Figure 16E) or 12 week old mice fed the HFFC diet followed by 4 weeks of recovery under a chow diet (Figure 16F) did not show any differences in total liver cholesterol between both genotypes. [Figure 17A](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) Representative images of 18-30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet), scale bar: 20 μm; (FIG. 17I) Graphical comparison of quantification of lipid droplets (LDs) by Oil Red O staining (FIG. 17I) and number of LDs (FIG. 17J) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT; n=10; KO: n=10); (FIG. 17K) (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet) in PCSK7+ / + (WT) mice after (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet). Representative images of 18–30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17L–M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10), *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17B](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17C](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17D](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17E](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17F](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17G](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17H](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17I](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17J](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17K](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17L](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 17M](FIG. 17A) Comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after (i) 20 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17B) (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). , followed by a comparison of weight gain between PCSK7+ / + (WT), PCSK7- / - (KO) mice after a 4-week normal diet recovery period (WT: n=8; KO: n=6); (FIG. 17C) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of a high fat / sugar (fructose) / cholesterol diet (NASH diet) (WT: n=10; KO: n=10); (FIG. 17D) (i) after 24 weeks Comparison of liver weight / body weight between PCSK7+ / + (WT), PCSK7- / - (KO) mice (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6); (FIG. 17E) (i) after 20 weeks of normal diet (chow diet), and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4-week normal diet recovery period (NASH diet). (FIG. 17F) Comparison of gonadal circumference weights between PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of a normal diet (chow diet), and (ii) 8 weeks of a normal diet followed by a 12 week high fat / sugar (fructose) / cholesterol (HFFC) diet followed by a 4 week normal diet recovery period (NASH diet) (WT: n=8; KO: n=6);(FIG. 17G) Percentage of weight loss excluding non-responders (WT: n=8; KO: n=6) after (i) 24 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet restoration; (FIG. 17H) (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). Representative images of 18–30 images of Oil Red O staining in PCSK7+ / + (WT) and PCSK7− / − (KO) mice, scale bar: 20 μm; (FIG. 17I) Oil Red O staining (FIG. 17I) and lipid droplet (LD) numbers (FIG. 17I) between PCSK7+ / + (WT) and PCSK7− / − (KO) mice after (i) 20 weeks of normal diet (chow diet), and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet). 17J) Graphical comparison of quantification of LDs by PCSK7+ / + (WT; n=10; KO: n=10); (Fig. 17K) Representative images of 18-30 images of quantification of lipid droplet (LD) staining in PCSK7+ / + (WT) and PCSK7- / - (KO) mice after (i) 24 weeks of normal diet (chow diet) and (ii) 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet followed by 4 weeks of normal diet (NASH diet), scale bar: 20 μm; (Fig. 1 7L-M) Graphical comparison of LD quantification between PCSK7+ / + (WT) and PCSK7- / - (KO) mice (i) after 24 weeks of normal diet (chow diet) and (ii) after 8 weeks of normal diet followed by 12 weeks of high fat / sugar (fructose) / cholesterol (HFFC) diet (NASH diet) followed by 4 weeks of normal diet (WT; n=10; KO: n=10); *p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001, *****p<0.000001; [Figure 18A]Liver histology images from WT and Pcsk7− / − mouse livers after 12 weeks on the HFFC diet (FIG. 18A) and steatosis scores, lobular inflammation, and hepatocellular ballooning (characteristics of NASH-associated pathology) (FIG. 18B). [Figure 18B] Liver histology images from WT and Pcsk7− / − mouse livers after 12 weeks on the HFFC diet (FIG. 18A) and steatosis scores, lobular inflammation, and hepatocellular ballooning (characteristics of NASH-associated pathology) (FIG. 18B). [Figure 18C] Liver histology images (FIG. 18C) and steatosis scores, lobular inflammation, and hepatocellular ballooning (FIG. 18D) from WT and Pcsk7− / − mouse livers after 12 weeks of the HFFC diet followed by a 4-week recovery period on a chow diet. [Figure 18D] Liver histology images (FIG. 18C) and steatosis scores, lobular inflammation, and hepatocellular ballooning (FIG. 18D) from WT and Pcsk7− / − mouse livers after 12 weeks of the HFFC diet followed by a 4-week recovery period on a chow diet. [Figure 18E] Liver tissue images (FIG. 18E) and fibrosis scores (FIG. 18F) from WT and Pcsk7− / − mouse livers after 12 weeks of HFFC diet. [Figure 18F] Liver tissue images (FIG. 18E) and fibrosis scores (FIG. 18F) from WT and Pcsk7− / − mouse livers after 12 weeks of HFFC diet. [Figure 18G] Liver histology images (FIG. 18G) and fibrosis scores (FIG. 18H) from WT and Pcsk7− / − mouse livers after 12 weeks of the HFFC diet followed by a 4-week recovery period on a chow diet. [Figure 18H] Liver histology images (FIG. 18G) and fibrosis scores (FIG. 18H) from WT and Pcsk7− / − mouse livers after 12 weeks of the HFFC diet followed by a 4-week recovery period on a chow diet. [Figure 18I]Hepatotoxicity as assessed by levels of circulating ALT (alanine aminotransferase) from WT and Pcsk7− / − mouse livers after 12 weeks of a chow diet or HFFC diet (Figure 18I) or after 16 weeks of a chow diet or 12 weeks of a HFFC diet followed by a 4-week recovery period on the chow diet (Figure 18J). [Figure 18J] Hepatotoxicity as assessed by levels of circulating ALT (alanine aminotransferase) from WT and Pcsk7− / − mouse livers after 12 weeks of a chow diet or HFFC diet (Figure 18I) or after 16 weeks of a chow diet or 12 weeks of a HFFC diet followed by a 4-week recovery period on the chow diet (Figure 18J). [Figure 18K] Hepatic TG content in WT and Pcsk7− / − mice after 12 weeks of a chow diet or HFFC diet (FIG. 18K) or after 16 weeks of a chow diet or 12 weeks of a HFFC diet followed by a 4-week recovery period on the chow diet (FIG. 18L). [Figure 18L] Hepatic TG content in WT and Pcsk7− / − mice after 12 weeks of a chow diet or HFFC diet (FIG. 18K) or after 16 weeks of a chow diet or 12 weeks of a HFFC diet followed by a 4-week recovery period on the chow diet (FIG. 18L). [Figure 18M] Log2-transformed averaged signal intensities were performed (https: / / software.broadinstitute.org / morpheus). The lipid changes observed under the HFFC diet in a WT background are not observed in Pcsk7- / - mice. Overall, the latter findings suggest that loss of Pcsk7 expression reversed the hepatic lipid perturbations induced by the HFFC diet. [Figure 19A] mRNA levels of the ER stress marker IRE1α (Figure 19A), the ER chaperones GRP78 (Figure 19A), PERK (Figure 19B), sXBP1 (Figure 19B), or ATF6 (Figure 19C) measured in WT and Pcsk7- / - (KO) mice at 12 and 16 week periods on a chow diet (12 or 16 weeks) or a HFFC diet (12 or 12 weeks, followed by a 4 week recovery period on a chow diet) (NASH). [Figure 19B] mRNA levels of the ER stress marker IRE1α (Figure 19A), the ER chaperones GRP78 (Figure 19A), PERK (Figure 19B), sXBP1 (Figure 19B), or ATF6 (Figure 19C) measured in WT and Pcsk7- / - (KO) mice at 12 and 16 week periods on a chow diet (12 or 16 weeks) or a HFFC diet (12 or 12 weeks, followed by a 4 week recovery period on a chow diet) (NASH). [Figure 19C] mRNA levels of the ER stress marker IRE1α (Figure 19A), the ER chaperones GRP78 (Figure 19A), PERK (Figure 19B), sXBP1 (Figure 19B), or ATF6 (Figure 19C) measured in WT and Pcsk7- / - (KO) mice at 12 and 16 week periods on a chow diet (12 or 16 weeks) or a HFFC diet (12 or 12 weeks, followed by a 4 week recovery period on a chow diet) (NASH). [Figure 19D] Protein levels of the ER stress marker IRE1α measured in WT and Pcsk7− / − (KO) mice at 12 and 16 weeks on the HFFC diet (12 weeks or 12 weeks followed by a 4-week recovery period on a chow diet) (NASH). [Figure 19E] Protein levels of the ER stress marker IRE1α measured in WT and Pcsk7− / − (KO) mice at 12 and 16 week periods on a chow diet (12 or 16 weeks). [Figure 19F] A schematic proposed model in which PCSK7 functions as an apoB100 chaperone to enable efficient exit from the ER and its absence leads to increased UPR activation, resulting in enhanced autophagy and lipid β-oxidation, leading to reduced lipid accumulation in the liver (lower levels of LD). [Figure 20A](FIG. 20A) Western blot analysis of MTP and TM6SF2 levels in livers of WT and Pcsk7− / − mice after 12 weeks of chow or HFFC (NASH) diet, and (FIG. 20B) after 12 weeks of chow or HFFC (NASH) diet followed by a 4-week recovery period. Levels of control α-tubulin are also shown. [Figure 20B] (FIG. 20A) Western blot analysis of MTP and TM6SF2 levels in livers of WT and Pcsk7− / − mice after 12 weeks of chow or HFFC (NASH) diet, and (FIG. 20B) after 12 weeks of chow or HFFC (NASH) diet followed by a 4-week recovery period. Levels of control α-tubulin are also shown. [Figure 21A] Association between a gain-of-function PCSK7 SNP (rs236918) and cardiometabolic traits. [Figure 21B] Association between a loss-of-function PCSK7 SNP (rs201598301) resulting in proline-777-leucine and lipid profiles in humans, including P777L heterozygous individuals B007 and B041. V. Sachan & R. Essalmani: collaboration with May Faraj (IRCM). [Figure 22A] FIG. 1 is a schematic diagram of the human PC7 P777L variant. [Figure 22B]Western blot analysis of apoB and PC7 in IHH cells overexpressing either a control empty vector or a cDNA expressing human PC7 in the absence or presence of its protease inhibitors (+RVKR-cmk). The relative levels of apoB100 to those of the control empty vector are shown after normalization to tubulin levels (Figure 22C). IHH cells were co-transfected with a cDNA encoding a short apoB construct (s-apoB; 965aa) tagged with V5 at the C-terminus and either an empty vector or one expressing PC7 or its variant P777L in the absence or presence of 10 mM NH4Cl. Western blot analysis of cells and media for apoB (V5) and PC7 is shown. Levels normalized to actin are presented as bar graphs below. [Figure 22C] Western blot analysis of apoB and PC7 in IHH cells overexpressing either a control empty vector or a cDNA expressing human PC7 in the absence or presence of its protease inhibitors (+RVKR-cmk). The relative levels of apoB100 to those of the control empty vector are shown after normalization to tubulin levels (Figure 22C). IHH cells were co-transfected with a cDNA encoding a short apoB construct (s-apoB; 965aa) tagged with V5 at the C-terminus and either an empty vector or one expressing PC7 or its variant P777L in the absence or presence of 10 mM NH4Cl. Western blot analysis of cells and media for apoB (V5) and PC7 is shown. Levels normalized to actin are presented as bar graphs below. [Figure 22D] FIG. 1 is a table showing the plasma profile of five female (F) and three male (M) subjects exhibiting low levels of TG, apoB, apoB / PCSK9 and transferrin receptor 1 (TfR1). [Figure 23A] FIG. 1 is a schematic diagram of the primary structure of the active form of human PC7 (92 kDa), showing the catalytic, transmembrane domain (M), and C-terminal cytoplasmic tail (C), with the locations of mutant P777L, four N-glycosylation sites, and two Cys-palmitoylation sites. [Figure 23B] HEK293 cells were co-transfected with cDNAs encoding hTfR1 and either WT PC7 or its cytoplasmic tail (CT) P777L variant. The next day, cell lysates and media were analyzed for shedding of full-length hTfR1 to the soluble, secreted form sTfR1. The P777L form results in 50% LOF for shedding of human transferrin receptor 1 (hTfR1) to the soluble form shTfR1 compared to WT. For comparison, the other CT variants are shown. These include soluble forms of PC7 (no shedding activity), forms lacking CT (delta-CT, 80% LOF), partially lacking (C699A+C704A; 20% LOF) or completely lacking (C673A+C699A+C704A; 50% LOF) Cys-palmitoylation (Durand L. et al. JBC 2019; PMID:31915245). [Figure 24A] Fast protein liquid chromatography (FPLC) profiles. For each man, 300 mL of plasma from either normal (RE) or B041 patients