Methods for treating liver fibrosis and nonalcoholic fatty liver disease

Abstract

This article discloses compositions and methods for treating subjects with non-alcoholic fatty liver disease or at risk of developing non-alcoholic fatty liver disease.

Description

[0001] sequence list

[0002] This application contains a sequence list, which has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on February 12, 2025, is named "51595-006WO2_Sequence_Listing_2_12_25", and has a size of 77,059 bytes. Technical Field

[0003] This disclosure relates to the treatment of non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). Background Technology

[0004] Nonalcoholic fatty liver disease (NAFLD) (also known as fatty liver disease (SLD), metabolic dysfunction-associated fatty liver disease (MAFLD), or metabolic dysfunction-associated fatty liver disease (MASLD)) is a growing health problem affecting approximately one-third of adults and a growing population of children in developed countries. The early stages of the disease are characterized by the accumulation of lipids in hepatocytes during a process called hepatic steatosis. As lipids (e.g., triglycerides) continue to accumulate in hepatocytes, the early stages of nonalcoholic fatty liver (NAFLD) can progress to nonalcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH), a further condition characterized by hepatocyte damage and liver inflammation. Both NAFLD and NASH can lead to the activation of hepatic stellate cells, which stimulates the deposition of extracellular matrix (ECM) components. This deposition of ECM components characterizes liver fibrosis, which causes the liver tissue to scar and thicken, often leading to further liver damage that can result in cirrhosis, where damaged and dying liver cells are replaced by scar tissue in the later stages of cirrhosis. Current treatment options include dietary modifications, weight loss, bariatric surgery, treatment of comorbidities, and additionally, oral administration of the thyroid hormone receptor-beta agonist resmetirom (CAS No. 920509-32-6) for NASH. For individuals with advanced liver inflammation, steatosis, and / or fibrosis, liver transplantation may be the only viable treatment. Therefore, therapeutic interventions remain necessary to treat NAFLD (e.g., NAFL and NASH). Summary of the Invention

[0005] In one aspect, this disclosure is characterized by a method for treating a human subject identified as having non-alcoholic fatty liver disease (NAFLD), the method comprising the step of administering to the subject an inhibitor of a target listed in Table 1 (e.g., an effective amount of the inhibitor).

[0006] In some embodiments, the targets listed in Table 1 are hepatocyte type A receptor 3 (EPHA3). In some embodiments, the targets listed in Table 1 are integrin subunit αV (ITGAV). In some embodiments, the targets listed in Table 1 are neuronal navigator 2 (NAV2).

[0007] On the other hand, this disclosure features a method for treating a human subject identified as being at risk of developing NAFLD, the method comprising administering to the subject an inhibitor (e.g., an effective amount of the inhibitor) of a target listed in Table 1. In some embodiments, the target listed in Table 1 is EPHA3. In some embodiments, the target listed in Table 1 is ITGAV. In some embodiments, the target listed in Table 1 is NAV2.

[0008] In some embodiments, the subject has liver fibrosis. In some embodiments, the liver fibrosis is stage 0 fibrosis (F0), stage 1 fibrosis (F1), stage 2 fibrosis (F2), stage 3 fibrosis (F3), or stage 4 fibrosis (F4).

[0009] In some embodiments, the NAFLD is non-alcoholic fatty liver disease (NAFL). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the subject has previously been diagnosed with NAFL or NASH. In other embodiments, the subject has not previously been diagnosed with NAFL or NASH.

[0010] In some embodiments of any of the foregoing aspects, the inhibitor is delivered to the liver of the subject. In some embodiments, the inhibitor is delivered to the hepatic stellate cells of the subject.

[0011] In another aspect, this disclosure features a method for reducing the deposition of one or more extracellular matrix (ECM) components in the liver of a human subject, the method comprising the step of administering to the subject an inhibitor (e.g., an effective amount of the inhibitor) of a target listed in Table 1. In some embodiments, the target listed in Table 1 is EPHA3. In some embodiments, the target listed in Table 1 is ITGAV. In some embodiments, the target listed in Table 1 is NAV2.

[0012] In some embodiments, the one or more extracellular matrix components are selected from the group consisting of collagen, hyaluronic acid, laminin, fibronectin, fibrodysregulatory kinase, and elastin.

[0013] In some embodiments, the method further reduces hepatic steatosis. In some embodiments, the method further reduces the number of lipid droplets in the liver cells (e.g., hepatocytes) of the human subject. In some embodiments, the method reduces the lipid droplet area in the liver cells (e.g., hepatocytes) of the human subject. In some embodiments, the liver cells are hepatocytes. In some embodiments, the method further reduces liver inflammation.

[0014] On the other hand, this disclosure features a method for treating liver fibrosis in a human subject identified as having NAFLD (e.g., NAFL or NASH) or at risk of developing NAFLD, the method comprising administering to the subject an inhibitor (e.g., an effective amount of the inhibitor) of a target listed in Table 1. In some embodiments, the target listed in Table 1 is EPHA3. In some embodiments, the target listed in Table 1 is ITGAV. In some embodiments, the target listed in Table 1 is NAV2.

[0015] In some embodiments, the subject has NASH or NAFL.

[0016] In some embodiments, the subject has >5% hepatic steatosis. In some embodiments, the subject has ≤5% hepatic steatosis. In some embodiments, the subject has liver fibrosis. In some embodiments, the subject does not have liver fibrosis. In some embodiments, the liver fibrosis is F0, F1 (e.g., F1A, F1B, or F1C), F2, F3, or F4.

[0017] In some embodiments of any of the foregoing aspects, the inhibitor is an agent listed in Table 2. In some embodiments, the agents listed in Table 2 are small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotides (ASO), gapmeR, mRNA-encoded proteins, soluble proteins or antibodies, small molecules, CRISPR-associated (CAS) proteins, transcription activator-like effector nucleases (TALEN), or zinc finger nucleases. In some embodiments, the inhibitor is selected from the group consisting of: (i) siRNA; (ii) ASO; (iii) small molecules listed in Table 3 (e.g., AWL-II.38.3 or bexetograst); and (iv) antibodies listed in Table 3 (e.g., ifabotuzumab). In some embodiments, the siRNA is a siRNA listed in Table 4 (e.g., siRNA having the nucleotide sequence of any one of SEQ ID NO: 9-32).

[0018] In some embodiments of any of the foregoing aspects, the method further includes the step of administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of: (i) siRNAs listed in Table 4; (ii) ASO; (iii) small molecules listed in Table 3 (e.g., AWL-II.38.3 or bexetograst); and (iv) antibodies listed in Table 3 (e.g., isofluruzumab). In some embodiments, the additional therapeutic agent is an inhibitor of a different target (e.g., EPHA3, ITGAV, or NAV2) listed in Table 1, such that two or more of EPHA, ITGAV, and NAV2 are targeted by the inhibitor. In some embodiments of any of the foregoing aspects, the method includes the step of administering two or more therapeutic agents (e.g., two or more inhibitors) to the subject to target two or more of EPHA3, ITGAV, and NAV2. In some embodiments, the method includes the step of administering two or more therapeutic agents to the subject to target EPHA3 and ITGAV. In some embodiments, the method includes administering two or more therapeutic agents to the subject to target EPHA3 and NAV2. In some embodiments, the method includes administering two or more therapeutic agents to the subject to target ITGAV and NAV2. In some embodiments, the method includes administering three or more therapeutic agents to the subject to target all three of EPHA3, ITGAV, and NAV2.

[0019] In some embodiments of any of the foregoing aspects, the inhibitor is a repressive nucleic acid molecule targeting a target, such as siRNA that reduces the expression or activity of the target by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more relative to a reference). In some embodiments, the repressive nucleic acid molecule targets EPHA3. In some embodiments, the repressive nucleic acid molecule targets ITGAV. In some embodiments, the repressive nucleic acid molecule targets NAV2.

[0020] In some embodiments of any of the foregoing aspects, the inhibitor is administered parenterally (e.g., intramuscularly, subcutaneously, or intravenously). In some embodiments, the inhibitor is administered in an amount sufficient to reduce the subject's liver fibrosis (e.g., a reduction of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more relative to a reference). In some embodiments, administration of the inhibitor further reduces the subject's hepatic steatosis, liver inflammation, and / or hepatocellular ballooning degeneration (e.g., a reduction of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more relative to a reference).

[0021] In some embodiments of any of the foregoing aspects, the inhibitor includes a target portion of a delivery medium (e.g., lipid nanoparticles (LNP)) or a conjugate (e.g., covalently or non-covalently conjugate).

[0022] definition

[0023] Unless otherwise defined herein, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Unless the context otherwise requires, singular terms shall include plural and plural terms shall include singular. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including”, and other forms such as “includes” and “included”, is not restrictive.

[0024] As used herein, unless otherwise stated or clearly indicated from the context, when applied to one or more target values, the term “about” means a value falling within 10% of either direction (greater or less) of the stated reference value (except where this number would exceed 100% of the possible value).

[0025] As used herein, “administration” means the delivery or administration of a drug to a subject by any effective route (e.g., the drugs listed in Tables 2, 3, or 4). Exemplary routes of administration are oral, parenteral, intravenous, subcutaneous, or intramuscular administration.

[0026] As used herein, the term "antagonist" refers to an agent (e.g., antibody, small molecule, or soluble protein) that reduces or inhibits the activity of a target molecule (e.g., targets in Table 1) by directly binding to or directly modulating the target molecule. For example, in embodiments where the target molecule is a receptor, the antagonist may reduce receptor activity by directly binding to the receptor, by blocking the receptor binding site, or by modulating the receptor conformation (e.g., maintaining the receptor in a closed or inactive state). The antagonist reduces the activity of the target molecule by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a reference.

[0027] As used herein, the term "antibody" refers to a molecule that specifically binds to or responds to an immune response to a particular antigen, and includes at least a variable domain of the heavy chain, and typically includes at least variable domains of both the heavy and light chains of an immunoglobulin. Antibodies and their antigen-binding fragments, variants, or derivatives include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2), Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and antibodies containing V... L or V H Fragments of structural domains, fragments generated via Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. The antibody molecules of this invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) means comprising the complete molecule and an antibody fragment capable of specifically binding to a target protein (e.g., Fab and F(ab')2 fragments). Fab and F(ab')2 fragments lack the Fc fragment of the complete antibody.

[0028] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin can be achieved by fragments of a full-length antibody. Antibody fragments can be Fab, F(ab')2, scFv, SMIP, biantibodies, triantibodies, affinity molecules, nanobodies, aptamers, or domain antibodies. Examples of binding fragments covered by the term "antigen-binding fragment" of antibodies include, but are not limited to: (i) Fab fragments, which are composed of V... L V H C L and C H(ii) A monovalent segment composed of a structural domain; (iii) A bivalent segment consisting of two Fab segments connected by a disulfide bridge in the hinge region; (iv) A segment composed of V H and C H (iv) Fd fragment composed of 1 domain; V of antibody single arm L and V H The Fv segment, composed of structural domains, (v) dAb (Ward et al., Nature [Nature] 341:544-546, 1989), includes V H and V L Structural domain; (vi) by V H The dAb segment composed of structural domains; (vii) composed of V H or V L (viii) dAb composed of structural domains; and (ix) separate complementarity-determining regions (CDRs); and (ix) combinations of two or more separate CDRs, which may optionally be connected by a synthetic joint. Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by different genes, but they can be linked together via adapters using recombination methods, enabling them to be made into a single protein chain, where V L and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs)). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the use of the fragments can be screened in the same manner as for intact antibodies. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0029] As used herein, in the context of a disease or condition (e.g., NAFLD, NAFL, or NASH), the term “risk of developing…” refers to a state in which a human subject has an increased likelihood or probability of developing a disease or condition during their lifetime. For example, a subject has an increased risk of developing NAFLD (e.g., NAFL or NASH) if one or more of the following characteristics are present (e.g., 1, 2, 3, 4, 5, 6 or more): the subject has a metabolic or hormonal disorder (e.g., type 2 diabetes, insulin resistance, hypothyroidism, hypopituitarism, polycystic ovary syndrome, growth hormone deficiency, hyperuricemia, or dyslipidemia); the subject has hypertension; the subject has gout; the subject has had their gallbladder removed; the subject has completed menopause; the subject has elevated serum levels of type III procollagen peptide (PICP) (e.g., greater than 5.0 ng / mL), hyaluronic acid (e.g., greater than 40 ng / mL), tissue inhibitor of metalloproteinases 1 (TIMP-1) (e.g., greater than 2.1 ng / mL), deintegrin and metalloproteinases and platelet-reactive protein motif-like protein 2 (ADAMTSL2) (e.g., greater than 1.5 μg / mL), and aldo-keto reductase family 1 member B10 (AKR1B10) (e.g., greater than 90 μg / mL). The following substances are present in the subject: complement factor H-related protein 4 (CFHR4) (e.g., greater than 1.1 μg / mL), trigger receptor 2 expressed on myeloid cells (TREM2) (e.g., greater than 65 ng / mL), chitinase 3-like protein 1 (YKL-40) (e.g., greater than 80 ng / mL), retinol-binding protein 4 (RBP4) (e.g., greater than 1.7 μg / mL), leptin (LEP) (e.g., greater than 24 ng / mL), resistin (RETN) (e.g., greater than 23 ng / mL), or gastroglucan (GHRL) (e.g., greater than 170 fmol / mL), or reduced levels of adiponectin (ADIPOQ) (e.g., less than 7 μg / mL); the subject has altered liver transaminase levels (e.g., alanine transaminase (ALT) greater than about 25 U / L), such as an elevated or altered AST / ALT ratio (e.g., less than 1 or greater than or equal to 2); the subject has altered cholesterol levels, such as high circulating levels of free fatty acids (e.g., about 1 pg / mL). Subjects with high circulating levels of low-density lipoprotein (LDL) cholesterol (e.g., 1200 mg / L or higher) or low circulating levels of high-density lipoprotein (HDL) cholesterol (e.g., 500 mg / L or lower); subjects with high triglyceride levels (e.g., greater than 150 mg / dL); subjects with high uric acid levels (e.g., ≥ 4).The following criteria must be met: 1) The subject is taking amiodarone (CORDARONE®), diltiazem (CARDIZEM®), tamoxifen (NOLVADEX®), or a steroid; 2) The subject has a genetic predisposition, such as mutations or polymorphisms in genes associated with NAFLD (e.g., PNPLA3, TM6SF2, SAMM-50, FDFT1, COL13A1, NCAN, GCKR, SREBF2, MBOAT7-TMC4, and / or HSD17B13); 3) The subject has poor eating habits, poor exercise habits, sleep deprivation, or obstructive sleep apnea; 4) The subject experiences symptoms of fatigue, loss of appetite, weight loss, or jaundice; 5) The subject has a body weight ≥ 30 kg / m². 2 Body mass index (BMI); subjects had large waist circumferences (e.g., approximately ≥80 cm for men and ≥78 cm for women), with most body fat located in the abdomen; and Parameswaran et al., Cureus Other risk factors disclosed in , 13(12):e20776(2021), which are incorporated herein by reference and described herein. The more of these factors a patient has, the greater the patient is “at risk of developing…”. Although subjects identified as “at risk of developing NAFL” have not been clinically diagnosed with NAFLD, they may have additional signs of NAFLD in addition to those mentioned above, such as those determined by biopsy of one or more tissues (e.g., from the liver) to detect or measure fibrosis, inflammation, ballooning degeneration, and / or steatosis; magnetic resonance elastography (MRE) to detect or measure fibrosis; ultrasound (e.g., FIBROSCAN®) to detect liver fibrosis and / or steatosis; or magnetic resonance imaging (MRI) to detect or measure steatosis. Subjects “at risk of developing NASH” may have previously been diagnosed with NAFL. The diagnosis of NAFL can be based on diagnostic methods known in the art, including, for example, biopsy of one or more tissues (e.g., from the liver), magnetic resonance imaging (MRI) to detect or measure steatosis, magnetic resonance elastography (MRE) to detect or measure fibrosis, ultrasound (e.g., FIBROSCAN® to detect fibrosis and / or steatosis), or evaluation of one or more biomarkers (e.g., from enhanced liver fibrosis (ELF) tests, FibroMeter tests, FIBROTEST®, or other biomarkers described herein).

[0030] As used herein, the term "biological sample" refers to a sample obtained from any one or more cells, tissues (e.g., biopsy), organs, or extracellular fluids of a subject. Biological samples may be obtained, for example, from the following: blood, plasma, serum, liver, kidney, lung, skin, muscle, heart, brain, saliva, mucus, hepatic stellate cells, hepatocytes, bile epithelial cells, bile duct cells, Kupffer cells, macrophages, hepatic sinusoidal endothelial cells, erythrocytes, leukocytes, parietal epithelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, neurons, glial cells, osteoblasts, adipocytes, oocytes, and / or sperm.

[0031] As used herein, the term "hepatic stellate cell" refers to resident hepatocytes that play a central role in liver injury and fibrosis. In healthy liver tissue, hepatic stellate cells comprise approximately 10-15% of all hepatocytes. Unbound by any particular theory, activation of hepatic stellate cells (e.g., via signal transduction induced by pro-inflammatory mediators) contributes to fibrosis in the liver (i.e., the deposition of extracellular matrix (ECM) components). Activated hepatic stellate cells exhibit a relative increase in the expression of biomarkers or receptors (e.g., intracellular or surface expression) compared to quiescent hepatic stellate cells. Biomarkers or receptors with increased expression in activated hepatic stellate cells include, for example, α-smooth muscle actin (α-SMA), type VI collagen receptor, retinol-binding protein (RBP) receptor, platelet-derived growth factor receptor (PDGFR), synaptic growth proteins (e.g., major synaptic vesicle protein p38), insulin-like growth factor-II receptor (IGFIIR), low-density lipoprotein receptor (LDLR), TNF-associated apoptosis-inducing ligand receptor (TRAILR), integrins (e.g., αvβ3), and / or differentiation cluster 44 (CD44). Relative expression levels can be measured by nucleotide expression (e.g., DNA or RNA) or protein expression using conventional methods in the art (e.g., RT-PCR, qPCR, RNA-seq, RNA blot analysis, protein blot analysis, ELISA, flow cytometry, immunofluorescence, in situ hybridization, antibody-based detection, nucleic acid-based detection using hybridized nucleotides (e.g., aptamer-based detection), microscopy, mass spectrometry, and other conventional methods). Hepatic stellate cells can undergo morphological changes upon activation, as observed by Puche et al. Compr Physiol. [Comprehensive Physiology] 3(4):1473-92, 2013 and Hoffmann et al., Sci Rep. [Scientific Report] ,Those described in 10(1):3850, 2020, each of which is incorporated herein by reference in its entirety. Activated hepatic stellate cells may appear larger or flatter, become highly contractile (e.g., develop stress fibers or contractile filaments), and have a reduced number of vacuoles compared to quiescent hepatic stellate cells. Hepatic stellate cell morphology can be measured or quantified by microscopy.

[0032] As used herein, an “effective amount” of a pharmaceutical agent or composition means an amount sufficient to produce a beneficial or desired outcome (including clinical outcomes) when administered to a subject (e.g., in vivo, for example, a human) or to the subject’s cells (e.g., ex vivo) and therefore, “effective amount” or its synonyms depend on the context in which they are applied. For example, in the context of treating NAFLD (e.g., NAFL or NASH), it is the amount of the pharmaceutical agent or composition described herein sufficient to achieve a therapeutic remission compared to a remission achieved without the administration of the composition. The amount of a given composition described herein corresponding to this amount will vary depending on a variety of factors, such as the given pharmaceutical agent, pharmaceutical formulation, route of administration, type of disease or disorder, the subject’s identity (e.g., age, sex, weight), or the host receiving treatment, but can still be routinely determined by those skilled in the art. Similarly, as used herein, a “therapeutic effective amount” of a composition disclosed herein is an amount that produces a beneficial or desired outcome in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition disclosed herein can be readily determined by those skilled in the art using conventional methods known in the art. Dosage regimens can be adjusted to provide the best therapeutic response.

[0033] As used herein, “experimental sample” refers to a sample (e.g., a biological sample or clinical image) to be compared with a reference sample. Experimental samples may be obtained from subjects who are at risk of developing NAFLD (e.g., developing NAFL or NASH), at risk of NAFLD progression (e.g., developing NASH), and / or who already have NAFLD (e.g., NAFL or NASH).

[0034] As used herein, the term “inhibitor” refers to an agent that inhibits or reduces gene expression, protein activity, and / or signal transduction of a target (e.g., targets in Table 1). Inhibitors include antagonists that interact directly with the target molecule and other therapeutic agents that reduce the expression, activity, or signal transduction of a target molecule through interactions with its binding chaperone, upstream regulator, or molecules that mediate downstream signal transduction. Exemplary inhibitors include repressive nucleic acid molecules (e.g., small interfering RNA (siRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotides (ASO), or gapmeR); repressive antibodies; soluble proteins (e.g., proteins that disrupt the activity of a target, such as dominant-negative proteins corresponding to the target or one of its binding chaperones); RNA molecules (e.g., mRNA) encoding repressive proteins that can disrupt the function of a target molecule; small molecule inhibitors; and programmable nucleases (e.g., CRISPR-Cas endonucleases programmed to cleave DNA or RNA encoding a target) for editing and inhibiting gene expression. Inhibitors of specific targets can disrupt the expression, function and / or signal transduction of the target by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more).

[0035] As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than within an organism (e.g., an animal, plant, or microorganism).

[0036] As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, plant, or microorganism, or its cells or tissues).

[0037] As used herein, “modified” and “modified nucleic acid molecule” refer to an altered state or structure of the nucleic acid molecule described herein. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one embodiment, the repressive nucleic acid molecule of this disclosure (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, or gapmeR) is modified by introducing a non-natural nucleoside and / or nucleotide. In other embodiments, the repressive nucleic acid molecule of this disclosure (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, or gapmeR) is modified by conjugating an auxiliary portion (e.g., a targeting portion as described herein). In other embodiments, the RNA molecule of this disclosure (e.g., a protein-encoding mRNA molecule) is modified by introducing a non-natural nucleoside and / or nucleotide. Modified nucleic acid molecules in this disclosure are presented as having the nucleotides adenine (A), thymine (T), cytosine (C), and guanine (G). Where appropriate, i.e., when a given nucleic acid is an RNA molecule, thymine can be understood as uracil (U).

[0038] As used herein, in the context of a target (e.g., proteins in Table 1), the term "repressive nucleic acid molecule" refers to a nucleic acid molecule that is sufficiently complementary to bind to a nucleic acid encoding a target (e.g., a sequence sufficiently complementary to bind to transcripts (e.g., genes or splice variants thereof) listed in Table 1) and to inhibit or reduce the expression, function, or activity of the target. Exemplary repressive nucleic acid molecules are siRNA, dsRNA, miRNA, shRNA, ASO, and gapmeR. Repressive nucleic acid molecules can reduce the expression or activity of a target by 10% or more relative to a reference (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more). Repressive nucleic acid molecules are presented in this disclosure as having the nucleotides adenine (A), thymine (T), cytosine (C), and guanine (G). Where appropriate, i.e., when the given nucleic acid is an RNA molecule, thymine can be understood as uracil (U).