with heterozygous PC7-P777L mutation was separated by FPLC (Pharmacia, Sweden) on a Superose™ 6HR10 / 30 column (Pharmacia Biotech) at a flow rate of 0.3 ml / min. In each fraction (Figure 24A) the total cholesterol (TC) and (Figure 24B) triglyceride (TG) profiles were estimated from the measurement of total cholesterol (TC) and triglyceride (TG) using a commercial kit (Sobioda). Elution positions of VLDL, LDL, and HDL shown on the chromatogram. Optical density was obtained on a SpectraMax™ i3 plate reader (Molecular Devices). [Figure 24B]Fast protein liquid chromatography (FPLC) profiles. For each man, 300 mL of plasma from either normal (RE) or B041 patients with heterozygous PC7-P777L mutation was separated by FPLC (Pharmacia, Sweden) on a Superose™ 6HR10 / 30 column (Pharmacia Biotech) at a flow rate of 0.3 ml / min. In each fraction (Figure 24A) the total cholesterol (TC) and (Figure 24B) triglyceride (TG) profiles were estimated from the measurement of total cholesterol (TC) and triglyceride (TG) using a commercial kit (Sobioda). Elution positions of VLDL, LDL, and HDL shown on the chromatogram. Optical density was obtained on a SpectraMax™ i3 plate reader (Molecular Devices). [Figure 25A] Schematic diagram of the genomic organization of PCSK7. Exons, introns, and miRNA target sites on the PCSK7 sequence. The presence of two putative sites targeted by miR-125-5p on exons 14 and 15 is indicated by horizontal bars below the outlined E14 and E15 exons. Target sequences of four miRNAs (SEQ ID NOs: 16-21) in the 3'-UTR of PCSK7 (hsa-miR-125-5p (SEQ ID NO: 22), hsa-miR-143-3p (SEQ ID NO: 23), hsa-miR-409-3p (SEQ ID NO: 24), and hsa-miR-320a-3p (SEQ ID NO: 25) and their nucleotide hybridization status are shown. miR-125-5p and miR-143-3p each target two sites in the 3'-UTR (see PMID: 35888711). [Figure 25B] Schematic diagram of the genomic organization of PCSK7. Expression levels of PCSK7 mRNA and predicted microRNAs in three tested cell lines, HEK293T, HepG2, and Huh7, assessed by qPCR analysis. Expression levels of PCSK7 mRNA are highest in Huh7, while miR-143-3p and miR-409-3p have the lowest expression in all tested cell lines (see PMID:35888711). [Figure 26A] The expression levels of PCSK7 after miRNA overexpression in Huh7 (Figure 27A) and HEK293T (Figure 27B) cell lines are shown compared to their mock vector counterparts. The expression levels of PCSK7 mRNA in Huh7 cells were significantly downregulated by miR-143-3p and miR-409-3p. In both Huh7 and HEK293T cell lines, miR-125a-5p downregulated PCSK7 expression, but this difference did not reach statistical significance in Huh7 cells (P=0.06). In addition, miR-320a-3p was unable to downregulate the expression of PCSK7 in either Huh7 or HEK293T cells. *p<0.05, and **p<0.01 (see PMID:35888711). [Figure 26B] The expression levels of PCSK7 after miRNA overexpression in Huh7 (Figure 27A) and HEK293T (Figure 27B) cell lines are shown compared to their mock vector counterparts. The expression levels of PCSK7 mRNA in Huh7 cells were significantly downregulated by miR-143-3p and miR-409-3p. In both Huh7 and HEK293T cell lines, miR-125a-5p downregulated PCSK7 expression, but this difference did not reach statistical significance in Huh7 cells (P=0.06). In addition, miR-320a-3p was unable to downregulate the expression of PCSK7 in either Huh7 or HEK293T cells. *p<0.05, and **p<0.01 (see PMID:35888711). [Figure 27]Luciferase assay results after co-transfection of miR overexpression vector and wild-type PCSK7 3'-UTR in HEK293T cells. The relative luciferase activity was significantly decreased after overexpression of mir-125a-5p, miR-143-3p, and miR-409-3p, while there was no significant change in the relative luciferase activity for miR-320a-3p and miR-224-5p used as controls. *p<0.05, and **p<0.01, ***p<0.001 (see PMID:35888711). [Figure 28A] Luciferase assay analysis of cells co-transfected with miR overexpression vector and miRNA target site deleted (Mut) psiCHECK-2 vector in HEK293T cells. Results demonstrated direct interaction of miR-125a-5p, miRNA-143-3p, and miR-409-3p with the 3'-UTR of PCSK7. Both target sites of miR-125a-5p in the 3'-UTR were functional. There was no significant change in relative luciferase activity in cells transfected with miR-125a-5p, Mut-a,b, and both target sites in miR-125a-5p were omitted in the 3'-UTR of the psiCHECK-2 vector. The relative luciferase activity was significantly decreased after overexpression of miR-125a-5p in Mut-a,b in the 3′-UTR of the psiCHECK-2 vector, in which only one target site of miR-125a-5p was deleted. [Figure 28B]Luciferase assay analysis of cells co-transfected with miR-overexpression vector and miRNA target site deleted (Mut) psiCHECK-2 vector in HEK293T cells. Results demonstrated direct interaction of miR-125a-5p, miRNA-143-3p, and miR-409-3p with the 3'-UTR of PCSK7. The two miRNA-143-3p target sites in the 3'-UTR of PCSK7 are functional. No significant change was observed in the relative luciferase activity in cells transfected with miR-143-3p-overexpression vector and Mut-a,b in the 3'-UTR of psiCHECK-2 vector with both miR-143-3p target sites deleted. However, the relative luciferase activity was significantly decreased after cotransfection of the miR-143-3p-overexpression vector with Mut-a,b in the 3′-UTR of the psiCHECK vector, in which only one target site was missing. [Figure 28C] Luciferase assay analysis of cells co-transfected with miR overexpression vector and miRNA target site deleted (Mut) psiCHECK-2 vector in HEK293T cells. Results demonstrated direct interaction of miR-125a-5p, miRNA-143-3p, and miR-409-3p with the 3'-UTR of PCSK7. No significant change in luciferase activity was observed after deletion of the only target site of miR-409-3p in the 3'-UTR of PCSK7. Mut-a represents deletion of the first target site, Mut-b represents deletion of the second target site, and Mut-a,b represents deletion of both miRNA target sites. *p<0.05, and **p<0.01 (see PMID:35888711). [Figure 29A]Western blot analysis (Figure 29A) and quantitative analysis (Figure 29B) of human PC7 expression in miR-125a-5p and miR-224-5p overexpressed Huh7 cells. PC7 protein expression was significantly decreased by miR-125a-5p at 48 hours post-transfection compared to mock vector (PEGFP-C1), while miR-224-5p did not change the protein level of PC7. **p<0.01 (see PMID:35888711). [Figure 29B] Western blot analysis (Figure 29A) and quantitative analysis (Figure 29B) of human PC7 expression in miR-125a-5p and miR-224-5p overexpressed Huh7 cells. PC7 protein expression was significantly decreased by miR-125a-5p at 48 hours post-transfection compared to mock vector (PEGFP-C1), while miR-224-5p did not change the protein level of PC7. **p<0.01 (see PMID:35888711). [Figure 30A] Western blot analysis (Figure 30A) and quantitative analysis (Figure 30B) of PCSK7 protein expression after co-transfection of PC7 overexpression vector with different ratios of miR-125a-5p overexpression vector (1:2 [2x], 1:3 [3x], and 1:5 [5x]) in HuH7 cells. PC7 protein expression was significantly decreased (***p<0.001) in all three ratios of miR-125a-5p-overexpression plasmid, but there was no significant difference in the reduction of PCSK7 at different amounts of miR-125a-5p (see PMID:35888711). [Figure 30B]Western blot analysis (Figure 30A) and quantitative analysis (Figure 30B) of PCSK7 protein expression after co-transfection of PC7 overexpression vector with different ratios of miR-125a-5p overexpression vector (1:2 [2x], 1:3 [3x], and 1:5 [5x]) in HuH7 cells. PC7 protein expression was significantly decreased (***p<0.001) in all three ratios of miR-125a-5p-overexpression plasmid, but there was no significant difference in the reduction of PCSK7 at different amounts of miR-125a-5p (see PMID:35888711). [Figure 31A] Western blot analysis (FIG. 31A) and quantitative analysis (FIG. 31B) of cellular expression of human TfR1 after co-transfection of miR-125a-5p and TfR1 overexpression vector in Huh7 cells. No direct effect of miR-125a-5p on TfR1 expression was observed. Here, miR-125a-5p was overexpressed at different plasmid DNA ratios of PCSK7:miRNA:1:2 (2x), 1:3 (3x), and 1:5 (5x). × represents the vector DNA fold of miR-125-5p compared to vector-expressing PCSK7. [Figure 31B] Western blot analysis (FIG. 31A) and quantitative analysis (FIG. 31B) of cellular expression of human TfR1 after co-transfection of miR-125a-5p and TfR1 overexpression vector in Huh7 cells. No direct effect of miR-125a-5p on TfR1 expression was observed. Here, miR-125a-5p was overexpressed at different plasmid DNA ratios of PCSK7:miRNA:1:2 (2x), 1:3 (3x), and 1:5 (5x). × represents the vector DNA fold of miR-125-5p compared to vector-expressing PCSK7. [Figure 32A]Western blot analysis (Figure 32A) and quantitative analysis (Figure 32B) of human PC7 and TFR1 expression in miR-125-5p overexpressed Huh7 cells. Figure 32A shows that the level of shed soluble sTfR1-V5 in the medium was significantly decreased (***p<0.001) due to the reduction of PC7 protein level detected by Western blot in cells. miR-125a-5p was overexpressed at different plasmid DNA ratios of PCSK7:miRNA:1:2 (2x), 1:3 (3x), and 1:5 (5x). × represents the vector DNA fold of miR-125-5p compared to vector-expressed PC7. Because both PC7-V5 and hTfR1-V5 were V5 tagged, WB using mAb-V5 reflects the expression of both proteins. However, WB using a PC7-specific antibody clearly demonstrated that miR-125-5p reduced cellular PC7 protein levels without any effect on hTfR1 levels (see PMID:35888711). [Figure 32B] Western blot analysis (Figure 32A) and quantitative analysis (Figure 32B) of human PC7 and TFR1 expression in miR-125-5p overexpressed Huh7 cells. Figure 32A shows that the level of shed soluble sTfR1-V5 in the medium was significantly decreased (***p<0.001) due to the reduction of PC7 protein level detected by Western blot in cells. miR-125a-5p was overexpressed at different plasmid DNA ratios of PCSK7:miRNA:1:2 (2x), 1:3 (3x), and 1:5 (5x). × represents the vector DNA fold of miR-125-5p compared to vector-expressed PC7. Because both PC7-V5 and hTfR1-V5 were V5 tagged, WB using mAb-V5 reflects the expression of both proteins. However, WB using a PC7-specific antibody clearly demonstrated that miR-125-5p reduced cellular PC7 protein levels without any effect on hTfR1 levels (see PMID:35888711). [Figure 33A]1 is a schematic diagram of the human PCSK7 gene with positions where eight antisense oligonucleotides (ASOs) were designed and synthesized to target the RNA. [Figure 33B] Design of antisense oligonucleotides targeting human PCSK7 mRNA. The sequences for each of the tested human targeting PCSK7 ASO1-8 (SEQ ID NOs:27-34) (in that order from top to bottom) are shown along with the location of the chemical modifications at each nucleotide: 56-FAM, fluorophore; m,2'O-methyl (protection against nuclease degradation and immune response); *, phosphorothioate (PS) (enhanced stability and increased binding to serum proteins preserves oligonucleotide in circulation, delays clearance by the liver, and increases the time available for uptake into target tissues). [Figure 33C] 1 is a diagram of an antisense oligonucleotide design targeting human PCSK7 mRNA. 2 is an exemplary structure of phosphorothioate and phosphodiester linkages. [Figure 33D] Design of antisense oligonucleotides targeting human PCSK7 mRNA.Endogenous expression levels of PCSK7 RNA in four cell lines normalized to IHH as assessed by RT-qPCR. [Figure 34A] RNA and protein knockdown efficiency of ASO targeting human PCSK7. Human PCSK7 mRNA levels (Figure 34A-B) in human hepatocyte IHH cells were assessed by RT-qPCR, and protein levels (Figure 34C) were assessed by Western blot (using PC7 antibody) 48 hours after transfection with 25 nM ASO (FAM-labeled) or siRNA pools (siRNA sequences 1, 2, 3, and 4 in Figure 6A). Tubulin was used as a loading control. For RT-qPCR, RNA levels were assessed in unsorted bulk cells (Figure 34A) as well as FAM-positive sorted cells (Figure 34B). Expression values ​​were normalized to the transfection reagent only condition. [Figure 34B]RNA and protein knockdown efficiency of ASO targeting human PCSK7. Human PCSK7 mRNA levels (Figure 34A-B) in human hepatocyte IHH cells were assessed by RT-qPCR, and protein levels (Figure 34C) were assessed by Western blot (using PC7 antibody) 48 hours after transfection with 25 nM ASO (FAM-labeled) or siRNA pools (siRNA sequences 1, 2, 3, and 4 in Figure 6A). Tubulin was used as a loading control. For RT-qPCR, RNA levels were assessed in unsorted bulk cells (Figure 34A) as well as FAM-positive sorted cells (Figure 34B). Expression values ​​were normalized to the transfection reagent only condition. [Figure 34C] RNA and protein