[0039] As used herein, the term "pharmaceutical composition" refers to a mixture comprising a pharmaceutical agent (optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers) to be administered to a subject.

[0040] As used herein, the term “pharmaceutically acceptable” means, within reasonable medical judgment, compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0041] As used herein, the “sequence identity percentage (%)” of a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after sequence alignment and gap introduction (if necessary) to achieve the maximum sequence identity percentage. Alignment for determining the nucleic acid or amino acid sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. For example, the sequence comparison computer program BLAST can be used to generate sequence identity value percentages. As an illustration, the sequence identity percentage of a given nucleic acid or amino acid sequence A with, compared to, or relative to a given nucleic acid or amino acid sequence B (which can alternatively be expressed as the phrase: a given nucleic acid or amino acid sequence A having a certain sequence identity percentage with, compared to, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows: 100 × (fraction X / Y) Where X is the number of nucleotides or amino acids that are scored as identical matches in the A and B alignments by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It should be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percentage of sequence identity of A relative to B will not be equal to the percentage of sequence identity of B relative to A.

[0042] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to polymers of nucleosides. Typically, polynucleotides consist of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds and naturally occurring in DNA or RNA. The term encompasses molecules containing nucleosides or nucleoside analogues that contain chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. In the case of polynucleotides referred to herein, it should be understood that both DNA and RNA are provided, and in each case both single-stranded and double-stranded forms (and the complement sequence for each single-stranded molecule) are provided. As used herein, “polynucleotide sequence” refers to the polynucleotide material itself or the sequence information (i.e., a series of letters used as a base abbreviation) that characterizes a particular nucleic acid in a biochemical manner. Unless otherwise stated, polynucleotide sequences presented herein are presented in a 5' to 3' orientation. Polynucleotides are presented in this disclosure as having the following nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). Where appropriate, i.e., when the given nucleic acid is an RNA molecule, thymine can be understood as uracil (U).

[0043] As used herein, the terms “reference,” “reference sample,” and “control sample” are used interchangeably to describe predetermined reference values ​​(e.g., diagnostic reference levels) for samples (e.g., biological samples or clinical images) obtained from subjects. In some cases, the reference is derived from individuals who have been determined not to have NAFLD (e.g., NAFL and / or NASH) and / or have a hepatic steatosis value of less than 5% and a value of less than 30 kg / m². 2 The reference is obtained from the subject's BMI. In some cases, the reference is obtained from a subject who had or exhibited signs and symptoms of NAFLD prior to undergoing the treatment described herein. In this case, the reference serves as an initial reference value for the future value to be compared (e.g., from the experimental sample). The reference may be derived from a sample, such as a biological sample (e.g., a biopsy) or a clinical image (e.g., an image from MRE, ultrasound (e.g., FIBROSCAN®), or MRI). The reference may be obtained from a reference value or set of reference values ​​in a given reference chart (e.g., one or more MRE values, ultrasound values, biomarker levels (e.g., from the enhanced liver fibrosis (ELF) test, FibroMeter test, or FIBROTEST®), and / or histopathological values). The reference should be obtained from the same sample type as the experimental sample (e.g., blood, plasma, serum, liver, kidney, muscle, etc.). The reference will be used as a reference point to determine the efficacy of the treatment described herein.

[0044] As used herein, the terms “relative to reference,” “relative to reference sample,” and “relative to control sample” are used interchangeably to describe a comparison between an experimental sample and a reference sample (e.g., a reference value or reference). For example, a comparison between an experimental sample and a reference (e.g., fold change) can be constructed using the following formula and can be used to determine therapeutic efficacy.

[0045]

[0046] As used herein, the term "sample" refers to an organ (e.g., liver), a subset of its tissues (e.g., biopsy), a cellular or component portion (e.g., bodily fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, saliva, and hair), a homogenate, a lysate or extract prepared from an organism or a subset of its tissues, cells, or components, or an image (e.g., clinical image, such as MRI or ultrasound, or histopathological image, such as histological staining) of an organ (e.g., liver), tissue (e.g., biopsy), or cell (e.g., hepatic stellate cells; e.g., activated hepatic stellate cells). A sample further refers to a culture medium, such as a nutrient broth or gel, which may contain cellular components, such as protein or nucleic acid molecules.

[0047] As used herein, the terms “subject” and “patient” refer to an animal (e.g., a mammal, such as a human). A subject being treated according to the methods described herein may be a subject who has been diagnosed with NAFLD or a subject at risk of developing this condition. Diagnosis can be made by any method or technique known in the art or by the methods described herein. Those skilled in the art will understand that a subject to be treated according to this disclosure may have already undergone standard testing or may not require examination of a subject who has been identified as being at risk due to the presence of one or more risk factors associated with the disease or condition.

[0048] The term "synthesis" means artificially produced, prepared, and / or manufactured. The synthesis of polynucleotides, polypeptides, or other molecules disclosed herein can be chemical or enzymatic synthesis.

[0049] As used herein, the terms “non-alcoholic fatty liver disease,” “NAFLD,” “fatty liver disease,” “SLD,” “metabolic dysfunction-associated fatty liver disease,” “MAFLD,” “metabolic dysfunction-associated fatty liver disease,” and “MASLD” refer to conditions in which fat (e.g., lipids) accumulates in the liver due to causes other than excessive alcohol consumption. Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are two types of NAFLD. In some cases, in either of these conditions, fat accumulation in the liver (i.e., steatosis) is accompanied by liver inflammation, hepatocellular ballooning degeneration, and / or liver fibrosis. Steatosis (as defined by the presence of lipid droplets in more than 5% of hepatocytes (e.g., hepatocytes)) is the primary criterion for diagnosing NAFLD (e.g., NAFL or NASH).

[0050] As used in this article, the terms “non-alcoholic fatty liver disease” and “NAFL” refer to types of NAFLD characterized by the accumulation of fat in the liver (i.e., steatosis). In some cases, NAFL is accompanied by liver fibrosis.

[0051] As used in this article, the terms “non-alcoholic steatohepatitis” and “NASH” refer to a type of NAFLD characterized by the accumulation of fat in the liver (i.e., steatosis), liver inflammation, and ballooning degeneration of hepatocytes. In some cases, NASH is accompanied by liver fibrosis.

[0052] As used herein, the terms “liver fibrosis,” “hepatic fibrosis,” and “fibrosis” refer to the scarring of liver tissue caused by the deposition of extracellular matrix (ECM) components due to cellular processes (e.g., activated hepatic stellate cells and / or myofibroblasts). Exemplary ECM components include proteins and proteoglycans, such as collagen (e.g., type I, III, and IV collagen), hyaluronic acid, laminin, fibronectin, crude fibroregulatory substances, and elastin. Liver fibrosis can be classified into stages F0 to F4, where F0 represents no detectable fibrosis, F1 represents mild fibrosis, F2 represents significant or moderate fibrosis, F3 represents advanced fibrosis, and F4 represents cirrhosis. Alternatively, fibrosis stages can be classified based on the NAFLD Activity Score (NAS). According to the NAS score, stage 1 fibrosis indicates perisinusoidal or periportal fibrosis, and is further subclassified based on mild to moderate perisinusoidal fibrosis (e.g., stage 1A or 1B) or periportal fibrosis (e.g., stage 1C); stage 2 fibrosis indicates perisinusoidal and periportal or periportal fibrosis; stage 3 fibrosis indicates bridging fibrosis; and stage 4 indicates cirrhosis.

[0053] As used herein, the terms “pharmaceutical” and “therapeutic agent” refer to a pharmaceutical agent that can be used to treat patients with NAFLD or at risk of developing NAFLD (e.g., at risk of having NAFL, at risk of having NASH, having NAFL, or having NASH). Therapeutic agents used in the methods described herein are provided in Tables 2, 3, and 4 and are considered to be inhibitors of the targets listed in Table 1 (e.g., antibodies, small molecules, RNA molecules (such as inhibitory RNA molecules or mRNA molecules encoding proteins), components of gene editing systems, and protein therapeutic agents). When administered to a subject, the therapeutic agent has a therapeutic and / or prophylactic effect and / or induces the desired biological and / or pharmacological effect.

[0054] As used herein, “treatment” means a method for obtaining at least one of the following beneficial or desired outcomes in a subject (e.g., a human subject): slowing or inhibiting the progression of NAFLD in a subject identified as having NAFLD (e.g., NAFL or NASH); reversing the progression of NAFLD in a subject identified as having NAFLD (e.g., NAFL or NASH); promoting the maintenance of NAFLD in a subject identified as having NAFLD (e.g., NAFL or NASH); slowing or inhibiting the onset of NASH in a subject identified as having NAFLD (e.g., NAFL); alleviating or improving one or more symptoms of NAFLD (e.g., fatigue, weight loss, and / or jaundice) in a subject identified as having NAFLD (e.g., NAFL or NASH); reducing one or more clinical manifestations of NAFLD (e.g., NAFL or NASH) in a subject identified as having NAFLD (e.g., NAFL or NASH). The treatment aims to: reduce or suppress the onset of NAFL in subjects identified as being at risk of developing NAFLD; reduce or improve one or more symptoms of NAFLD (e.g., fatigue, weight loss, and / or jaundice) in subjects identified as being at risk of developing NAFLD (e.g., NAFL or NASH); reduce or suppress the progression of liver fibrosis, hepatic steatosis, and / or liver inflammation in subjects in need (e.g., subjects identified as having NAFLD (e.g., NASH) or at risk of developing NAFLD); reverse the progression of liver fibrosis, hepatic steatosis, and / or liver inflammation in subjects in need (e.g., subjects identified as having NAFLD (e.g., NASH) or at risk of developing NAFLD); and promote the maintenance of liver fibrosis, hepatic steatosis, and / or liver inflammation in subjects in need (e.g., subjects identified as having NAFLD (e.g., NASH) or at risk of developing NAFLD). "Relief," "improvement," "reduction," "reversal," or "slowing down" refers to a reduction in the severity, clinical manifestations, or symptoms of the disease, resulting in a slower progression of the disease compared to the absence of treatment. "Treatment" can also refer to prolonging liver function and / or survival in a subject compared to the absence of treatment. The treatments described herein can improve, reduce, reverse, or slow the secretion of extracellular matrix components in the liver of subjects (e.g., subjects at risk of developing NAFLD or subjects with NAFLD), as measured by liver biopsy or other biomarkers described herein.The treatments described herein can reduce the secretion of one or more extracellular matrix components (e.g., collagen, hyaluronic acid, laminin, fibronectin, crude fibroregulatory integrin, and elastin) in the liver of human subjects (e.g., subjects at risk of developing NAFLD or subjects with NAFLD), reduce liver fibrosis in subjects (e.g., subjects at risk of developing NAFLD or subjects with NAFLD), reduce the number (e.g., average number) and / or area (e.g., average area) of lipid droplets present in one or more hepatocytes (e.g., hepatocytes (HC)) in subjects (e.g., subjects at risk of developing NAFLD or subjects with NAFLD), reduce liver inflammation in subjects (e.g., subjects at risk of developing NAFLD or subjects with NAFLD), and / or reduce hepatocyte ballooning (e.g., in subjects at risk of developing NAFLD or subjects with NAFLD). Attached Figure Description

[0055] Figure 1 This is a representative graph showing the efficiency of siRNA knockdown of target genes. Compared to the control, knockdown of target hepatocyte glycoprotein receptor 3 (EPHA3) with siRNA resulted in reduced EPHA3 expression, among which... yes p Value < 0.05.

[0056] Figure 2 This is a representative diagram showing reduced hepatic stellate cell (HSC) activation after siRNA knockdown of the target gene. Knockdown of the target EPHA3 with siRNA resulted in decreased stellate cell activation, as measured by procollagen 1A1 (Pro-COL1A1) secretion, in which… yes p Value < 0.05.

[0057] Figure 3 This is a representative graph showing the reduction in hepatic stellate cell activation after treatment with a targeted small molecule inhibitor. Inhibition of EPHA3 with the small molecule inhibitor AWL-II.38.3 at increased concentrations resulted in decreased stellate cell activation, as measured by procollagen 1A1 secretion. yes p Value < 0.05, yes p Value < 0.01, and yes p Value < 0.001. Detailed Implementation

[0058] This article describes a method for treating subjects who have been diagnosed with non-alcoholic fatty liver disease (NAFLD) or are at risk of developing NAFLD, such as non-alcoholic fatty liver (NAFLD) or non-alcoholic steatohepatitis (NASH). For example, the methods described herein can be used to treat subjects at risk of developing NAFLD. If the subject has a family history of NAFLD, is obese (body mass index ≥ 30 kg / m²), or is other suitable candidate, this method may be used. 2 Subjects who have comorbidities affecting lipid metabolism may be at risk of developing NAFLD. Alternatively, the methods described herein can be used to treat subjects who have already developed some form of NAFLD, such as NAFL or NASH; such methods can slow, inhibit, or even reverse further disease progression. Standard clinical tests known in the art can be used to diagnose NAFLD (e.g., NAFL or NASH) in subjects, such as: liver biopsy, magnetic resonance imaging (MRI) detecting or measuring steatosis and / or hepatocellular ballooning, magnetic resonance elastography (MRE) detecting or measuring fibrosis, ultrasound detecting or measuring steatosis and / or fibrosis, or other biomarker laboratory results.

[0059] Additionally, this document provides compositions for use in the methods described herein. Specifically, the compositions described herein contain one or more therapeutic agents (e.g., pharmaceutical agents listed in Tables 2-4), which may include inhibitory nucleic acid molecules, RNA (e.g., mRNA) encoding proteins that can inhibit or activate the targets described herein, soluble proteins (e.g., dominant / negative proteins or soluble target-binding chaperones that can modulate the activity of the targets described herein), small molecules, antibodies, or components of gene editing systems.

[0060] NAFLD

[0061] NAFLD (also known as fatty liver disease (SLD), metabolic dysfunction-associated fatty liver disease (MAFLD), or metabolic dysfunction-associated fatty liver disease (MASLD)) is a condition in which steatosis (e.g., hepatic steatosis) occurs in the liver. Steatosis (also known as fatty changes) is the abnormal retention of lipids (i.e., fat) within cells (e.g., hepatocytes). A subject is diagnosed with NAFLD when the liver (the organ responsible for lipid metabolism) has diagnostic features of steatosis (e.g., hepatic steatosis) that are not caused by excessive alcohol consumption. The early stage of NAFLD (referred to as NAFL in this article) is characterized by hepatic steatosis (e.g., ≥ 5% hepatic steatosis) with little or no liver inflammation. The late stage of NAFLD (referred to as NASH in this article) is characterized not only by hepatic steatosis but also by hepatocyte ballooning and liver inflammation, which can lead to liver damage. Both early and late stages of NAFLD (e.g., NAFL or NASH) may coexist with fibrosis or scarring of tissues. Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins and proteoglycans (e.g., collagen (e.g., type I, III, and IV collagen), hyaluronic acid, laminin, fibronectin, crude fibrodylin, elastin) deposited by cells (e.g., activated hepatic stellate cells or myofibroblasts). In some cases, fibrosis occurs in response to fibrotic cytokines and pro-inflammatory mediators (e.g., transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), chitinase-3-like protein 1 (e.g., YKL-40), microfibrillary-associated protein 4 (MFAP4), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-4, IL-6, endothelin-I, angiotensin II, and reactive oxygen species) in the inflamed liver. The fibrotic environment further upregulates the expression and activity of pro-inflammatory mediators, as well as metalloproteinases (e.g., matrix metallopeptidase-1 (MMP1), MMP2, and MMP3) and protease inhibitors (e.g., tissue inhibitors of metalloproteinases (TIMPs); for example, TIMP-1). Without examination, excessive scarring from fibrosis can lead to cirrhosis, resulting in permanent liver damage and decreased liver function.

[0062] Subjects with NAFLD (e.g., subjects with NAFLD and with or without a diagnosis) may experience symptoms such as fatigue, weight loss, jaundice, fluid retention (e.g., swelling and / or lymphedema), nausea, confusion, pruritus, or a combination thereof. Current methods for identifying subjects as having NAFLD or at risk of having NAFLD include biopsy of one or more tissues (e.g., from the liver), magnetic resonance elastography (MRE) to detect or measure fibrosis, ultrasound to detect fibrosis and / or steatosis (e.g., FIBROSCAN®), and magnetic resonance imaging (MRI) to detect or measure steatosis. Other NAFLD biomarkers and diagnostic modalities have been described elsewhere, such as by Kleiner et al. Hepatology [Hepatology] 41(6):1313-1321, 2005, Martinou et al. Diagnostics [Diagnostics] 12(2):407, 2022, Canivet and Boursier, Diagnostics [Diagnostics] 13(1):91, 2023, and Vali et al. Lancet Gastroenterol. Hepatol.[The Lancet Gastroenterology & Hepatology] 8(8):714-725, 2023, each of which is hereby incorporated by reference. Furthermore, liver fibrosis or its risk can be evaluated in subjects with NAFLD (e.g., subjects with NAFLD and with or without a diagnosis). Liver fibrosis is typically classified into stages based on the severity or accumulation of scar tissue resulting from ECM deposits. Fibrosis can be classified as F0 (no detectable fibrosis), F1 (mild fibrosis), F2 (significant or moderate fibrosis), F3 (advanced fibrosis), or F4 (cirrhosis). Fibrosis stages can be classified based on the NAFLD Activity Score (NAS). According to the NAS scoring method, Stage 1 fibrosis represents perisinusoidal or periportal fibrosis, which is further subdivided based on mild to moderate perisinusoidal fibrosis (e.g., Stage 1A or 1B) or periportal fibrosis (e.g., Stage 1C). Stage 2 fibrosis, based on the NAS score, indicates perisinusoidal and portal vein or periportal fibrosis; Stage 3 fibrosis indicates bridging fibrosis; and Stage 4 indicates cirrhosis. The presence or severity of fibrosis can be based on liver stiffness (e.g., as measured by MRE), histopathological diagnostic methods or other biomarkers (including the fibrotic and pro-inflammatory mediators described herein), and other blood tests routinely used to detect and measure liver fibrosis (e.g., ELF test, FibroMeter, and / or FIBROTEST®). The fibrotic and pro-inflammatory mediators described herein can also be measured in samples (e.g., liver biopsy or blood samples) to determine whether a subject has liver fibrosis or NAFLD (e.g., NAFL or NASH) or is at risk of developing liver fibrosis or NAFLD. Additional factors that can be measured to determine whether a subject has liver fibrosis or NAFLD (e.g., NAFL or NASH) or is at risk of developing liver fibrosis or NAFLD include the expression level or activity of one or more fibrosis byproducts (e.g., type III procollagen peptide (PICP), procollagen III N-terminal propeptide (PIIINP), hyaluronic acid, and TIMP-1), the expression level or activity of one or more coagulation factors (e.g., prothrombin time and platelet count), and / or the expression level of other blood factors that indicate liver function (e.g., α-2 macroglobulin, haptoglobin, apolipoprotein A1, and bilirubin).

[0063] The secretion levels of one or more extracellular matrix components in the liver of subjects with liver fibrosis and / or NAFLD (e.g., NAFL or NASH, with or without a diagnosis) can be evaluated. For example, tissue staining (e.g., immunohistochemistry (IHC) or immunofluorescence) followed by imaging analysis (e.g., with ImageJ or FIJI) of a test sample (e.g., a liver biopsy from a subject) can be used to measure the levels of one or more extracellular matrix components (e.g., collagen (e.g., type I, III, and / or IV collagen), hyaluronic acid, laminin, fibronectin, crude fibroregulatory substances, elastin) relative to a reference sample. In some embodiments, liver fibrosis can be measured via one or more staining agents or probes, such as trichrome staining agents, Sirius red staining agents, histological staining, or probes specific to one or more ECM components (e.g., fluorescent probes or enzyme probes). Imaging analysis can be used to measure: (1) the number (e.g., mean or median) of ECM positive spots adjacent to one or more cells (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more cells, e.g., hepatic stellate cells), and / or (2) the area (e.g., mean or median) of one or more (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more) of ECM positive spots adjacent to one or more cells (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more) of ECM positive spots adjacent to one or more cells (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more) of ECM positive spots adjacent to one or more cells (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more) of ECM positive spots. If the number and / or area of ​​ECM positive spots increases by 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more relative to the reference, an increase in the level of one or more extracellular matrix components can be identified.

[0064] Further evaluation can be performed on the number of lipid droplets and / or lipid area in the liver of subjects with liver fibrosis and / or NAFLD (e.g., NAFL or NASH, with or without a diagnosis). For example, dyes, staining agents, or fluorescent probes (e.g., Biotium LIPIDSPOT) can be used for IHC tissue staining or immunofluorescence and detection of lipids. TM 488, BODIPY493 / 503, Nile Red, or Monodanylpentane can be used to detect the number of cell nuclei and lipid droplets, respectively. Lipid droplet accumulation can be measured by fluorescence microscopy and by analysis, for example, ImageJ or FIJI, to measure: (1) Biotium LIPIDSPOT in one or more cells (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more cells, e.g., hepatocytes).TM The number of 488 positive spots (e.g., mean or median), and / or (2) the area of ​​one or more (e.g., one, five, ten, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more) of stained or fluorescent spots (e.g., mean or median). An increase in the level of lipid droplets or lipid area (e.g., mean or median) can be determined if the number and / or area of ​​stained or fluorescent spots increases by 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more relative to a reference. Alternatively, changes in lipid droplet accumulation can be represented by, for example, a Z-score. A baseline for lipid droplet accumulation corrected for cell density should be calculated. The standard Z-test can determine whether lipid droplet accumulation is significantly higher or lower than that of a reference sample. The Z-score is interpreted as the amount of standard deviation by which the mean of the experimental sample (e.g., a liver biopsy of a subject) deviates from the reference. A negative Z-score reflects a decrease in lipid accumulation or area, while a positive Z-score reflects an increase in lipid accumulation or area.