knockdown efficiency of ASO targeting human PCSK7. Human PCSK7 mRNA levels (Figure 34A-B) in human hepatocyte IHH cells were assessed by RT-qPCR, and protein levels (Figure 34C) were assessed by Western blot (using PC7 antibody) 48 hours after transfection with 25 nM ASO (FAM-labeled) or siRNA pools (siRNA sequences 1, 2, 3, and 4 in Figure 6A). Tubulin was used as a loading control. For RT-qPCR, RNA levels were assessed in unsorted bulk cells (Figure 34A) as well as FAM-positive sorted cells (Figure 34B). Expression values ​​were normalized to the transfection reagent only condition. [Fig. 34D] RNA and protein knockdown efficiency of ASO targeting human PCSK7. Transfection of 25 nM ASO in human hepatocyte IHH cells. Protein knockdown efficiency of ASO targeting human PCSK7 was assessed by Western blot 72 hours (FIG. 34D) and 96 hours (FIG. 34E) after transfection, respectively. Tubulin was used as a loading control. [Figure 34E]RNA and protein knockdown efficiency of ASO targeting human PCSK7. Transfection of 25 nM ASO in human hepatocyte IHH cells. Protein knockdown efficiency of ASO targeting human PCSK7 was assessed by Western blot 72 hours (FIG. 34D) and 96 hours (FIG. 34E) after transfection, respectively. Tubulin was used as a loading control. [Figure 35A] 1 is a schematic diagram of the mouse Pcsk7 gene with positions where eight antisense oligonucleotides (ASOs) were designed and synthesized to target the RNA. [Figure 35B] Design of antisense oligos targeting mouse Pcsk7 mRNA. The sequences for each of the mouse PCSK7-targeting ASOs 1-8 (SEQ ID NOs: 35-42) tested (in that order) are shown along with the location of the chemical modifications at each nucleotide: 56-FAM, fluorophore; m,2'O-methyl (protection against nuclease degradation and immune response); *, phosphorothioate (PS) (enhanced stability and increased binding to serum proteins preserves oligonucleotides in circulation, delays clearance by the liver, and increases the time available for uptake into target tissues). [Figure 36A] RNA knockdown efficiency of ASO targeting mouse Pcsk7. Mouse Pcsk7 mRNA levels (FIG. 36A) and viability (FIG. 36B) in mouse FL83B hepatocytes were assessed by real-time qPCR 48 hours after transfection with 25 nM ASO Dharmafect™. RNA levels were assessed in unsorted bulk cells as well as FAM-positive sorted cells (not shown). Expression values ​​were normalized to transfection reagent only condition. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Cell viability of FL83B cells is shown in FIG. 27B. [Figure 36B]RNA knockdown efficiency of ASO targeting mouse Pcsk7. Mouse Pcsk7 mRNA levels (FIG. 36A) and viability (FIG. 36B) in mouse FL83B hepatocytes were assessed by real-time qPCR 48 hours after transfection with 25 nM ASO Dharmafect™. RNA levels were assessed in unsorted bulk cells as well as FAM-positive sorted cells (not shown). Expression values ​​were normalized to transfection reagent only condition. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Cell viability of FL83B cells is shown in FIG. 27B. [Figure 37A] Comparison of various ASOs and vitamin E on lipid droplet accumulation in mouse FL38B hepatocytes. Immunocytochemistry analysis of mouse FL38B hepatocytes incubated with oleic acid for 48 hours in the presence of either fluorescent mouse mASO-1, mASO-2 or mASO-7 (not shown) or α-tocopherol (vitamin E) (arrows pointing to transfected cells, red staining indicates lipid droplets). [Figure 37B] Comparison of various ASOs and vitamin E on lipid droplet accumulation in mouse FL38B hepatocytes. Quantitative analysis of results presented in bar graphs. [Figure 37C] Comparison of various ASOs and Vitamin E on lipid droplet accumulation in mouse FL38B hepatocytes. Effect of eight ASOs on Pcsk7 mRNA levels measured by quantitative PCR (qPCR). Experiments were repeated independently twice. *p<0.05 (FIG. 37A) Effect of mASO-1, mASO-2, mASO-7 and Vitamin E on reducing intracellular fluorescent triglyceride accumulation as estimated by FACS measurement of lipid droplets (LD) by LipidTox™ staining. [Figure 37D]Comparison of various ASOs and Vitamin E on lipid droplet accumulation in mouse FL38B hepatocytes. Effect of eight ASOs on Pcsk7 mRNA levels measured by quantitative PCR (qPCR). Experiments were repeated independently twice. *p<0.05 (FIG. 37A) Effect of mASO-1, mASO-2, mASO-7 and Vitamin E on reducing intracellular fluorescent triglyceride accumulation as estimated by FACS measurement of lipid droplets (LD) by LipidTox™ staining. [Figure 37E] Comparison of various ASOs and vitamin E on lipid droplet accumulation in mouse FL38B hepatocytes. Modification of ASOs by trivalent binding to N-acetyl-galactosamine (GlcNac). [Figure 38A] Effect of eight different ASOs (see FIG. 35B) in mouse FL38B hepatocytes. Dose-dependent efficacy of these ASOs in reducing total hepatic Pcsk7 mRNA after subcutaneous injection in mice at 2, 5, 10 and 15 mg / kg twice a week for one month. [Figure 38B] Effect of eight different ASOs (see Figure 35B) on mouse FL38B hepatocytes. Livers and partially purified primary hepatocytes from mice injected with either GalNac-ASO-7 or GalNac-ASO-2 at 5 mg / kg twice weekly for one month and analyzed by RT-qPCR for levels of Pcsk7 mRNA. [Figure 38C] Effects of eight different ASOs (see FIG. 35B) in mouse FL38B hepatocytes. Schematic diagram of an experiment in which 8-week-old mice were fed a HFFC diet for 16 weeks (until 24 weeks of age) and then switched to a normal chow diet for an additional 8-week recovery period (until 32 weeks of age). During the 24-week period prior to the recovery period, mice were divided into three groups of 12 mice each: (1) a control untreated group; (2) two GalNac-ASO groups that were subcutaneously injected with either GalNac-ASO-7 or GalNac-ASO-2 at 5 mg / kg twice a week for 8 weeks; and (3) a group that was fed a chow diet containing vitamin E (α-tocopherol) at 500 IU / kg of food. [Figure 38D] Effects of eight different ASOs (see FIG. 35B) in mouse FL38B hepatocytes. Analysis of apoB levels in plasma from 24- (pre-recovery) and 32-week-old mice (post-recovery) revealed that after recovery, both GalNac-ASO2 and GalNac-ASO7 reduced plasma apoB100 by >60%, very similar to the levels observed in Pcsk7- / - mice, supporting the notion that hepatocyte-derived PCSK7 is the primary regulator of circulating apoB levels. In each of FIGS. 38A-B and D, the top-to-bottom order of the legend corresponds to the left-to-right order in the histograms for each test condition (e.g., in FIG. 38A, the results obtained with ASO NT in whole liver are illustrated in the first column, those obtained with ASO2 in the second column, and those obtained with ASO7 in the third column). [Figure 39A] Top panels - Oil Red O staining of liver sections from control (untreated) and ASO-2, ASO-7 and α-tocopherol treated mice. Bottom panels; Total LD ​​area, average LD area and LD number in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2 or α-tocopherol. [Figure 39B] Liver morphology and fat accumulation measured in terms of liver inflammation, hepatocyte balloon expansion, and steatosis grade in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2, or α-tocopherol. [Figure 39C] Extent of fibrosis measured by Sirius red staining in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2, or α-tocopherol. [Figure 39D]Levels of Pcsk7 mRNA in liver after 16 weeks of HFFC diet followed by 8 weeks of recovery in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2, or alpha-tocopherol. In each of Figures 39A and D, the top-to-bottom order of the legend corresponds to the left-to-right order in the histograms for each test condition (e.g., in Figure 39A, the results obtained with ASO NT in total stained area are shown in the first column, those obtained with ASO-2 in the second column, those obtained with ASO-7 in the third column, and those obtained with alpha-tocopherol in the fourth column). [Figure 40A] Measurements of liver or toxicity in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2, or α-tocopherol, as measured by levels of either (FIG. 40A) AST (aspartate aminotransferase) or (FIG. 40B) ALT (alanine aminotransferase). In each of FIG. 40A and B, the top-to-bottom order of the legend corresponds to the left-to-right order in the histograms for each test condition (e.g., in FIG. 40A, results obtained with untreated animals are illustrated in the first column, results obtained with ASO2 are illustrated in the second column, results obtained with ASO7 are illustrated in the third column, and results obtained with tocopherol are illustrated in the fourth column). [Figure 40B] Measurements of liver or toxicity in untreated mice or mice treated with GalNac-ASO-7, GalNac-ASO-2, or α-tocopherol, as measured by levels of either (FIG. 40A) AST (aspartate aminotransferase) or (FIG. 40B) ALT (alanine aminotransferase). In each of FIG. 40A and B, the top-to-bottom order of the legend corresponds to the left-to-right order in the histograms for each test condition (e.g., in FIG. 40A, results obtained with untreated animals are illustrated in the first column, results obtained with ASO2 are illustrated in the second column, results obtained with ASO7 are illustrated in the third column, and results obtained with tocopherol are illustrated in the fourth column). [Figure 41A]1 is the amino acid sequence of human PC7 (SEQ ID NO:1). The signal peptide is highlighted in grey, the propeptide is in bold, the extracellular domain is underlined, the transmembrane domain is in italics, and the cytoplasmic domain is in lowercase. [Figure 41B] The amino acid sequence in FIG. 41A is reproduced, further indicating the known naturally occurring polymorphisms (highlighted) and variants (in bold) disclosed herein below the relevant lines in the sequence. [Figure 42A] The nucleotide sequence of human PC7 (SEQ ID NO:2) is disclosed in Figure 41A. In Figure 42A, the nucleotide sequence encoding the protein is underlined (SEQ ID NO:26) and its nucleotide sequence encoding the signal peptide is highlighted in grey. The fragment corresponding to the siRNA of Figure 6A is in bold (wherein the uracils of the siRNA are shown as thymines). [Figure 42B] Another human PC7 isoform (SEQ ID NO:3). [Figure 42C] Another human PC7 isoform (SEQ ID NO:3). [Fig.42D] Another human PC7 isoform (SEQ ID NO: 4). [Diagram 43] 5 is the amino acid sequence of rat PC7 (SEQ ID NO:5). The signal peptide is highlighted in grey, the propeptide is in bold, the extracellular domain is underlined, the transmembrane domain is in italics, and the cytoplasmic domain is in lowercase. [Diagram 44] The nucleotide sequence of rat PC7 (SEQ ID NO:6) is disclosed in Figure 43. The nucleotide sequence encoding the protein sequence is shown underlined with the nucleotide sequence encoding the signal peptide highlighted in grey. [Diagram 45] 7 is the amino acid sequence of mouse PC7 (SEQ ID NO:7). The signal peptide is highlighted in grey, the propeptide is in bold, the extracellular domain is underlined, the transmembrane domain is in italics, and the cytoplasmic domain is in lowercase. [Figure 46A]The nucleotide sequence of mouse PC7 (SEQ ID NO:8) is disclosed in Figure 44. The nucleotide sequence encoding the protein sequence is shown underlined with the nucleotide sequence encoding the signal peptide highlighted in grey. [Figure 46B] Another mouse PC7 isoform (SEQ ID NO: 9). [Figure 46C] Another mouse PC7 isoform (SEQ ID NO: 9). [Figure 46D] Another mouse PC7 isoform (SEQ ID NO: 10). [Figure 46E] Another mouse PC7 isoform (SEQ ID NO: 10). [Figure 46F] Another mouse PC7 isoform (SEQ ID NO: 11). [Figure 46G] Another mouse PC7 isoform (SEQ ID NO: 11). [Figure 47A] Schematic diagram of ASO mechanism of action (prior art - https: / / www.nature.com / articles / nrneurol.2017.148). [Figure 47B] Schematic diagram of ASO mechanism of action (prior art - https: / / www.nature.com / articles / nrneurol.2017.148). [Figure 47C] Schematic diagram of ASO mechanism of action (prior art - https: / / www.nature.com / articles / nrneurol.2017.148). [Figure 47D] Schematic diagram of ASO mechanism of action (prior art - https: / / www.nature.com / articles / nrneurol.2017.148). [Figure 47E] Schematic diagram of ASO mechanism of action (prior art - https: / / www.nature.com / articles / nrneurol.2017.148). [Figure 48] Schematic of GalNAc-conjugated ASO. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0112] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0113] Example 1: Pcsk7 - / - Mice show reduced circulating and hepatic apoB along with reduced hepatic lipid accumulation. To understand the role of PC7 in vivo, we used mouse (Pcsk7 - / - ) generated a whole-body knockout of the Pcsk7 gene ( Wetsel et al., 2013 ).