[0065] Risk factors for NAFLD include other diseases or conditions. In some embodiments, a subject is at risk of developing NAFLD if they have a metabolic or hormonal disorder, such as type 2 diabetes or insulin resistance, hypothyroidism, hypopituitarism, polycystic ovary syndrome, growth hormone deficiency, or dyslipidemia. In some embodiments, a subject may be at risk of developing NAFLD if they have low circulating levels of high-density lipoprotein (HDL) cholesterol of about 500 mg / L or lower (e.g., about 500 mg / L, about 490 mg / L, about 480 mg / L, about 470 mg / L, about 460 mg / L, about 450 mg / L, about 440 mg / L, about 430 mg / L, about 420 mg / L, about 410 mg / L, about 400 mg / L, or below 400 mg / L). In some embodiments, a subject may be at risk of developing NAFLD if they have high circulating levels of low-density lipoprotein (LDL) cholesterol of about 1200 mg / L or higher (e.g., about 1200 mg / L, about 1250 mg / L, about 1300 mg / L, about 1350 mg / L, about 1400 mg / L, about 1450 mg / L, or higher than 1450 mg / L). In some embodiments, a subject at risk of developing NAFLD may have altered liver transaminase levels (e.g., an elevated or altered AST / ALT ratio; for example, an AST / ALT ratio less than 1 or greater than or equal to 2). In some embodiments, a subject may be at risk of developing NAFLD if they have gout or high uric acid levels. In other embodiments, a subject may be at risk of developing NAFLD if they have gallstones or have undergone cholecystectomy. In some embodiments, a subject may be at risk of developing NAFLD if they have a genetic predisposition, such as mutations or polymorphisms in genes associated with NAFLD (e.g., PNPLA3, TM6SF2, SAMM-50, FDFT1, COL13A1, NCAN, GCKR, SREBF2, MBOAT7-TMC4, and / or HSD17B13). In other embodiments, a subject may be at risk of developing NAFLD if they are taking certain medications, such as amiodarone (CORDARONE®), diltiazem (CARDIZEM®), tamoxifen (NOLVADEX®), or steroids. Other risk factors for NAFLD or liver fibrosis are related to a subject's body type or lifestyle. In some embodiments, a subject may be at risk of developing NAFLD if they are over 40 years of age or have completed menopause. In some embodiments, a subject may be at risk of developing NAFLD if they have sarcopenia or skeletal muscle atrophy.In some embodiments, subjects may be at risk of developing NAFLD if they have poor diet, exercise, and sleep habits, or if they have obstructive sleep apnea or sleep deprivation. In some embodiments, subjects may be at risk of developing NAFLD if they have high oxidative stress or gut microbiome dysbiosis. In some embodiments, subjects may be at risk of developing NAFLD if they have ≥ 30 kg / m². 2 Subjects with a high BMI or large waist circumference (e.g., approximately 80 cm or larger for men and approximately 78 cm or larger for women), with most body fat located in the abdomen, may be at risk of developing NAFLD. In some embodiments, subjects with hypertension or blood pressure ≥ 130 / 85 mm Hg may also be at risk of developing NAFLD. In addition to the aforementioned risk factors, subjects with NAFLD or at risk of developing NAFLD who also have NASH or are at risk of developing NASH (i.e., steatosis with liver inflammation and hepatocellular ballooning degeneration), are older than 45 years, or have low levels of circulating platelets (e.g., approximately 230 x 10⁻⁶). 9 Those with liver fibrosis may be at risk of developing liver fibrosis if they have a liver blood alcohol content of 1 / 2 liter or lower (or smoke).

[0066] Targets for the treatment of NAFLD

[0067] This disclosure is based in part on the findings that proteins listed in Table 1 regulate liver fibrosis. While not bound by theory, this can occur via ECM components (e.g., proteins and proteoglycans; such as collagen, hyaluronic acid, and laminin), activation of hepatic stellate cells, and / or transdifferentiation of hepatic stellate cells into myofibroblasts.

[0068] Table 1. Targets for the treatment of NAFLD

[0069] Inhibitors

[0070] Agents that inhibit the targets listed in Table 1 can be used to treat subjects who have NAFLD (e.g., NAFL or NASH) or are at risk of developing NAFLD. The agents described herein (e.g., see Tables 2, 3, and 4) can provide therapeutic effects by inhibiting the targets listed in Table 1. A list of therapeutic platforms and agents for inhibiting the targets listed in Table 1 is provided in Table 2 below.

[0071] Table 2. Treatment platforms and agents used to treat NAFLD

[0072] Inhibitors (see, for example, Tables 3 and 4) include repressive nucleic acid molecules (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, and gapmeR), RNA molecules encoding repressive proteins, soluble proteins that inhibit the activity of a target or the binding of a target to a binding chaperone, small molecule inhibitors, repressive antibodies, and programmable nucleases for editing and repressing gene expression (e.g., CRISPR-Cas-mediated gene repression). Each inhibitor is described in further detail below.

[0073] Table 3. Exemplary Targeting Agents

[0074] EPHA3 = type A liver ligand receptor 3; ITGAV = integrin subunit αV

[0075] Inhibitory nucleic acid molecules

[0076] In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule that reduces the expression or activity of one or more targets shown in Table 1. In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule that reduces the expression or activity of EPHA3. In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule that reduces the expression or activity of ITGAV. In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule that reduces the expression or activity of NAV2.

[0077] Exemplary repressive nucleic acid molecules are siRNA, dsRNA, miRNA, shRNA, ASO, and gapmeR; however, any nucleic acid molecule capable of reducing the expression of a target mRNA and / or protein is contemplated for use in the methods described herein. The repressive nucleic acid molecules disclosed herein may be referred to as RNA repressive (RNAi) molecules (e.g., siRNA, dsRNA, miRNA, and shRNA).

[0078] In some embodiments, the repressive RNA has a nucleotide sequence containing a portion of at least 8 consecutive nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) that is at least 70% complementary to an equal-length portion of the target region of a human gene mRNA transcript encoding the target to be repressed (e.g., the mRNA transcript or variant thereof cited in Table 1). In some embodiments, the repressive RNA has a nucleotide sequence containing a portion of at least 8 consecutive nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) that is 100% complementary to an equal-length portion of the target region of the mRNA transcript or its variants cited in Table 1.

[0079] In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least eight consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least nine consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least ten consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least eleven consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least twelve consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least thirteen consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least fourteen consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 15 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 16 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 17 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 18 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 19 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 20 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 21 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 22 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 23 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1.In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 24 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 25 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 26 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 27 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 28 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 29 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 30 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 31 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 32 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 33 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 34 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 35 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 36 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 37 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 38 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 39 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1.In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 40 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 41 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 42 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 43 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 44 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 45 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 46 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 47 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 48 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 49 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1. In some embodiments, the repressive nucleic acid molecule comprises a sequence complementary to at least 50 consecutive nucleotides shown in at least one of the mRNA transcripts or variants thereof referenced in Table 1.

[0080] In some embodiments, the repressive nucleic acid is a siRNA that inhibits the target of Table 1. In some embodiments, the repressive nucleic acid is a dsRNA that inhibits the target of Table 1. In some embodiments, the repressive nucleic acid is an ASO that inhibits the target of Table 1. In some embodiments, the repressive nucleic acid is a miRNA that inhibits the target of Table 1. In some embodiments, the repressive nucleic acid is a shRNA that inhibits the target of Table 1. In some embodiments, the repressive nucleic acid is a gapmeR that inhibits the target of Table 1. Each of these repressive nucleic acid molecules is further described in detail below.

[0081] Small interfering RNA

[0082] The siRNA molecules disclosed herein are single-stranded (ss) or double-stranded (ds) nucleic acid molecules made of DNA, RNA, or DNA and RNA (e.g., chimeric) nucleosides (these nucleosides are complementary to a transcript encoding a target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing) or a portion or variant thereof), and prevent the translation of mRNA into protein. Once the siRNA molecule enters the cell, it is incorporated into the RNA-induced silencing complex (RISC). After the antisense strand of the siRNA molecule hybridizes with the target mRNA (e.g., mRNA transcripts cited in Table 1), the RISC complex cleaves the target mRNA, thereby inactivating the target mRNA and resulting in reduced levels of the target mRNA and protein.

[0083] In some embodiments, the siRNA molecule disclosed herein may include a nucleotide sequence of about 10 to about 30 nucleotides (e.g., about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or 31 nucleotides).

[0084] In some embodiments, the siRNA molecule disclosed herein may include a nucleotide sequence of 10 to 30 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length).

[0085] Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention.

[0086] In some embodiments, the siRNA molecule disclosed herein contains a sequence complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides as shown in at least one of the mRNA transcripts or variants thereof cited in Table 1.

[0087] In some embodiments, the siRNA molecule disclosed herein contains an antisense strand. In some embodiments, the length of the antisense strand is between 10 and 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 15 and 2 The antisense strand can be between 5 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), or between 18 and 23 nucleotides (e.g., 18, 19, 20, 21, 22, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0088] In some embodiments, the siRNA molecule disclosed herein contains a sense strand. In some embodiments, the sense strand is between 10 and 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), or between 14 and 23 nucleotides (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

[0089] In some embodiments, the sense and antisense strands of the disclosed siRNA molecule are completely complementary to each other. In some embodiments, the sense and antisense strands of the disclosed siRNA molecule are completely complementary in the extent to which their lengths overlap. Depending on the sequences of the sense and antisense strands, complementarity does not need to be complete or perfect, meaning that the sense and antisense strands are not 100% base-paired due to mismatches. One or more (e.g., 1, 2, 3, 4, or 5) mismatches may be present within the ds siRNA molecule without affecting the siRNA molecule's ability to reduce the expression of the target mRNA transcript and / or protein.

[0090] The nucleotide sequence of the siRNA molecule disclosed herein may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts cited in Table 1 or variants thereof) such that the siRNA molecule can hybridize with one or more transcripts encoding a target. In some embodiments, the siRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts cited in Table 1 or variants thereof) or a portion thereof. In some embodiments, the siRNA molecule is 100% complementary to one or more transcripts encoding a target (e.g., transcripts cited in Table 1 or variants thereof) or a portion thereof.

[0091] In some embodiments, the nucleotide sequence of the siRNA molecule disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of the siRNA molecule may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target or a portion thereof. In some embodiments, the siRNA molecule disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript encoding any of the targets listed in Table 1 (e.g., transcripts cited in Table 1 or variants thereof).

[0092] In some embodiments, the nucleotide sequence of the siRNA molecule disclosed herein may contain a sense strand and / or an antisense strand having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one or more of SEQ ID NO: 9-32 (e.g., see Table 4).

[0093] In some embodiments, the siRNA molecule described herein may have a 3' overhang of 0-7 nucleotides or a 5' overhang of 0-4 nucleotides. In some embodiments, the siRNA molecule has a single uracil overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has two uracil overhangs at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a single thymine overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has two thymine overhangs at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a single cytosine and a single thymine (e.g., CT) overhang at one or more 3' ends of the siRNA.

[0094] For any of the methods described herein, different siRNA molecules disclosed herein may be combined to reduce one or more transcripts of one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more siRNA molecules may be used in the methods of the present invention, such as two different siRNA molecules, three different siRNA molecules, four different siRNA molecules, five different siRNA molecules, or more, to inhibit the same target transcript (e.g., transcripts cited in Table 1 or variants thereof). Alternatively, two different siRNA molecules, three different siRNA molecules, four different siRNA molecules, five different siRNA molecules, or more may be used in the methods of the present invention to inhibit different target transcripts (e.g., two or more transcripts listed in Table 1 or variants thereof).

[0095] Table 4. Exemplary siRNA sequences

[0096] A = adenine; T = uracil; C = cytosine; G = guanine; EPHA3 = type A liver ligand receptor 3; ITGAV = integrin subunit αV; NAV2 = neuronal navigator 2

[0097] Double-stranded RNA

[0098] The double-stranded RNA (dsRNA) molecules disclosed herein are double-stranded nucleic acid molecules made from both DNA, RNA, or DNA and RNA (e.g., chimeric) nucleosides (these nucleosides are complementary to a transcript of the target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing) or a portion thereof or variants thereof), and prevent the translation of mRNA into protein. Typically, dsRNA molecules are longer than siRNA molecules and are processed intracellularly to form siRNA molecules. The antisense strand of the siRNA molecule is then incorporated into a RISC, which cleaves the target mRNA when the siRNA hybridizes with the target mRNA (e.g., mRNA transcripts cited in Table 1), thereby inactivating the target mRNA and resulting in reduced levels of the target mRNA and protein.