[0114] They first examined the hepatic and circulating apoB levels in these mice.

[0115] On a normal chow diet, the inventors found no significant differences between WT mice and Pcsk7 in circulating TC or TG. - / - We found no significant differences between Pcsk7-deficient mice and those without Pcsk7 (Figure 2A-B). Upon closer examination, Western blot (WB) analysis revealed that mice lacking Pcsk7 exhibited approximately 50-60% reductions in circulating (Figure 2C) and hepatic apoB100 protein levels (Figure 2D), without changes in hepatic apoB mRNA levels (Figure 3B) or apoB or microsomal triglyceride transfer protein (MTP) levels (Figure 2E).

[0116] Furthermore, Pcsk7 - / - Livers of Pcsk7 mice showed a significant reduction in the accumulation of lipid droplet size (LDs, TG-rich vesicles) (Figure 2G). Image analysis revealed approximately 40-50% lower Oil Red O staining (Figure 2H) and average area of ​​LDs (Figure 2I) as well as an approximately 30% reduction in total hepatic TG (Figure 2K), with no change in the total number of LDs (Figure 2F-2J), suggesting that the absence of PC7 does not alter the formation of LDs but rather reduces their TG content. This is because, compared to WT mice, Pcsk7 - / -This is consistent with approximately 40% lower hepatic TG levels in Pcsk7 compared to WT (Figure 4A). Finally, we measured protein levels of key proteins in lipid metabolism by Western blot (WB) in liver, including total acetyl-coenzyme A carboxylase 1, Acc1 (tAcc1), the ratio of inhibitory phosphorylated Acc1 (pAcc1c) to total Acc (tAcc1), and carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for β-oxidation of fatty acids (FAs) (Figure 4B). Protein levels of Cpt1a were significantly lower in Pcsk7 compared to WT. - / - Note that the FA concentration was approximately 2.1-fold higher in the KO mice, suggesting that the KO mice exhibit a greater potential for β-oxidation of FA.

[0117] In summary, these data suggest that lack of Pcsk7 is associated with approximately 50% lower apoB levels and TG accumulation in the liver.

[0118] To investigate the timing behind the approximately 50% reduction in apoB, we investigated the expression of WT and Pcsk7 - / - Pulse-chase analysis was performed on primary hepatocytes isolated from mice. Data showed that Pcsk7 - / - Enhanced cotranslational degradation of apoB in hepatocytes ( Fig. 5A ) and Pcsk7 over the chase time period. - / - We found a reduction in apoB100 secretion associated with increased generation of a secreted ∼50 kDa fragment of apoB in hepatocytes (Figure 5B–D), likely due to ER-60, an endoplasmic reticulum (ER)-resident apoB quality control protease (Adeli et al., 1997). Indeed, Western blot analysis revealed that the level of ER60 protein was two-fold higher in Pcsk7 KO compared to WT hepatocytes (Figure 5E–F). This points to an ER-associated degradation (ERAD) mechanism of apoB that is increased in the absence of PC7.

[0119] Example 2: Enhanced apoB degradation in cells lacking PC7: evidence for a chaperone function of PC7 favoring apoB secretion Regulation of human PC7 by siRNA To determine whether the reduction in PC7 levels is associated with a substantial decrease in apoB secretion from human hepatocytes, we analyzed endogenous apoB levels in the culture medium of human HepG2 cells and immortalized human hepatocyte (IHH) cells obtained from immortalized human primary hepatocytes. The use of a mixture of four siRNAs optimized against PCSK7 (Dharmacon) (Figure 6A) revealed an almost complete reduction in endogenous PC7 protein levels, associated with a significant reduction in apoB levels in the culture medium of both cell lines (Figure 6B). Thus, it can be concluded that in humans, as in mouse hepatocytes, PC7 positively regulates apoB levels.

[0120] The effect of overexpression of human PC7 was also examined on the levels of apoB-associated secreted triglycerides secreted from these cells as TG-rich very low density lipoprotein (VLDL) particles. The data showed that transient transfection of human PC7 cDNA in these cells significantly increased the levels of secreted de novo synthesized triglycerides within VLDL particles. 14 We show that overexpression of PC7 resulted in an approximately 1.5-fold increase in the levels of C-TG (Figure 6C). Thus, overexpression of PC7 enhances the levels of apoB secreted from hepatocytes, which is the opposite result to that observed with siRNA-mediated silencing of PC7 in IHH cells (Figure 6B).

[0121] Silencing human PC7 using CRISPR-Cas9 To explain the mechanism underlying the lack of PCSK7 in apoB and hepatic TG levels in mice and extend this observation to human cells, we used CRISPR-Cas9 to delete PCSK7 expression from IHH cells by incubating CRISPR-mediated knockout PCSK7 IHH cells with oleate for 72 h. As previously observed in mice, human PCSK7 (PCSK7 - / -Absence of IFN-γ-γ (IHH cells) resulted in a ∼60% reduction in endogenous intracellular apoB100 protein levels (Figure 7A), reduced the levels and size of intracellular lipid droplets (as revealed by Oil Red O labeling) and enhanced cell proliferation based on cell number compared to control IHH cells (Figures 8, 10G).

[0122] To define the time course of apoB degradation, these IHH cells were incubated with cycloheximide (CHX) to block de novo mRNA translation. - / - We show that cells exhibit a much faster post-translational degradation of apoB, with a significant reduction occurring within the first 15 min after CHX addition (Figure 7B), consistent with the early apoB100 degradation observed in primary hepatocytes after a 15 min pulse (Figures 5A-B).

[0123] To identify the degradation pathway, WT and PCSK7 - / - IHH cells were incubated with either brefeldin A (BFA), an inhibitor of endoplasmic reticulum (ER) exit, the autophagy inhibitor 3-methyladenine (3MA), or the proteasome inhibitors MG132 or lactacystin, and cellular apoB levels were compared to DMSO or non-treated (NT) controls. The data clearly point to enhanced cotranslational early proteasomal degradation of apoB in the absence of PC7 (MG132 and lactacystin) (Figure 7C-D), as confirmed by increased polyubiquitination of apoB20 (Figure 7E) and the absence of BFA inhibition (Figure 7C-D).

[0124] Because PC7 is a secreted serine protease related to yeast kexin, naive IHH cells overexpressing PC7 and a secreted short N-terminal fragment of apoB (s-apoB; aa1-965aa) were incubated with decanoyl-RVKR-cmk, a cell-permeable pan-protein convertase inhibitor that blocks PC7 shedding activity of TfR1 (Guillemot J et al., 2013). Notably, overexpression of PC7 enhanced secretion of s-apoB (apoB21), suggesting that PC7 binds to the N-terminal domain of apoB, likely providing a "chaperone-like" effect, preventing apoB degradation and enhancing its escape from the ER and secretion into the medium. Moreover, decanoyl-RVKR-cmk did not inhibit this chaperone-like activity of PC7 on apoB, suggesting that it occurs via a nonenzymatic pathway (Figure 7F), as also observed for apoA-V10 and major histocompatibility complex-I (MHC-I) (PMID:20164418).

[0125] Next, we investigated the possibility that PC7 may bind apoB in the ER. Thus, rat PC7 and its soluble KDEL variant (retained in the ER) were co-expressed in naive IHH cells with s-apoB. The data showed that, similar to human PC7, rat PC7 (rPC7) was also enhanced by approximately two-fold secretion of s-apoB (apo21). However, rPC7-KDEL significantly (approximately 30%) reduced s-apoB (apo21) secretion compared to rPC7 by retaining it in the ER (Figure 7G, 6C). Supporting this observation, overexpressed PC7 specifically co-immunoprecipitated with endogenous apoB100 (Figure 7H) in IHH cells (Figure 7H). Co-immunoprecipitation (co-IP) experiments also showed that endogenous apoB from IHH cells co-IPs with membrane-bound PC7 (tagged with V5) but not with the control membrane-bound converter enzyme SKI-1 (tagged with V5) ( Abifadel et al., 2003 ) ( Figure 7I ).

[0126] The above data revealed that PC7 functions as a molecular chaperone-like molecule that binds to apoB in the ER and promotes its secretion, and its absence leads to rapid degradation of apoB by the proteasome.

[0127] Example 3: Pcsk7 - / - Mechanism of hepatic TG reduction in mouse and human IHH cells Pcsk7 - / - To explore the mechanism behind the reduction of hepatic TG in mice, we extensively analyzed the mRNA levels of 23 hepatic genes known to be involved in fatty acid (FA) oxidation, esterification, adipogenesis, inflammation, and cholesterol metabolism. However, we did not detect any significant mRNA expression differences (Figures 9A-W).

[0128] According to the literature, Pcsk7 - / - The low hepatic accumulation of lipids observed in mice (Figure 2G-J) could be due to a number of factors: (1) increased secretion of TG-enriched very low density lipoproteins (VLDL); (2) decreased de novo synthesis of FAs; (3) reduced levels of circulating albumin-bound nonesterified FAs (NEFAs) that would be taken up by the liver and converted to TGs; (4) reduced levels of hepatic receptors involved in the uptake of TG-enriched lipoproteins; (5) increased lipophagy; or (6) enhanced β-oxidation of FAs. We address each of these possibilities below.

[0129] To determine whether increased hepatic VLDL secretion was responsible for the reduction in LD observed in the liver (Figure 2C-J), plasma TG levels were measured in vivo after administration of the detergent poloxamer-407 (P-407), which blocks TG lipolysis. Six hours after injection of P-407, Pcsk7 - / - Plasma TG levels were reduced by approximately 50% in mice (Figure 10A), and Pcsk7 - / - We found a significant reduction in hepatic secretion of VLDL-TG in WT and Pcsk7 mice, thereby ruling out possibility #1. To look for de novo changes in adipogenesis, we compared the WT and Pcsk7 mice with the Pcsk7 - / -Primary mouse hepatocytes were incubated with 3H-acetate for 2 h, 3H-TG was extracted and newly incorporated radioactivity was counted as reported (PMID: 26382148). Apparently, possibility #2 was also ignored, since no significant difference in de novo adipogenesis between the two genotypes was detected (Figure 10B). Pyruvate kinase (PKLR), the hepatic receptor for TG-enriched VLDL (LDL receptor-related protein 1, LRP1) and albumin-bound fatty acids (CD36), as well as plasma nonesterified free FAs (NEFAs) (Figure 10C-D) are unchanged in both genotypes, thus ruling out possibilities #3 and #4. In contrast, possibility #5 is the first to suggest that we may be able to explain the role of Pcsk7 in the pathogenesis of de novo adipogenesis. - / - This is likely because we observed a significant increase in a functional macroautophagy-lysosomal pathway in primary mouse hepatocytes, whereby levels of LC3-II were higher both in the absence and presence of chloroquine (Figure 10E), suggesting enhanced functional lipophagy, as previously reported also in the absence of apoB (PMID:27599291).

[0130] To address possibility #6, we compared the FA oxidation potential in human hepatic IHH cells expressing or completely lacking PCSK7 (IHH-CRISPR PCSK7 KO). Using a published protocol (PMID: 26382148), the cells 14 C-oleate and released 14 CO2 was captured at different time points, thereby enhancing FA β-oxidation ( 14 CO2 increase) (Figure 10F).

[0131] We also analyzed the protein levels of key hepatic regulators involved in de novo β-oxidation by Western blot. Under normal diet, the protein levels of Cpt1a and Pcsk7 were significantly higher than those of WT. - / -2.1-fold higher in KO mice, further suggesting that the KO mice exhibit a greater potential for β-oxidation (Figure 4B). Therefore, it is concluded that the absence of PC7 leads to enhanced degradation of apoB by the ERAD pathway, ultimately resulting in enhanced autophagy and hydrolysis of ER-associated TGs, and ultimately resulting in the release of fatty acids and their β-oxidation in mitochondria (model Figure 11).

[0132] Pcsk7 - / - To extend the observation of reduced LD accumulation in the liver of mice (Figure 2G-J) to human cells, we incubated IHH and IHH-CRISPR PCSK7 KO cells with 0.3 mM oleic acid (OA) overnight, then washed the cells and incubated them in medium lacking OA for 88 h. The cells were then stained with Oil Red O and the observed Pcsk7 - / - As in mice, we revealed a loss of LD staining in the absence of PCSK7 ( Fig. 10G ).

[0133] They hypothesized that PC7 is chaperone-like for apoB (Example 2), and since the absence of mouse apoB has been reported to enhance ER stress (PMID:27599291), they compared the protein levels of ER stress markers in IHH with IHH-CRISPR PCSK7 KO cells. The data showed higher levels of phospho-IRE1α and phospho-PERK in cells lacking PCSK7 (Figure 10H), suggesting activated unfolded protein response (UPR) signaling in the absence of PCSK7.

[0134] In summary, upregulated UPR signaling leads to the observation of lower apoB, loss of the potential apoB chaperone PC7, and lower lipid accumulation in hepatocytes.

[0135] Example 4: Pcsk7 - / - Mechanism of TG level fluctuation in mice To understand the mechanism behind PC7-induced changes in TG levels, primary hepatocytes were isolated from WT and KO mice as previously described ( Essalmani et al., 2013 ). These cells were then cultured with radiolabeled 14 C-oleic acid and analyzed the TG content at different time points (0, 6 h, 12 h, 24 h, and 36 h) by lipid extraction and separation on thin-layer chromatography plates. - / - Far fewer primary hepatocytes were isolated from mice compared to WT 14 C-oleic acid (fat) accumulated in the liver of KO mice (Figure 12A), consistent with the reduced TG levels observed in the liver of KO mice (Figure 4A).

[0136] To determine whether the lack of PC7 leads to reduced lipid synthesis, we cultured primary hepatocytes. 13 A novel lipogenesis assay was performed by incubating with C-acetate and measuring lipid synthesis. The data did not reveal any changes in lipogenesis (Figure S12B), suggesting that the lower TG observed in Pcsk7 KO livers may be due to enhanced lipid metabolism (fat burning) rather than reduced synthesis.

[0137] To further explore this fat burning mechanism, we investigated the mechanism of fat burning in WT and Pcsk7 mice. - / - We measured the levels of the autophagosome marker microtubule-associated protein 1A / 1B-light chain 3 (LC3-II) by Western blot in primary hepatocytes isolated from mice. Surprisingly, the data showed that LC3-II levels were significantly increased in Pcsk7 mice compared to WT. - / - The results showed that the expression level was approximately 6 to 7 times higher in primary hepatocytes (Figure 13).

[0138] Additionally, treatment with the lysosomal inhibitor chloroquine reduced the expression of WT and Pcsk7 - / -Both hepatocytes and peritoneal spleen showed accumulation of LC3-II, with the latter showing greater accumulation, indicating that the surge in LC3-II is associated with increased autophagosome biogenesis. It has been concluded that under normal diet, the absence of PC7 leads to enhanced ER-associated autophagy and hydrolysis of TGs, ultimately resulting in enhanced fatty acid release and mitochondrial β-oxidation, as reported in mice treated with antisense oligonucleotides targeting apolipoprotein B (apoB) (Conlon et al., 2016).

[0139] Example 5: Pcsk7 recovery from a high fat / sugar (fructose) / cholesterol (HFFC) diet - / - Hepatic fat accumulation in WT versus mice Next, we explored the effect of loss of PC7 expression on the extent of fat accumulation in the liver following a Western diet high in fat, fructose, and cholesterol (HFFC), which has been reported to induce a robust NAFLD-like phenotype in mice (PMID:32924526). Thus, we compared 20 WT and 20 Pcsk7 mice with HFFC and HFFC, respectively. - / - Four groups of mice (2 months old) were fed a chow diet or a HFFC diet for 12 weeks, after which half of them were sacrificed for evaluation of the development of NAFLD and the other half were allowed to recover under the chow diet for another 4 weeks (Figure 14A). This recovery strategy was elaborated based on our evidence that no changes in LD or apoB were observed under a high-fat diet (HFD) (Figure 35C-D). Indeed, Western blot analysis revealed that apoB levels before recovery were unchanged over the 12-week period in plasma and liver (Figure 15A-B). In contrast, apoB levels were reduced at the end of the 16th week under a chow diet or a HFFC diet followed by recovery in plasma (Figure 14B-C) and liver (Figure 14D-E). The data show that apoB100 levels were significantly higher in both plasma and liver than in WT, consistent with those shown in Figure 2C-D. - / - This indicates that the levels of apoB48 in mice are approximately 50-60% lower, with no significant changes in apoB48.

[0140] Oil Red O (ORO) staining of liver sections revealed robust accumulation of LDs following the HFFC diet in both genotypes, suggesting that the absence of PC7 does not prevent the development of diet-induced NAFLD (Figure 14F-G). In contrast, after a recovery period, the absence of PC7 dramatically reduced the extent of LD accumulation, evidenced by a reduction in the total area stained by ORO, a decrease in the average area, and an increase in the number of LDs (Figure 14H-I). This suggests that the absence of PC7 is associated with a higher metabolism of LDs into smaller ones, as also observed in human IHH cells (Figure 10G). 16-week-old mice fed a chow diet showed significantly higher Pcsk7 accumulation compared to WT. - / - The mice showed a significant reduction of LD of approximately 20% (FIGS. 16C-D).

[0141] We did not observe any significant differences in body weight gain under the chow or NASH diets (Figure 44A) or after a 4-week recovery period (Figure 44B). Similarly, no changes were observed in the liver / body weight or perigonadal adipose tissue weight ratios (Figures 17C-F). The only significant difference observed (p-0.0113) was in Pcsk7 compared to WT (Figure 17G). - / - There was a five-fold reduction in body weight after a recovery period from the HFFC diet in mice.

[0142] Next, we examined the pathology that developed in the livers of these mice after the HFFC diet, before and after a 4-week recovery period (Figures 18A-L). After 12 weeks on the HFFC diet, WT and Pcsk7 - / - Mice exhibited similar grades of steatosis, lobular inflammation, and hepatocellular ballooning (characteristics of NASH-associated pathology) (Figure 18A-B). In contrast, this difference was significantly accentuated after the recovery period, especially for steatosis scores (Figure 18C-D) and lobular inflammation, and Pcsk7 - / - Mice show a lower grade (Figure 18C-D). Furthermore, the improvement in fibrosis score was consistent with the upregulation of Pcsk7 in both the pre- and post-recovery periods. - / - Furthermore, hepatotoxicity, as assessed by the levels of circulating ALT (alanine aminotransferase), was observed in WT and Pcsk7 mice during the pre-recovery period (Figure 18E-H).- / - Although Pcsk7 was increased in both mice and rats (Fig. 18I), - / - Mice recovered significantly after 4 weeks of normal diet compared to WT (Figure 18J). Finally, total liver cholesterol levels were unchanged in both periods (Figure 16E-F), whereas Pcsk7 mice showed significantly higher cholesterol levels compared to WT. - / - TG levels in mice were reduced by approximately 25% after the recovery period (Figures 18K-L).

[0143] Next, we used untargeted lipidomics analysis to assess the effect of Pcsk7 on hepatic lipid bodies in mice fed a chow diet followed by a period of recovery from the NASH diet. - / - The effect of Pcsk7 was addressed (data not shown). Mass spectrometry (MS) results of the chow diet show 1168 features obtained after data processing (data not shown). Using a threshold of P value <0.05, 30 features distinguished KO from WT mice, of which 12 features were annotated using tandem MS analysis (data not shown). The most prominent changes concerned the triglyceride (TG) class, with 9 TGs annotated as downregulated by up to -36%. This reduction in several individual TGs is consistent with our findings supporting lower lipid accumulation in the liver through reduced LD average area. A similar strategy was then used to characterize the Pcsk7 - / - Mice were compared to WT but under a HFFC diet for 12 weeks followed by a 4-week recovery period, yielding 1168 features (data not shown). Using a threshold of P value <0.018, 149 features were identified as Pcsk7 from WT mice. - / - of which 31 features were annotated using tandem MS analysis (data not shown). - / -The major significant changes in hepatic lipid classes in the HFFC diet can be summarized as follows: (1) upregulation: 6 glycerophospholipids (1.44-2.64 fold), 1 TG (1.97 fold), coenzyme Q9 (1.23 fold), and (2) downregulation: 3 free fatty acids (0.30 fold), 1 TG (0.74 fold), and 19 lysoglycerophospholipids (0.18-0.41 fold). Having completed this analysis, the lipid observed as affected by the HFFC diet was Pcsk7. - / - To visualize the extent to which Pcsk7 mice were normalized, a heatmap (Figure 18M) was performed showing the log2-transformed mean signal intensity (https: / / software.broadinstitute.org / morpheus). Notably, the lipid changes observed under the HFFC diet in the WT background were significantly higher than those observed in the Pcsk7 mice. - / - Overall, the latter findings suggest that loss of Pcsk7 expression prevents hepatic lipidomic perturbations induced by the HFFC diet.

[0144] Finally, at the mRNA level, a significant increase in the ER stress marker IRE1α and the ER chaperone GRP78 was detected only during the 12-week period (Figure 19C), but not for PERK, sXBP1, or ATF6 (Figures 19D-E). No changes in mRNA were observed after the recovery period (16 weeks) for either marker (Figures 19C-E). At the protein level, enhanced UPR signaling was observed in both periods, especially after the recovery period, with higher levels of phoho-IRE1α (P-IRE1α) (Figure 19A). Notably, higher UPR signaling was also observed in mice fed the chow diet for 12 and 16 weeks (Figure 19F).

[0145] In summary, the data presented above indicate that PC7 likely functions as an apoB chaperone, allowing efficient escape from the ER, whose absence leads to increased ER stress, leading to enhanced autophagy and lipid β-oxidation, resulting in reduced lipid accumulation in the liver (reduced LD levels), as outlined in the proposed model (Figure 18B).

[0146] Example 6: Pcsk7 - / - The mice show no differences in the levels of MTP or TM6SF2. The above data indicate that the absence of hepatic PC7 suppresses the upregulation of Pcsk7 without affecting the protein levels of microsomal triglyceride transfer protein (MTP) (PMID: 22121032) or transmembrane 6 superfamily member 2 (TM6SF2) (PMID: 34923175) (Figures 20A-B), both of which are recognized as major apoB chaperones. - / - As observed in mice, PC7 resulted in lower levels of apoB in the liver and circulation. Altogether, the data presented herein suggest that PC7 may function as a novel molecular chaperone for apoB, enhancing its escape from the ER and secretion from hepatocytes.