[0099] In some embodiments, the dsRNA molecule disclosed herein may include a sense strand and an antisense strand, each containing a nucleotide sequence of about 25 to about 5,000 nucleotides or longer.

[0100] Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention.

[0101] In some embodiments, the dsRNA molecule disclosed herein contains a sequence of at least 25 consecutive nucleotides complementary to at least one of the mRNA transcripts or variants thereof referenced in Table 1.

[0102] In some embodiments, the sense and antisense strands of the dsRNA molecule disclosed herein are completely complementary to each other. In some embodiments, the sense and antisense strands of the dsRNA molecule disclosed herein are completely complementary to each other in the extent to which their lengths overlap. Depending on the sequences of the sense and antisense strands, complementarity does not need to be complete or perfect, meaning that the sense and antisense strands are not 100% base-paired due to mismatches. One or more mismatches (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) may be present within the dsRNA molecule without affecting the ability of the dsRNA to reduce the expression of target mRNAs (e.g., transcripts or variants thereof cited in Table 1) and / or proteins (e.g., proteins cited in Table 1).

[0103] The nucleotide sequence of the dsRNA molecule disclosed herein may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts or variants thereof cited in Table 1) such that the dsRNA molecule can hybridize with one or more transcripts encoding a target. In some embodiments, the dsRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof. In some embodiments, the dsRNA molecule is 100% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof.

[0104] In some embodiments, the nucleotide sequence of the dsRNA molecule disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of the dsRNA molecule may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the dsRNA molecule disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript (e.g., transcripts listed in Table 1 or variants thereof) encoding the targets described herein (e.g., targets listed in Table 1).

[0105] For any of the methods described herein, different dsRNA molecules disclosed herein can be combined to reduce one or more transcripts of one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more dsRNA molecules can be used in the methods of the present invention, such as two different dsRNA molecules, three different dsRNA molecules, four different dsRNA molecules, five different dsRNA molecules, or more, to inhibit the same target transcript (e.g., transcripts cited in Table 1 or variants thereof). Alternatively, two different dsRNA molecules, three different dsRNA molecules, four different dsRNA molecules, five different dsRNA molecules, or more can be used in the methods of the present invention to inhibit different target transcripts (e.g., two or more transcripts cited in Table 1 or variants thereof).

[0106] microRNA

[0107] The microRNA (miRNA) molecules disclosed herein are short ss nucleic acid molecules made from both DNA, RNA, or DNA and RNA (e.g., chimeric) nucleosides (these nucleosides are complementary to a transcript of the target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing) or a portion thereof) and prevent the translation of mRNA into protein. Once the miRNA molecule enters the cell, it is incorporated into a RISC, which cleaves the target mRNA when the miRNA hybridizes with the target mRNA (e.g., mRNA transcripts cited in Table 1), thereby inactivating the target mRNA and resulting in reduced levels of the target mRNA and protein.

[0108] In some embodiments, the miRNA molecule disclosed herein may include a nucleotide sequence of about 6 to about 30 nucleotides (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or 31 nucleotides).

[0109] In some embodiments, the miRNA molecule disclosed herein may include a nucleotide sequence of 6 to 30 nucleotides in length (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length).

[0110] Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention.

[0111] In some embodiments, the miRNA molecule disclosed herein contains a sequence complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides as shown in at least one of the mRNA transcripts or variants referenced in Table 1.

[0112] The nucleotide sequence of the miRNA molecule may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts or variants thereof cited in Table 1) such that the miRNA molecule can hybridize with one or more transcripts encoding a target. In some embodiments, the miRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof. In some embodiments, the miRNA molecule is 100% complementary to a transcript encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof.

[0113] In some embodiments, the nucleotide sequence of the miRNA molecule disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of the miRNA molecule may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target or a portion thereof. In some embodiments, the miRNA molecule disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript encoding any of the targets listed in Table 1 (e.g., transcripts cited in Table 1 or variants thereof).

[0114] For any of the methods described herein, different miRNA molecules disclosed herein can be combined to reduce one or more transcripts of one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more miRNA molecules can be used in the methods of the present invention, such as two different miRNA molecules, three different miRNA molecules, four different miRNA molecules, five different miRNA molecules, or more, thereby inactivating the same target transcript (e.g., transcripts or variants thereof cited in Table 1). Alternatively, two different miRNA molecules, three different miRNA molecules, four different miRNA molecules, five different miRNA molecules, or more can be used in the methods of the present invention to inactivate different target transcripts (e.g., two or more transcripts or variants thereof cited in Table 1).

[0115] Short hairpin RNA

[0116] The short hairpin RNA (shRNA) molecules disclosed herein are ss or ds nucleic acid molecules made from both DNA, RNA, or DNA and RNA (e.g., chimeric) nucleosides (these nucleosides are complementary to a transcript of the target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing) or a portion thereof) and prevent the translation of mRNA into protein. Once the shRNA molecule enters the cell, it is incorporated into a RISC complex, which cleaves the target mRNA when the shRNA hybridizes with the target mRNA (e.g., mRNA transcripts cited in Table 1), thereby inactivating the target mRNA and resulting in reduced levels of the target mRNA and protein.

[0117] In some embodiments, the shRNA molecule disclosed herein may include a nucleotide sequence of about 60 to about 100 nucleotides (e.g., about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105 or 110 nucleotides).

[0118] In some embodiments, the shRNA molecule disclosed herein may include a nucleotide sequence of 60 to 100 nucleotides in length (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length).

[0119] In some embodiments, the shRNA molecule disclosed herein may contain a variable hairpin loop structure and a stem sequence. In some embodiments, the stem sequence may be 10 to 50 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the hairpin size is between 4 and 50 nucleotides in length (e.g., lengths of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides), although the loop size can be larger without significantly affecting the silencing activity. The shRNA molecules disclosed herein may contain mismatches, such as GU mismatches between the two strands of the shRNA stem, without reducing efficacy. In some embodiments, the shRNA molecule is designed to include one or more GU pairs in the hairpin stem to stabilize the hairpin, for example, during replication in bacteria.

[0120] The nucleotide sequence of the shRNA molecule disclosed herein may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts cited in Table 1 or variants thereof) such that the shRNA molecule can hybridize with one or more transcripts of the target. In some embodiments, the shRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts cited in Table 1 or variants thereof) or a portion thereof. In some embodiments, the shRNA molecule is 100% complementary to one or more transcripts encoding a target (e.g., transcripts cited in Table 1 or variants thereof) or a portion thereof.

[0121] In some embodiments, the nucleotide sequence of the shRNA molecule disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of the shRNA molecule may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the shRNA molecule disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript (e.g., transcripts listed in Table 1 or variants thereof) encoding the targets described herein (e.g., targets listed in Table 1).

[0122] For any of the methods described herein, different shRNA molecules can be combined to reduce one or more transcripts (e.g., transcripts listed in Table 1) encoding one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more shRNA molecules can be used in the methods of the present invention, such as two different shRNA molecules, three different shRNA molecules, four different shRNA molecules, five different shRNA molecules, or more, to reduce the same target transcript (e.g., transcripts cited in Table 1 or variants thereof). Alternatively, two different shRNA molecules, three different shRNA molecules, four different shRNA molecules, five different shRNA molecules, or more, can be used in the methods of the present invention to reduce different target transcripts (e.g., two or more transcripts cited in Table 1 or variants thereof).

[0123] antisense oligonucleotides

[0124] The antisense oligonucleotides (ASOs) disclosed herein are ss nucleic acid molecules containing DNA nucleosides that are complementary to a portion or variant thereof of a transcript encoding a target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing). They prevent the translation of mRNA into protein. When an ASO hybridizes with a target mRNA (e.g., mRNA transcripts cited in Table 1), RNase H degrades the mRNA through hydrolysis, resulting in reduced levels of both the target mRNA and protein.

[0125] In some embodiments, the ASO disclosed herein may include a nucleotide sequence of about 12 to about 50 nucleotides (e.g., about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 or 51 nucleotides).

[0126] In some embodiments, the ASO disclosed herein may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0127] Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention.

[0128] In some embodiments, the ASO disclosed herein contains a sequence complementary to at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49 or at least 50 consecutive nucleotides as shown in at least one of the mRNA transcripts or variants referenced in Table 1.

[0129] The nucleotide sequence of the ASO disclosed herein may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts or variants thereof cited in Table 1) such that the ASO can hybridize with one or more transcripts encoding the target. In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof. In some embodiments, the ASO is 100% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof.

[0130] In some embodiments, the nucleotide sequence of the ASO disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of the ASO may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target or a portion thereof. In some embodiments, the ASO disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript encoding any of the targets listed in Table 1 (e.g., transcripts cited in Table 1 or variants thereof).

[0131] For the methods described herein, different ASOs can be combined to reduce one or more transcripts of one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more ASOs can be used in the methods of the present invention, such as two different ASOs, three different ASOs, four different ASOs, or five different ASOs, to degrade the same target transcript (e.g., transcripts cited in Table 1 or variants thereof). Alternatively, two different ASOs, three different ASOs, four different ASOs, or five different ASOs can be used in the methods of the present invention to degrade different target transcripts (e.g., two transcripts cited in Table 1 or variants thereof).

[0132] GapmeR

[0133] The gapmeR disclosed herein is a single-stranded nucleic acid molecule containing an internal DNA region (i.e., a gap segment) flanked by one or two external RNA regions (i.e., flanking segments). At least, the gap segment contains a sequence complementary to a transcript of the target mRNA (e.g., transcripts cited in Table 1; e.g., transcripts of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or splice variants of any of the foregoing), and typically, the flanking segments contain modified RNA; the modified RNA (and DNA) are further described below. When gapmeR hybridizes with the target mRNA (e.g., mRNA transcripts encoding the target cited in Table 1), RNase H degrades the mRNA through hydrolysis, resulting in reduced levels of the target mRNA and protein.

[0134] In some embodiments, the gapmeR disclosed herein may include a nucleotide sequence of about 10 to about 25 nucleotides (e.g., about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 or 26 nucleotides).

[0135] In some embodiments, the gapmeR disclosed herein may include a nucleotide sequence of 10 to 25 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length).

[0136] Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention.

[0137] In some embodiments, the gapmeR disclosed herein contains at least one (e.g., one or two) wing regions. In some embodiments, the gapmeR contains one wing region located at 5' of the gap region. In some embodiments, the gapmeR contains one wing region located at 3' of the gap region. In some embodiments, the gapmeR contains two wing regions, one located at 5' of the gap region and the other located at 3' of the gap region.

[0138] In some embodiments, the gapmeR disclosed herein contains at least one (e.g., one or two) flanking regions of about 1 to about 7 nucleotides in length (e.g., about 1-8, about 1-7, about 1-6, about 1-5, about 1-4, about 1-3, or about 1-2 nucleotides in length). In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 1 nucleotide in length. In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 2 nucleotides in length. In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 3 nucleotides in length. In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 4 nucleotides in length. In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 5 nucleotides in length. In some embodiments, the gapmeR contains at least one (e.g., one or two) flanking regions of 6 nucleotides in length. In some embodiments, gapmeR contains at least one (e.g., 1 or 2) flanking regions of 7 nucleotides in length. In some embodiments, gapmeR contains at least one (e.g., 1 or 2) flanking regions of 8 nucleotides in length.