[0147] Example 7: Human Gain of Function (GOF) of PC7 Human genetic evidence from a large biobank supports an association between PC7 levels and circulating lipids and apolipoproteins (Figure 21A). The rs236918 variant is a polymorphism that affects PC7 activity (PMID:21149283) and levels (PMID:30918065). On average, for each additional GOF PCSK7 C allele, carriers have 0.09% higher TG levels (p=4.6×10 -119 ), 0.04 mg / dl higher total cholesterol (TC) level (p=1.9×10 -24 ), and 0.54 mg / dl higher apoB level (p=8.1×10 -13 ), and 0.56 mg / dl higher apolipoprotein A1 levels (p=2.5×10 -10In contrast, the carriers had a lower high density lipoprotein cholesterol (HDLc) of 0.02 mg / dl (p=2.7×10 -5 ) (FIG. 21A). These data confirm the association of PCSK7 GOF with increased cardiometabolic risk factors such as TG and apoB, resulting in relatively small changes in their levels.

[0148] Example 8: Human Loss of Function (LOF) of PC7 We then searched for human PCSK7 variants that may be associated with loss of function of PCSK7 and therefore lead to lower TG levels. Therefore, we selected individuals that showed low apoB and TG levels (with the help of Dr. May Faraj, IRCM). This led to the identification of a man (B041) and a woman (B007) with low TG and apoB at the 5th percentile (Figure 21B). In the case of the man (B041), this phenotype is associated with low plasma LDL cholesterol at the 10th percentile (Figure 21B). DNA sequencing revealed that both exhibited the heterozygous P777L variant of PCSK7. This modification is a Val-Pro 777 The sequence is dialiphatic Val-Leu 777 (Durand et al., 2020), resulting in the cytoplasmic tail of PC7, potentially regulating its intracellular trafficking and localization. Herein, we speculate that trafficking of [WT PC7-apoB] vs. [PC7-P777L-apoB] complexes is affected, with the latter possibly being more efficiently targeted to lysosomes for apoB degradation (Figure S2A-C), thereby providing the basis for the observed low circulating levels of apoB (Figure S21B).

[0149] In cell-based assays, this variant resulted in a 50% reduction in the ability of PC7 to clear its only known specific membrane-bound substrate, transferrin receptor 1 (hTfR1) (Guillemot et al., 2013; Durand et al., 2020), revealing the first validated loss-of-function (LOF) variant of PC7, likely due to reduced PC7 concentration in early endosomes where cleavage of hTfR1 occurs (Figure 23A-B) (Durand et al., 2020). Surprisingly, FPLC analysis of plasma from male subject B041 showed low concentrations of both LDL-cholesterol and VLDL-TG, but no changes in HDL-cholesterol or HDL-TG, compared to a control male (RE) with a similar body weight (Figure 24A-B). Thus, in men, LOF of PC7 may result in low apoB, TG and LDL cholesterol. The genome aggregate database (gnomADv2.1.1 and v3.1.2 combined, gnomAD:P777L from broadinstitute; RS201598301) revealed that 8,115 heterozygous and 175 homozygous P777L have been reported (see Tables 1 and 2 below). Thus, homozygous P777L LOF is not apparently associated with morbidity, as also observed in Pcsk7 KO mice. [Table 1] [Table 2]

[0150] Example 9: Materials and methods for testing the effect of miRNAs on PCSK7 expression and function Computational prediction of miRNAs targeting the PCSK7 gene The following publicly available bioinformatics algorithms were used to predict potential miRNAs targeting the PCSK7 gene: TargetScan (http: / / www.targetscan.org / vert_71 / ), DIANA tools (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php), miRDB (http: / / mirdb.org / ), miRanda (http: / / www.microrna.org / microrna / home.do), and the UCSC website (http: / / genome.ucsc.edu).

[0151] cell culture Human embryonic kidney 293 (HEK293T), hepatocellular carcinoma (HepG2), and hepatoblastoma (Huh7) cells were selected for further functional experiments. HEK293T and Huh7 cell lines were cultured in Gibco's Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, USA) supplemented with penicillin 100 U / mL, streptomycin 100 μg / mL (Sigma, USA), and 10% fetal bovine serum (FBS) (Invitrogen, USA), and then incubated at 37°C with 5% CO2. HepG2 cells were cultured in DMEM-F12 (Invitrogen, USA) containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0152] RNA extraction, complementary DNA (cDNA) synthesis, and quantitative polymerase chain reaction (qPCR) RNA was isolated from cells using TRIzol™ Reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNaseI (Fermentas, USA) to eliminate any possible DNA contamination. Reverse transcription was performed using the PrimeScript™ First Strand cDNA Synthesis Kit (Takara, Japan) according to the manufacturer's protocol. Briefly, one unit of DNaseI enzyme, 1 μL of buffer, and 1 μg of total RNA were incubated at 37° C. for 30 min. Then, 1 μL of 50 mM EDTA was added for enzyme inactivation and incubated at 65° C. for 10 min. Then, 5 μL of DNase-treated RNA was added to a mixture of 0.5 μL of reverse transcriptase, 2 μL of reverse transcriptase buffer, and 1 μL of random hexamers and incubated at 37° C. for 15 min, followed by incubation at 85° C. for 5 s to inactivate the enzyme. To assess miRNA expression, stem-loop RT-qPCR was applied ( Kramer, 2011 ).

[0153] qPCR tests were performed in 20 μL PCR reaction mixtures in an Applied Biosystems StepOne™ instrument (Applied Biosystems, USA) using SYBR GreenI (Takara, Japan). Briefly, cDNA equivalent to 50 ng of RNA was added to a mixture of 10 μL SYBR Green, 0.5 μM of each primer, 0.4 μL Rox reference dye, and sufficient water. The real-time thermal programs were as follows: 40 cycles of 95°C for 30 s, 95°C for 20 s, and 60°C for 35 s for PCSK7, and 40 cycles of 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, and 72°C for 15 s for miR-125a-5p, miR-143-3p, miR-409-3p, miR-320a-3p, and miR-244, using RNU48 small nuclear RNA, β2-microglobulin (β2m), and GAPDH mRNA as internal controls.

[0154] All reactions were repeated in duplicate. Melting curves were then analyzed with the average threshold cycle used for further analysis. The relative expression of miRNAs and PCSK7 to RNU48 small nuclear RNA and / or GAPDH and β2m, respectively, was calculated via the 2-ΔΔCt method. PCR products were sequenced (3500 ABI) to verify the accuracy of amplification. All primers for PCSK7 and recommended miRNAs are listed in Table 3 below. [Table 3-1] [Table 3-2]

[0155] Plasmids and cell transfection MiR overexpression vectors The effects of selected miRNAs on the mRNA expression level of PCSK7 were examined 48 hours after transfection of overexpressed miRNAs in Huh7 and HEK293T cell lines. Plasmids encoding pEGFP-C-miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p and their corresponding controls (miR-NC) were constructed. The nominated miRNA genes were amplified and cloned downstream of the GFP gene into the pEGFP-C1 vector (Clontech, Japan). All primer sequences used are available in Table 3 above.

[0156] Pre-miR miRNA precursor overexpression vectors (300 ng) were transfected into Huh7 and HEK293T cell lines in 12-well plates using FuGENE™ HD (Promega Corporation, Madison, WI, USA). Transfections were performed in triplicate, and mock-related counterpart vectors served as controls.

[0157] Vectors containing wild-type and mutant forms of PCSK7 3'-UTR The interaction between miRNAs and their possible targets on PCSK7 was explored by cloning the potential target regions in psiCHECK-2™ (Promega, USA), a luciferase reporter vector. In psiCHECK-2™, hRluc, a Renilla luciferase gene, is located upstream of the target region of interest cloned into the psiCHECK-2™ vector downstream of the Renilla gene. A region corresponding to the 3'-UTR of PCSK7 (a 926-nt long sequence) constituting the predicted miRNA response element was PCR amplified and cloned downstream of the Renilla luciferase gene in the psiCHECK-2™ vector (Promega, USA). To confirm whether the miRNA response element (miRNA target site) on the 3'-UTR of PCSK7 is active and has a direct interaction with miRNA, different mutants (plasmids) were constructed by splicing by overhang extension (SOEing) PCR. Three different mutant constructs were constructed for miR-125a-5p and miR-143-3p, each of which has two miRNA response elements on PCSK7 3'-UTR. Two constructs were made by deleting the predicted miRNA target sites, and the third construct was made by omitting both predicted miRNA target sites in the 3'-UTR sequence of PCSK7. In the miR-409-3p mutant construct, the predicted miRNA target sites were deleted from the 3'-UTR sequence. The sequences of the primers are listed in Table 3 above.

[0158] Luciferase reporter assay HEK293T cells were co-transfected through application of wild-type psiCHECK-2, wild-type PCSK7 3'-UTR, mutant PCSK7 3'-UTR, and miR overexpression vectors, allowing direct interaction of the designated miRNAs with PCSK7 3'-UTR to be investigated. In summary, 150 ng of wild-type or mutant 3'-UTR constructs and 300 ng of miRNA expression vectors were co-transfected in HEK293T culture 48-well plates using FuGENE™ (Invitrogen, USA). In addition, psiCHECK-2 and pEGFP-C1 mock vectors were transfected and served as controls for luciferase assay and transfection, respectively. Transfection efficiency was monitored by fluorescence microscopy (Nikon TE2000S, Japan) 36 hours after the procedure.

[0159] The PsiCHECK-2 reporter construct plasmid contained a Renilla luciferase gene upstream of the PCSK7 3'-UTR and an independent firefly luciferase gene as an internal control for normalization. Forty-eight hours after co-transfection of HEK293T, luciferase reporter assays were performed using a luminometer (Titertek-Berthold, Germany) with the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's protocol. Each sample was performed in triplicate, and the experiment was biologically repeated at least three times. Briefly, after removing the cell medium, lysis buffer was added to each well. Then, LARII reagent was added, and after 20 minutes, firefly luciferase activity was measured as a control. Renilla activity was then determined using Stop&Glo reagent. Relative luciferase activity was calculated using the following formula: Δ activity fold of luciferase (Renilla / Firefly) = average Renilla / Firefly from samples A / B.

[0160] Western blot analysis In the next step, the effect of miR-125a-5p on PC7 function was determined. First, the miR-125a-5p-overexpression vector and its related mock plasmid were co-transfected with a pIRES vector (Invitrogen, USA) containing a full-length cDNA encoding PCSK7 mRNA with a complete 3'-UTR in Huh7 cells. Western blot analysis was performed 48 hours after transfection. The effect of miR-125a-5p on PC7 enzymatic function was evaluated by co-transfecting Huh7 cells with a miR-125a-5p-overexpression vector encoding the full-length PCSK7 and a plasmid encoding hTfR1 (Guillemot et al., 2013; Durand et al., 2020). Cell lysates and medium were harvested for Western blot analysis 48 hours after transfection.

[0161] Proteins were then isolated in ice-cold RIPA buffer (1×) containing 50 mM Tris hydrochloride (pH 8), 150 mM sodium chloride, 0.1% sodium dodecyl sulfate, 1% Nonidet P40, 0.25% sodium deoxycholate, and a cocktail of protease inhibitors (Roche, Oakville, ON, Canada). Proteins were subjected to electrophoresis on a 12% polyacrylamide sodium dodecyl sulfate gel and blotted onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was then blocked with nonfat 5% milk powder dissolved in Tris-buffered saline (0.1 M Tris hydrochloride [pH 8] and 1.5 M sodium chloride) containing 0.1% Tween-100 (TBS-T). Both PC7 and TfR1 were C-terminally tagged with V5 and detected with V5 monoclonal antibody (Invitrogen), and membranes were incubated with appropriate primary and secondary antibodies as reported (Guillemot et al., 2013). Immunoreactive bands (signals) were subsequently visualized and recorded via chemiluminescence using an enhanced chemiluminescence reaction kit (Bio-Rad, USA). Bands were analyzed and quantified using NIH ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

[0162] statistical analysis The 2-(ΔΔCt) method was applied for qPCR data analysis and gene expression determination. GraphPad™ Prism, version 8 (GraphPad Software, Inc, La Jolla, CA, USA) was used to analyze the data obtained by qPCR, dual luciferase, and Western blot analysis, and P-value calculation. A P-value of less than 0.05 was considered statistically significant in all experiments.