[0139] In some embodiments, the gapmeR disclosed herein includes a notch region. In some embodiments, the gapmeR includes a notch region located at 5' of a side wing region. In some embodiments, the gapmeR includes a notch region located at 3' of a side wing region. In some embodiments, the gapmeR includes a notch region adjacent to two side wing regions.

[0140] In some embodiments, the gapmeR disclosed herein contains at least one gap region of about 8 to about 24 nucleotides in length (e.g., about 8-24, about 8-23, about 8-22, about 8-21, about 8-20, about 8-19, about 8-18, about 8-17, about 8-16, about 8-15, about 8-14, about 8-13, about 8-12, about 8-10, or about 8-9 nucleotides in length).

[0141] The nucleotide sequence of gapmeR may contain sufficient complementarity to a portion of a transcript encoding a target (e.g., transcripts or variants thereof cited in Table 1) such that gapmeR can hybridize with one or more transcripts encoding the target. In some embodiments, the gapmeR is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to one or more transcripts encoding a target (e.g., transcripts or variants thereof cited in Table 1) or a portion thereof. In some embodiments, gapmeR is 100% complementary to one or more transcripts encoding a target or a portion thereof.

[0142] In some embodiments, the nucleotide sequence of gapmeR disclosed herein may contain sufficient complementarity to the exon sequences within one or more transcripts encoding a target (e.g., transcripts listed in Table 1) or a portion thereof. In some embodiments, the nucleotide sequence of gapmeR may contain sufficient complementarity to the intron sequences within one or more transcripts encoding a target or a portion thereof. In some embodiments, the nucleotide sequence of gapmeR disclosed herein may contain sufficient complementarity to the mRNA precursor transcript or mRNA transcript encoding any of the targets listed in Table 1 (e.g., transcripts cited in Table 1 or variants thereof).

[0143] For the methods described herein, different gapmeRs can be combined to reduce one or more transcripts (e.g., transcripts listed in Table 1) encoding one or more targets described herein (e.g., one or more targets listed in Table 1). Combinations of two or more gapmeRs can be used in the methods of the present invention, such as two different gapmeRs, three different gapmeRs, four different gapmeRs, five different gapmeRs, or more, to degrade the same target transcript (e.g., transcripts cited in Table 1 or variants thereof). Alternatively, two different gapmeRs, three different gapmeRs, four different gapmeRs, or five different gapmeRs can be used in the methods of the present invention to degrade different target transcripts (e.g., two transcripts cited in Table 1 or variants thereof).

[0144] RNA that encodes proteins

[0145] In some embodiments, the agent is an RNA molecule (e.g., mRNA) encoding a protein that reduces the expression or activity of one or more targets listed in Table 1 (e.g., EPHA3, ITGAV, and / or NAV2). In some embodiments, the RNA molecule (e.g., mRNA) encodes a protein that reduces the expression or activity of one or more targets listed in Table 1. In some embodiments, the protein is soluble or membrane-bound. In some embodiments, the RNA molecule (e.g., mRNA) encodes an antibody or an antigen-binding fragment thereof, such as antibodies in Table 3 (e.g., ifatozumab). In some embodiments, the mRNA comprises a nucleotide sequence of any one of SEQ ID NO: 35-41 and / or any one of SEQ ID NO: 42-48. In some embodiments, the mRNA encodes an antibody having the light chain amino acid sequence of SEQ ID NO: 33 and / or the heavy chain amino acid sequence of SEQ ID NO: 34.

[0146] soluble protein

[0147] In some embodiments, the agent is a soluble protein that reduces the expression or activity of one or more targets listed in Table 1 (e.g., EPHA3, ITGAV, and / or NAV2).

[0148] In some embodiments, the soluble protein is an agent that disrupts the binding of a target to its binding chaperone. For example, the soluble protein may be a variant of a target lacking the ability to bind to its binding chaperone (e.g., a variant of a target where the binding domain has been mutated or missing) or a variant of a target's binding chaperone lacking the ability to bind to the target (e.g., a variant of a binding chaperone where the binding domain has been mutated or missing). In some embodiments, the soluble protein is an agent that disrupts downstream signaling, such as a variant of a transmembrane target lacking an intracellular domain but retaining the ability to bind to an extracellular binding chaperone, or a variant of a target where one or more of its signaling domains have been mutated.

[0149] Small molecule inhibitors

[0150] In some embodiments, the agent is a small molecule inhibitor that reduces the expression or activity of one or more targets listed in Table 1 (e.g., EPHA3, ITGAV, and / or NAV2).

[0151] In some embodiments, the small molecule inhibitor is an antagonist that directly binds to a target to reduce or inhibit its function or activity. In some embodiments, the small molecule antagonist is selective to the target and does not exhibit substantial binding to other proteins.

[0152] Small molecules include organic and inorganic compounds (including heteroorganic and organometallic compounds) that typically have a molecular weight of less than about 5,000 g / mol, such as organic or inorganic compounds with a molecular weight of less than about 2,000 g / mol, such as organic or inorganic compounds with a molecular weight of less than about 1,000 g / mol, such as organic or inorganic compounds with a molecular weight of less than about 500 g / mol, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. Exemplary small molecule inhibitors that can be used to inhibit the targets described herein include AWL-II-38.3 (CAS No. 1135205-95-5) for inhibiting EPHA3 and bexetograst (e.g., PLN-74809) for inhibiting αv integrin subunits (e.g., ITGAV). Additional small molecule inhibitors may also be identified by screening based on their ability to reduce or inhibit the function or signaling of the targets listed in Table 1.

[0153] Antibody

[0154] In some embodiments, the inhibitor is an inhibitory antibody (e.g., mAb) that reduces the expression or activity of one or more targets listed in Table 1 (e.g., EPHA3, ITGAV, and / or NAV2). In one embodiment, the antibody is epfatuzumab (also known as KB004) targeting EPHA3. In some embodiments, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 33 and / or a light chain containing the amino acid sequence of SEQ ID NO: 34.

[0155] In some embodiments, the antibody targets one or more targets listed in Table 1 or their antigen-binding fragments, binding to the targets listed in Table 1. In some embodiments, the antibody is an antibody against a target or its antigen-binding fragment that reduces or inhibits the function of the target. The antibody may have one or more of the following functional properties: preventing the target from binding to a binding chaperone (e.g., spatially blocking the binding of the target to a binding chaperone), chelating soluble targets, and / or reducing or inhibiting the activity and / or function of the target. Once the desired functional properties are identified herein (e.g., by screening phage display or other antibody libraries), antibodies having one or more of these functional properties are routinely screened and selected.

[0156] The preparation and use of antibodies against target antigens (e.g., against targets described herein (e.g., proteins listed in Table 1)) are known in the art (see, for example, Zhiqiang An (ed.), *Therapeutic Monoclonal Antibodies: From Bench to Clinic*, 1st ed. Wiley, 2009, and Greenfield (ed.), *Antibodies: A Laboratory Manual* (2nd ed.), Cold Spring Harbor Laboratory Press, 2013, for methods of preparing recombinant antibodies, including antibody engineering, the use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques).

[0157] Gene editing related drugs

[0158] In some embodiments, the agent is a component of a gene editing system that reduces the expression or activity of one or more targets (e.g., EPHA3, ITGAV, and / or NAV2) listed in Table 1. For example, the gene editing-mediated agent introduces alterations (e.g., insertions, deletions (e.g., knockouts), translocations, inversions, single-point mutations, or other mutations) in a gene (e.g., a genomic locus or mRNA transcript) encoding a target (e.g., targets listed in Table 1). Exemplary gene editing systems (e.g., base editing, leader editing, or homology-directed repair (HDR) systems) may include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly spaced short palindromic repeats (CRISPR) systems listed in Table 2. ZFN, TALEN, and CRISPR-based methods have been described, for example, by Gaj et al. Trends Biotechnol. [Biotechnology Trends] 31.7(2013):397-405.

[0159] CRISPR refers to a collection (or system containing) of clustered, regularly spaced short palindromic repeats. A CRISPR system is a system derived from CRISPR and Cas (CRISPR-associated proteins) or another nuclease that can be used to silence or mutate genes encoding targets described herein (e.g., genomic loci or mRNA transcripts). CRISPR systems are naturally occurring systems found in bacterial and archaea genomes. CRISPR loci consist of alternating repeat sequences and spacer sequences. In naturally occurring CRISPR systems, spacers are typically sequences foreign to bacteria (e.g., plasmids or bacteriophage sequences). CRISPR systems have been modified for gene editing in eukaryotes (e.g., altering, silencing, and / or enhancing certain genes). See, for example, Wiedenheft et al. Nature . [nature] 482: 331, 2012. For example, this modification of the system involves introducing a plasmid containing a specially designed CRISPR and one or more suitable Cas proteins into a eukaryotic cell. The CRISPR locus is transcribed into RNA and processed by the Cas protein into a small RNA containing a repetitive sequence with side-joint spacers. The RNA acts as a guide to direct the Cas protein to silence a specific DNA / RNA sequence, depending on the spacer sequence. See, for example, Horvath et al. Science [Science] 327: 167, 2010; Makarova et al. Biology Direct [Biology Guide] 1:7, 2006; Pennisi, Science [Science] 341: 833, 2013. In some instances, the CRISPR system includes the Cas9 protein, a nuclease that cuts along both strands of DNA. See, for example, ibid.

[0160] In some embodiments, the CRISPR system may include one of the following nucleases that cleave both strands of a DNA sequence (e.g., the DNA sequence encoding a target referenced in Table 1): Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j. In other embodiments, the CRISPR system may include one of the following nucleases that cleave the strand of an mRNA sequence (e.g., the mRNA transcript or variants thereof referenced in Table 1): Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g.

[0161] In some embodiments, in a CRISPR system used for the purposes described herein, for example, according to one or more methods described herein, the CRISPR spacers are derived from target gene sequences, for example, from gene sequences encoding targets (e.g., targets listed in Table 1).

[0162] In some embodiments, gene editing-mediated agents include guide RNA (gRNA) or leader editing gRNA (pegRNA) for gene editing in a CRISPR system (e.g., HDR-mediated editing, leader editing, or base editing). In embodiments, gene editing-mediated agents include zinc finger nucleases (ZFNs) or mRNA encoding ZFNs that target (e.g., cleave) nucleic acid sequences (e.g., DNA sequences) encoding targets (e.g., targets listed in Table 1). In embodiments, gene editing-mediated agents include TALENs or mRNA encoding TALENs that target (e.g., cleave) nucleic acid sequences (e.g., DNA sequences) encoding targets (e.g., targets listed in Table 1).

[0163] In some instances, gRNAs can be used in a CRISPR system to engineer alterations in genes (e.g., genes encoding a target). In other instances, ZFNs and / or TALENs can be used to engineer alterations in genes (e.g., genes encoding a target). Exemplary alterations include frameshift insertions or deletions (insertion deletions) (e.g., knockout), translocations, inversions, single-point mutations, or other mutations. Alterations can be introduced into genes within cells, e.g., in vitro, ex vivo, or in vivo. In some embodiments, the alteration reduces the level and / or activity of a target (e.g., targets listed in Table 1) (e.g., knockdown or knockout), for example, the alteration is a negative functional regulator.

[0164] In some embodiments, the CRISPR system is used to edit (e.g., add or delete base pairs) target genes, such as genomic loci encoding targets (e.g., targets listed in Table 1) or mRNA transcripts. In other embodiments, the CRISPR system is used to introduce an early stop codon, thereby reducing the expression or function of the target gene (e.g., genomic loci or mRNA transcript). In still other embodiments, the CRISPR system is used to shut down the target gene in a reversible manner, such as similar to RNA interference. In embodiments, the CRISPR system is used to guide Cas to the promoter of the target gene (e.g., a genomic locus encoding a target (e.g., targets listed in Table 1)) to spatially block RNA polymerase.