[0163] Example 10: Bioinformatics prediction of PCSK7-targeting miRNAs The molecular mechanisms underlying the regulation of PCSK7 expression by miRNAs were explored via bioinformatics analysis of five different publicly available target prediction programs, namely TargetScan, DIANA-micro-T, miRDB, miRanda, and UCSC, to predict miRNAs targeting the 3'-UTR of PCSK7 transcript. Several miRNAs were identified by these programs, leading to the selection for further analysis of four different targeting miRNAs that were commonly predicted by all programs, namely miR-125a-5p, miR-143-3p, miR-409-3P, and miR-320a-3p. Among the selected miRNAs, miR-125a-5p has four target sites, two of which are located in the 3'-UTR and the rest are located in exons 14 and 15 of PCSK7, and miR-143-3p has two target sites within the 3'-UTR of PCSK7. In contrast, miR-409-3P and miR-320a-3p each have only one predicted target site (Figure 25A).

[0164] Example 11: Predicted miRNA and PCSK7 expression in HEK293T, HepG2, and Huh7 cell lines The next step was to evaluate the endogenous expression patterns of the four selected miRNAs (miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p) and PCSK7 mRNA by qPCR in three cell lines, namely, renal-derived HEK293T, hepatocyte-derived HepG2, and Huh7 cell lines (Figure 25B).

[0165] Huh7 cells had the highest relative expression of PCSK7 mRNA and very low RNA expression levels of miR-143-3p and miR-409-3p, whereas miR-125a-5p and miR-320a-3p expression levels were similar in all cell lines. In HepG2 and HEK293T cells, miR-125a-5p and miR-320-3p expression levels were comparable to PCSK7, whereas miR-143-3p and miR-409-3p expression levels were low. From these data, PCSK7 mRNA levels appear to be inversely correlated with miR-143-3p and miR-409-3p RNA levels in all cells, whereas miR-125a-5p may negatively regulate PCSK7 mRNA levels in Huh7 cells.

[0166] Example 12: Negative regulation of PCSK7 mRNA expression levels by overexpression of miR-125a-5p, miR-143-3p, and miR-409-3p Huh7 cells were transfected with miRNA overexpression vectors carrying precursors of miRNA sequences and mock-related corresponding vectors to evaluate the possible relationship between the expression patterns of miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p and PCSK7 expression at the transcriptional level. qPCR results obtained 48 h after transfection showed that overexpression of miR-143-3p and miR-409-3p in Huh7 cells significantly reduced the expression of PCSK7 mRNA by 40-50% compared to cells transfected with mock vector (P=0.0158 and P=0.0084) (Figure 26A). Overexpression of miR-125a-5p tended to reduce the expression of PCSK7 mRNA in these cells, but this reduction did not reach statistical significance (P=0.0693). In contrast, ectopic expression of miR-320a-3p did not downregulate PCSK7 expression in Huh7 cells (P = 0.5538).

[0167] The relationship between the expression levels of miR-125a-5p and miR-320a-3p and the expression levels of PCSK7 was further investigated by measuring the levels of PCSK7 mRNA in HEK293T cells. qPCR results showed that overexpression of miR-125a-5p resulted in a significant reduction of PCSK7 mRNA levels by about 25% 48 hours after transfection in these cells (P=0.0114) (Figure 26B). However, similar to Huh7 cells, no significant regulatory relationship was observed for miR-320a-3p in HEK293T cells (P=0.9972). Thus, overexpression of miR-143-3p, miR-409-3p and even less miR-125a-5p may downregulate PCSK7 expression.

[0168] Example 13: Direct interactions between predicted miRNAs and the 3'-UTR of PCSK7 Dual luciferase assay was applied to investigate the interaction between the predicted miRNAs cloned downstream of Renilla luciferase gene in psiCHEK-2 plasmid and the 3'-UTR of human PCSK7 mRNA. In addition, miR-224-5p, which does not have any target site on the 3'-UTR of PCSK7, served as a control. Overexpression of miR-125a-5p significantly reduced the relative luciferase activity by about 50% (P<0.0001) in HEK293T cells co-transfected with miR-125a-5p overexpression plasmid and psiCHECK-2 carrying wild-type PCSK7 3'-UTR (Figure 27). Similarly, miR-143-3p and miR-409-3p significantly decreased luciferase activity by about 20-30% by targeting PCSK7 3'-UTR (P=0.0013 and P=0.04, respectively). However, miR-320a-3p and miR-224-5p, used as controls, showed no significant effect on luciferase activity (P≧0.05, P>0.99, and P=0.91). Therefore, miR-125a-5p, miR-143-3p, and miR-409-3p were selected for further analysis of their possible direct interactions with PCSK7 3'-UTR.

[0169] Example 14: Direct interactions between miR-125a-5p, miR-143-3p, and miR-409-3p and the 3'-UTR of PCSK7 Luciferase reporter assays further confirmed that miR-125a-5p, miR-143-3p, and miR-409-3p directly targeted the 3'-UTR of the PCSK7 transcript, since deletion of the miRNA target sites (miR-125a-5p, miR-143-3p, and miR-409-3p) on the 3'-UTR of the PCSK7 reporter plasmid abolished negative regulation (Figure 28A-B). In this regard, three different mutant miR plasmids were constructed for both miR-125a-5p and miR-143-3p. A single target site for each miRNA was individually omitted in two separate plasmids, and two putative binding sites of the miRNA were deleted in the other plasmid. The data demonstrated that overexpression of miR-125a-5p still reduced the relative luciferase activity in HEK293T cells co-transfected with a plasmid carrying a mutant form of the PCSK7 3'-UTR containing a single deletion of the miR binding site (Mut-a and Mut-b) compared to cells co-transfected with the mock counterpart vector together with the same mutant vector (Mut-a and Mut-b) (P=0.04 and P=0.01). In contrast, a mutant form of the PCSK7 3'-UTR featuring deletion of both miRNA target sites (Mut-a, b) failed to significantly reduce the relative luciferase activity in these cells (P=0.24) (Figure 28A). Similar to what was observed for miRNA-125a-5p, overexpression of miR-143-3p did not significantly affect luciferase activity when the two putative target sites (Mut-a,b) were omitted from the 3'-UTR of PCSK7 in HEK293T cells (P=0.98). In comparison, luciferase activity was greatly reduced in these cells expressing the miR-143-3p plasmid and a mutant vector carrying only one miRNA response element sequence (Mut-a; P=0.03 and Mut-b; P=0.004) (Figure 28B). Finally, overexpression of miR-409-3p did not result in a reduction in luciferase activity when the only putative target site of miR-409-3p was absent from the 3'-UTR of PCSK7 (P=0.40) (Figure 28C).

[0170] Overall, the above results confirm two independent miRNA response elements for both miR-125a-5p and miR-143-3p, and one miRNA response element for miR-409-3p, as active elements, and thus, miR-125a-5p, miR-143-3p, and miR-409-3p can regulate PCSK7 mRNA expression through direct interaction with these miRNA binding target sites.

[0171] Example 15: Functional effect of miR-125a-5p on PCSK7 activity Western blot analysis was performed to determine whether miR-125a-5p affected the functional activity of endogenous PC7 in Huh7 cells. Wild-type human PC7 overexpression vector was co-transfected with pre-miR-125a-5p and pre-miR-224-5p overexpression plasmids in Huh7 cells. Western blot results 48 hours after transfection revealed that overexpressed PC7 protein levels were significantly reduced by approximately 80% (P=0.0025) after miR-125a-5p coexpression compared to mock-related (pEGFP-C1) and miR-224-5p controls (Figures 29A-B). As a control, human PCSK9 overexpression vector was co-transfected with miR-125a-5p and miR-224-5p vectors in Huh7 cells, and these miRNAs did not affect the expression levels of PCSK9 protein (not shown).

[0172] Huh7 cells were then co-transfected with vectors expressing PC7 and miR-125a-5p at DNA ratios of 1:2 (2x), 1:3 (3x), and 1:5 (5x). The results showed that all three ratios of miR-125a-5p significantly reduced PC7 protein levels by more than 80%, with no significant difference in PC7 silencing between different amounts of miR-125a-5p (Figure 30A-B).

[0173] PC7 is known to specifically cleave human type II membrane-bound hTfR1 into a soluble secreted form, thereby increasing its circulating level (Guillemot et al., 2013). Furthermore, bioinformatics analysis based on Targetscan and UCSC genome browser (miRcode predicted microRNA target site tracking) demonstrated that miR-125a-5p does not have any target site on the 3'-UTR of hTfR1. Consistently, miR-125a-5p, which reduced the level of PC7 protein (Figures 29A-B and 30A-B), did not affect the expression level of hTfR1 protein (Figures 31A-B). Therefore, to evaluate the functional effect of miR-125a-5p on PC7 activity, hTfR1-V5 was co-expressed with PC7 and different amounts of miR-125a-5p (2x, 3x, and 5x) expression vector in Huh7 cells (Figure 32A-B). The data show that the presence of miR-125a-5p resulted in a decrease in the ability of PC7 to shed hTfR1 into the medium (Figure 32A-B). These results demonstrated that miR-125a-5p not only reduced the expression level of PCSK7 mRNA, but also functionally suppressed its protease activity on hTfR1.

[0174] Example 16: Silencing of human PC7 with antisense oligonucleotides A series of antisense oligos (ASOs) were designed against the human (h) PC7 gene, each targeting a different exon. Two non-targeting ASOs were also designed as controls. From this series, eight were selected to be synthesized and their efficiency in knocking down PC7 was tested (Figure 33A-B) (see Figure 33B for sequences). All oligonucleotides were 20 nucleotides long and labeled with a FAM fluorophore at the 5' end, allowing their internalization in cells after transfection to be tracked. After gapmer design, the first five nucleotides at the 5' end and the last five nucleotides at the 3' end of each oligonucleotide were modified with 2'O-methyl moieties, while all nucleotides contained phosphorothioate modifications (Figure 33C).

[0175] The mRNA expression levels of PC7 were measured in three different human liver cell lines (IHH, HepG2, HuH7) and one renal cell line, HEK293. As shown in Figure 33D, the levels of PC7 were highest in HuH7, followed by IHH, HEK293 and HepG2. IHH cells were transfected in parallel with 25 nM of each ASO using Dharmafect™ transfection reagent.

[0176] Analysis of FAM fluorescence 24 h post-transfection by flow cytometry analysis revealed a transfection efficiency of 84-90%. The knockdown efficiency of each ASO was assessed by RT-qPCR and Western blotting of PC7 48 h post-transfection (Figure 34A). ASO#1, #7 and #8 targeting exons 12, 14 and 3, respectively, produced the strongest knockdown, achieving >85-90% reduction in PC7 mRNA levels and 89-94% reduction at the protein level, similar to siRNA-mediated knockdown of PC7 (Figure 34C). With the exception of ASO#6 targeting exon 9, all other ASOs tested reduced PC7 expression by 62-88% at the protein level (Figure 34D). Knockdown of PC7 persisted and became even stronger at 72 h post-transfection, resulting in a loss of PC7 of 92-100% for seven of the eight ASOs tested (Figure 34D). Even 96 hours after transfection, the protein levels of PC7 remained at 20-40% for ASO#1, #7, and #8 compared to non-targeting ASO control levels, while the levels returned to 70-90% normal levels with other ASOs (Figure 34E). This indicates that ASO#1, #7, and #8 are potent and can reduce PC7 protein levels as efficiently as siRNA up to 96 hours after transfection.

[0177] Example 17: Silencing of mouse Pcsk7 by ASO ASOs against different exons of mouse Pcsk7 gene were also designed (Figure 35A-B). Eight ASOs were selected and synthesized following the same chemistry as Example 16: 20 nt gapmer with 5'FAM label (see Figure 35B for sequences). 25 nM of each ASO was then transfected in parallel in mouse hepatocyte cell line FL83B, achieving 37-56% transfection efficiency 24 hours post-transfection as assessed by flow cytometry. Analysis of Pcsk7 RNA levels in bulk cells by RT-qPCR 48 hours post-transfection revealed 58-60% knockdown efficiency for ASO#2, #6, #7 and #8 targeting exons 6, 2, 4 and 5, respectively. When RT-qPCR was performed only on FAM-positive transfected cells, following cell sorting by FACS, the actual knockdown efficiency of ASO#2, #6, #7, and #8 was 80-95% (Figure 36A). Knockdown of Pcsk7 using ASO#1, #2, and #7 did not affect the viability of FL83B cells compared to non-targeting ASOs (Figure 36B).

[0178] Example 18: Effects of mASO-2 and mASO-7 and Vitamin E on reducing lipid droplet accumulation in mouse FL38B hepatocytes Effect of eight ASOs on Pcsk7 mRNA levels measured by quantitative PCR (qPCR) (Figure 37C). Mouse FL38B hepatocytes were incubated with oleic acid for 48 h in the presence of either fluorescent (green) mouse mASO-1, mASO-7, or a-tocopherol (vitamin E). The accumulation of lipid droplets (Figure 37A, visible as small dots around the nucleus) was significantly reduced in the presence of both ASOs and vitamin E, as shown in the bar graph quantitative analysis (Figures 37B-37D). Mouse FL38B hepatocytes were plated on day 1, ASOs were added on day 2, medium was replaced on day 3, and RNA was extracted on day 4. The same experiment was repeated twice independently, showing that mASO-1, -2, -7 and vitamin E achieved the best reduction in the accumulation of fluorescent triglycerides in cells, as estimated by FACS measurement of lipid droplets (LDs) by LipidTox™ staining (Figure 37D). This led the inventors to choose to modify ASO-2 and ASO-7 by attachment of trivalent N-acetyl-galactosamine (Figure 37E) to allow these ASOs to efficiently target hepatocytes in the liver via their binding to the hepatocyte-specific receptor ASGR1 / 2 (see PMIDs: 34508778 and 34645280).

[0179] Example 19: Treatment for diet-induced NAFLD Eight ASOs against mouse Pcsk7 were tested in mouse FL38B hepatocytes, and the best ASO2 and ASO7 were modified with covalent linkages to trivalent N-acetyl-galactosamine (GalNac) residues (Figure 37E) (Figure 37D). The dose-dependent efficacy of these ASOs after subcutaneous injection in mice was tested for reduction of total liver Pcsk7 mRNA at 2, 5, 10, and 15 mg / kg twice weekly for 1 month. ASO-7 and ASO-2 at 5 mg / kg, to a lesser extent, reduced total liver Pcsk7 mRNA by 40-60% (Figure 38A). Mice were then injected with either GalNac-ASO7 or GalNac-ASO-2 at 5 mg / kg twice weekly for 1 month, whereupon the livers and their partially purified primary hepatocytes were analyzed by RT-qPCR for levels of Pcsk7 mRNA. GalNac-ASO-7 reduced the level of Pcsk7 mRNA in hepatocytes by about 69% compared to about 60% in the whole liver, likely representing a reduction of Pcsk7 mRNA in hepatocytes by >98% (Figure 38B). For GalNac-ASO2, a reduction of about 69% in the level of Pcsk7 mRNA in hepatocytes was observed compared to about 34% in the whole liver (Figure 38B). Thus, both GalNac-ASO7 and GalNac-ASO-2 can reduce Pcsk7 mRNA in whole hepatocytes by >98%. Eight-week-old mice were then fed the HFFC diet for 16 weeks, followed by a program of switching to a normal chow diet for an additional 8-week recovery period. Mice were divided into three groups of 12 mice each and treated from the beginning of the recovery period as follows (Figure 38C): (1) a control untreated group; (2) two GalNac-ASO groups that were subcutaneously injected with either GalNac-ASO7 or GalNac-ASO2 at 5 mg / kg twice a week for 8 weeks; (3) a group fed a chow diet containing vitamin E (α-tocopherol) at 500 IU / kg of diet, in accordance with the current American Association for the Study of Liver Disease guidelines (PMID:28714183) for the use of vitamin E to treat patients with NAFLD.ApoB levels were first analyzed in the plasma of these mice, and both GalNac-ASO-2 and GalNac-ASO-7 upregulated Pcsk7. - / - We observed that it was possible to reduce circulating levels of apoB by >60% (Figure 38D), very similar to the plasma levels observed in mice (Figure 2C), supporting the idea that it is hepatocyte-derived PC7 that primarily regulates circulating apoB levels.

[0180] Example 20: ASO-7 and ASO-2 reduce hepatic steatosis and NAFLD much more effectively than α-tocopherol. Next, we analyzed the reduction in LD accumulation in mice treated with GalNac-ASO-7 or GalNac-ASO-2 compared to α-tocopherol (Figure 39A). - / - In agreement with the data obtained in mice (Fig. 2H, 14H-I), hepatocyte-specific reduction of Pcsk7 mRNA by GalNac-ASO-7 and GalNac-ASO-2 also significantly reduced LD levels after the recovery period, much more effectively than α-tocopherol (Fig. 39A). Thus, GalNac-ASO7 treatment led to an average 47% reduction in the total area of ​​LDs, which are now more abundant (+61%) but smaller (-67%), likely due to higher lipolysis of TGs induced by loss of Pcsk7, as previously described (Fig. 10A-F, 10B). This was also seen to a lesser extent with GalNac-ASO-2, but significantly less with α-tocopherol (Fig. 39A). These results were supported by the analysis of liver morphology and fat accumulation, graded blindly by a pathologist (Fig. 39B). Accordingly, GalNac-ASO-7 significantly reduced liver inflammation, hepatocyte ballooning, and steatosis grade (Figure 39B) to levels near those of normal liver (denoted stage 0). Finally, the degree of fibrosis that typically develops in NAFLD / NASH was also analyzed by Sirius Red staining, showing that GalNac-ASO-7 significantly reduced liver fibrosis to a similar extent as the antioxidant α-tocopherol (Figure 39C).

[0181] Example 21: ASO-7 and ASO-2 reduced hepatic Pcsk7 mRNA levels, whereas α-tocopherol enhanced them. We next compared the levels of Pcsk7 mRNA in the livers of mice after 16 weeks of HFFC diet followed by an 8-week recovery period. GalNac-ASO7 and GalNac-ASO2 reduced total liver Pcsk7 mRNA by 56% and 45%, respectively, whereas dietary α-tocopherol caused a 33% increase in Pcsk7 mRNA levels (Figure 39D). However, none of these treatments caused liver damage or toxicity, as assessed by unchanged levels of either AST (aspartate aminotransferase) (Figure 40A) or ALT (alanine aminotransferase) (Figure 40B).

[0182] Similar experiments were performed on Apovec-1, a mouse model with a lipid profile closer to that of humans. - / - and Ldlr - / - This will be carried out in mice (PMID: 8824235 and 9701246).

[0183] The scope of the claims should not be limited by the embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0184] References Abifadel M, et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat Genet 34,154-156 (2003). Adeli K, Macri J, Mohammadi A, Kito M, Urade R, Cavallo D. Apolipoprotein B is intracellularly associated with an ER-60 protease homologue in HepG2 cells. J Biol Chem 272, 22489-22494 (1997). Ashraf Y,Duval S,Sachan V,Essalmani R,Susan-Resiga D,Gerhardy S,Kirchhofer D,Tagliabracci VS,Prat A,Kiss RS,Seidah NG.“Proprotein Convertase 7(PCSK7)Reduces apoA-V Levels”.FEBS J.287:3565-3578(2020). Ben Djoudi Ouadda A,et al.Ser-Phosphorylation of PCSK9(Proprotein Convertase Subtilisin-Kexin 9)by Fam20C (Family With Sequence Similarity 20,Member C)Kinase Enhances Its Ability to Degrade the LDLR(Low-Density Lipoprotein Receptor).Arterioscler Thromb Vasc Biol 39,1996-2013(2019). Conlon DM,et al.Inhibition of apolipoprotein B synthesis stimulates endoplasmic reticulum autophagy that prevents steatosis.J Clin Invest 126,3852-3867 (2016). Durand L,et al.The motif EXEXXXL in the cytosolic tail of the secretory human proprotein convertase PC7 regulates its trafficking and cleavage activity.J Biol Chem 295,2068-2083(2020). Essalmani R,et al.Furin is the primary in vivo convertase of angiopoietin-like 3 and endothelial lipase in hepatocytes.J Biol Chem 288,26410-26418(2013). Guillemot J,Canuel M,Essalmani R,Prat A,Seidah NG.Implication of the Proprotein Convertases in Iron Homeostasis: Proprotein Convertase 7 Sheds Human Transferrin Receptor 1 and Furin Activates Hepcidin.Hepatology 57,2514-2524(2013). Kramer,M.F.(2011).Stem-loop RT-qPCR for miRNAs.Current protocols in molecular biology 95,15.10.11-15.10.15.doi.10.1002 / 0471142727.mb1510s95. Wetsel WC,et al.Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice.Proc Natl Acad Sci U S A 110,17362-17367(2013). Xiao,et al.The Guideline of the Design and Validation of MiRNA Mimics.J Cell Physiol 212:285-292,2007.

Claims

1. 1. A method for reducing hepatic triglyceride levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a proprotein convertase 7 (PC7) inhibitor.

2. The method of claim 1, wherein the inhibitor is an anti-PC7 antisense oligonucleotide (ASO), an anti-PC7 microRNA (miRNA) or an anti-PC7 small interfering RNA (siRNA).

3. The method of claim 2 , wherein the inhibitor is an ASO.

4. The method of claim 3 , wherein the ASO targets a human PC7 RNA DNA transcript, preferably an exon.

5. The method of claim 2 , wherein the inhibitor is an siRNA.

6. The method of claim 2 , wherein the inhibitor is a miRNA.

7. The method of claim 6, wherein the miRNA targets one or more of human PCSK7 3'-UTR, PCSK7 exon 14, or PCSK7 exon 15.

8. The method of any one of claims 1 to 7, wherein the subject has non-alcoholic fatty liver disease (NAFLD) or is a potential candidate for NAFLD.

9. 9. The method of claim 8, wherein the subject has or is a candidate for nonalcoholic steatohepatitis (NASH).

10. The method of any one of claims 1 to 7, wherein the method further reduces at least one of the following: steatosis score, liver inflammation, hepatocyte ballooning, and liver fibrosis score.

11. The method according to any one of claims 1 to 10, wherein the subject is a human.

12. 1. A kit for reducing hepatic triglyceride levels in a subject, comprising: (A) a proprotein convertase 7 (PC7) inhibitor; and (B)(i) another agent for the prevention or treatment of nonalcoholic fatty liver disease (NAFLD) or a symptom thereof; (ii) a pharma- ceutically acceptable carrier; (iii) instructions for using the kit to reduce liver triglyceride levels; or (iv) a combination of at least two of (i) to (iii).

13. The kit of claim 12, wherein the PC7 inhibitor is (a) an anti-PC7 antisense oligonucleotide (ASO), (b) an anti-PC7 microRNA (miRNA) against PC7, or (c) an anti-PC7 small interfering RNA (siRNA).

14. The kit of claim 13 , wherein the ASO or the siRNA is specific to human PC7.

15. 1. A composition comprising: (A) a proprotein convertase 7 (PC7) inhibitor; and (B) (i) another agent for the prevention or treatment of nonalcoholic fatty liver disease (NAFLD) or a symptom thereof; (ii) a pharma- ceutically acceptable carrier; or (iv) a combination of at least two of (i)-(iii).

16. The composition of claim 15, wherein the PC7 inhibitor is (a) an anti-PC7 antisense oligonucleotide (ASO) specific for PC7, (b) an anti-PC7 microRNA (miRNA), or (c) an anti-PC7 small interfering RNA (siRNA).

17. The composition of claim 16, wherein the ASO, miRNA, or siRNA is specific to human PC7.

18. The composition of claim 17 , wherein the ASO targets a human PC7 RNA transcript, preferably an exon.

19. The composition of claim 17 , wherein the inhibitor is an siRNA.

20. The method of claim 17, wherein the inhibitor is a miRNA.

21. 18. The method of claim 17, wherein the miRNA targets one or more of human PCSK7 3'-UTR, PCSK7 exon 14, or PCSK7 exon 15.

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