[0165] In some embodiments, the techniques described below can be used to generate a CRISPR system to edit genes encoding targets (e.g., targets listed in Table 1): e.g., U.S. Publication No. 20140068797; Cong, Science [Science] 339: 819, 2013; Tsai, Nature Biotechnol [Nature Biotechnology] 32:569, 2014; and U.S. Patent Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.

[0166] In some embodiments, CRISPR interference (CRISPRi) technology can be used for transcriptional repression of specific genes, such as genes encoding targets (e.g., targets listed in Table 1). In CRISPRi, an engineered Cas9 protein (e.g., nuclease-free dCas9, or a dCas9 fusion protein, such as dCas9-KRAB or dCas9-SID4X fusions) can pair with sequence-specific guide RNA (sgRNA). The Cas9-gRNA complex can block RNA polymerase, thereby interfering with transcriptional elongation. The complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method is specific, has minimal off-target effects, and is multiplexable, for example, allowing simultaneous repression of more than one gene (e.g., using multiple gRNAs). Moreover, the CRISPRi method allows for reversible gene repression.

[0167] In any of the aforementioned CRISPR systems, a suitable gRNA includes at least one crripr RNA (crRNA) region to enable specificity in each CRISPR reaction. A suitable gRNA may also include a trans-activating crRNA (tracrRNA) region that is fused to or hybridized with crRNA. Additionally, a suitable gRNA may contain multiple nucleotide restriction enzyme (NREM) recognition sites (e.g., Csy4) for multiplexing. Introducing NREM recognition sites between adjacent gRNA sequences allows transcription of more than one gRNA in a single expression cassette. Directly repeating sequences can also serve as NREM recognition sites for multiplexing.

[0168] The gRNA may contain a spacer sequence containing multiple bases and complementary to the original spacer sequence in the target DNA (e.g., the DNA sequence encoding the targets listed in Table 1) or the mRNA sequence (e.g., see Table 1). The gRNA spacer sequence may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, 99%, or 100% complementary to its intended target sequence (e.g., the targets listed in Table 1).

[0169] gRNA may contain one or more modified or non-naturally occurring nucleosides or nucleotides, and / or inter-nucleoside bonds. In some embodiments, gRNA may contain locked nucleic acids (LNA), bridging nucleic acids (BNA), and / or peptide nucleic acids (PNA). By further examples, modified nucleic acid molecules may contain modified or non-naturally occurring nucleosides, nucleotides, and / or inter-nucleoside bonds, such as 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluorine (2′-F) modified nucleosides, and phosphate thioester (PS) bonds (or any other nucleic acid molecule modification described herein).

[0170] Modification of nucleic acid molecules

[0171] It is conceivable that any nucleic acid molecule described herein (e.g., repressive nucleic acid molecules, protein-coding RNA, gRNA) can be used in the methods disclosed herein, either in an unmodified or modified form. Unmodified nucleic acid molecules contain nucleobases including purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below.

[0172] Modifications can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

[0173] Modifications can be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative nucleoside internucleotides. Typically, these types of modifications are introduced to optimize the efficacy or biophysical properties of a molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, reducing immunogenicity, and / or targeting specific sites or cell types). By way of example, modified nucleotides such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or bridging nucleic acids (BNAs) can be incorporated into any of the nucleic acid-based inhibitors described above. Further nucleic acid modifications are described below.

[0174] Modifications can be further achieved by covalently or non-covalently conjugating a portion (e.g., a targeting portion, a hydrophobic portion, a cell-penetrating peptide, or a polymer) to the 5' and / or 3' ends of an inhibitory nucleic acid molecule, as described in more detail below.

[0175] Nucleoside modification

[0176] The modifications of inhibitory nucleic acid molecules described herein include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil. Pyrimidines, cytosines, and thymines; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines; 5-halogenated, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 2-F-adenine; 2-aminoadenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine and / or 3-deazaguanine and 3-deazaadenine. Inhibitory nucleic acid molecules may also include nucleobases in which the purine or pyrimidine base is substituted by other heterocyclic compounds, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modifications to the inhibitory nucleic acid molecules described herein may include the nucleobases disclosed in the following: US 3,687,808; Kroschwitz, JI (ed.), The Concise Encyclopedia of Polymer Science and Engineering, New York; John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 30:613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ (ed.), 1993, pp. 289-302.

[0177] Sugar modification

[0178] Modifications to the inhibitory nucleic acid molecules described herein may also include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2′-modifications that can modify the repressive nucleic acid molecules described herein include all of the following possible orientations: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and ynyl groups. Other potential sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluorine (F). The 2′-sugar substituent group can be at the arabinose (top) position or the ribose (bottom) position. In some embodiments, the 2′-arabinose modification is 2′-F. Similar modifications can also be made at other positions on the interfering RNA molecule, particularly at the 3′ position of the sugar on the 3′ terminal nucleotide or at the 5′ position of the 5′ terminal nucleotide in 2′-5′ linked oligonucleotides. Oligonucleotides can also have sugar analogs, such as the cyclobutyl moiety replacing furanylpentose sugar.

[0179] Nucleoside inter-bond modification

[0180] Modifications of the repressive nucleic acid molecules described herein may include one or more of the following internucleotide modifications: thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methylphosphonates and other alkylphosphonates (including 3'-alkylphosphonides, 5'-alkylphosphonides), phosphonites, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thionophosphoramidates, thionoalkylphosphonates, thionoalkyl phosphate triesters, selenophosphates and boron phosphates having a normal 3'-5' bond, analogs of these with 2'-5' linkages, and those with opposite polarities, wherein one or more internucleotide bonds are 3' to 3', 5' to 5', or 2' to 2' bonds.

[0181] Conjugate

[0182] Any inhibitory nucleic acid molecule described herein can be modified by adding an auxiliary moiety (e.g., a cell-penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety). This auxiliary moiety can be present as a 5' end modification (e.g., covalently bonded to a 5' end nucleoside), a 3' end modification (e.g., covalently bonded to a 3' end nucleoside), or an inter-nucleoside bond (e.g., covalently bonded to a phosphate ester or thiophosphate ester in the inter-nucleoside bond).

[0183] CPP is known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, Expert Opinion. Drug Deliv [Expert Opinion on Drug Delivery] 2(1): 43-51, 2005. Specific examples of CPP are provided in WO 2011157713, which is incorporated herein by reference in its entirety.

[0184] The repressive nucleic acid molecules disclosed herein may include covalently attached auxiliary portions based on neutral polymers. Neutral polymers include poly(C1-6 epoxy), such as poly(ethylene glycol) and poly(propylene glycol), and copolymers thereof, such as diblock and triblock copolymers.

[0185] Compared to inhibitory nucleic acid molecules lacking a hydrophobic moiety, inhibitory nucleic acid molecules containing a hydrophobic moiety can exhibit superior cellular uptake. The hydrophobic moiety is a monovalent group (e.g., bile acids (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyllithocholic acid, or oleoylcholine), glycolipids, phospholipids, sphingolipids, isoprene, vitamins, saturated fatty acids, unsaturated fatty acids, fatty acid esters, triglycerides, pyrene, porphyrin, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas red, digoxin, dimethoxytriphenylmethyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, anthocyanin dyes (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently attached to the nucleic acid backbone (e.g., the 5' end) of the inhibitory nucleic acid molecule.

[0186] The targeting portion is selected based on its ability to target the oligonucleotide of the invention to a desired or selected cell population expressing the corresponding binding partner (e.g., the corresponding receptor or ligand) of the selected targeting portion. For example, the oligonucleotide of the invention can target hepatic stellate cells by selectively targeting ligands that target biomarkers or receptors expressed on the surface of hepatic stellate cells. Exemplary biomarkers or receptors for hepatic stellate cells include type VI collagen receptor, retinol-binding protein (RBP) receptor, platelet-derived growth factor receptor (PDGFR), synaptic growth protein (e.g., major synaptic vesicle protein p38), insulin-like growth factor-II receptor (IGFIIR), low-density lipoprotein receptor (LDLR), TNF-associated apoptosis-inducing ligand receptor (TRAILR), integrins (e.g., αvβ3), and / or differentiation cluster 44 (CD44).

[0187] The targeting portion may include one or more ligands (e.g., 1 to 9 ligands, 1 to 6 ligands, 1 to 3 ligands, 3 ligands, or 1 ligand). Ligands include any naturally occurring or synthetic binding partners targeting receptors or surface biomarkers present on the desired cellular target (e.g., hepatic stellate cells). Exemplary ligands include RGD peptides binding to type VI collagen receptors and integrins, vitamin A binding to RBP receptors, mannose (e.g., mannose-6-phosphate (M6P)) binding to IGFIIR, and hyaluronic acid binding to CD44.

[0188] The ligand can be a small molecule, such as a small molecule that targets one or more receptors or surface biomarkers on hepatic stellate cells. The ligand can be a lipid, such as cholesterol. Alternatively, the ligand can be an antibody or its antigen-binding fragment or engineered derivative (e.g., Fcab or a fusion protein (e.g., scFv)).

[0189] Pharmaceutical Composition

[0190] The pharmaceutical preparations described herein can be formulated into various compositions (e.g., pharmaceutical compositions) for administration to a subject in a biocompatible form suitable for in vivo administration. For example, the pharmaceutical preparations described herein may be administered in suitable diluents, carriers, stabilizers, or excipients, and may further contain preservatives, such as those to prevent microbial growth. Conventional procedures and ingredients for selecting and preparing suitable compositions are described, for example, in Remington, JP, *The Science and Practice of Pharmacy*, Easton, PA., Mack Publishers, 2012, 22nd edition, and in *The United States Pharmacopeial Convention*, *The National Formulary*, United States Pharmacopeia, 2015, USP 38 NF 33.

[0191] The mixtures of pharmaceutical preparations described herein can be prepared in water, suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Suitable forms of pharmaceutical preparations for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In any case, the formulation may be sterile and may be fluid enough for easy injection. The formulation may be stable under manufacturing and storage conditions and may be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. The effect of preventing microorganisms can be achieved by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Prolonged absorption of injectable compositions can be achieved by using agents with delayed absorption, such as aluminum monostearate and gelatin, in the composition.

[0192] For example, if necessary, the solution containing the pharmaceutical composition described herein may be appropriately buffered, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, in view of this disclosure, sterile aqueous media that may be used will be known to those skilled in the art. For example, a dose may be dissolved in 1 mL of isotonic NaCl solution and added to 1,000 mL of subcutaneous injection solution or injected at the recommended infusion site. Some variations in dosage will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for the individual subject. Furthermore, for human administration, the formulation may meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics Standards.

[0193] Although the description of the pharmaceutical compositions provided herein is primarily directed toward pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, or non-human mammals. Modifications to pharmaceutical compositions suitable for administration to humans are well known to make them suitable for administration to a variety of animals, and such modifications can be designed and / or performed by a general veterinary pharmacist through routine experiments (if any). We envision subjects to whom the pharmaceutical compositions are administered include, but are not limited to, humans and / or other primates and animals.

[0194] Compositions containing the pharmaceutical agents described herein may further comprise a second pharmaceutical agent (e.g., a nucleic acid molecule, peptide, or drug to be expressed intracellularly). For example, the second pharmaceutical agent may be a blood pressure medication, a steroid, or an immunosuppressant. In some embodiments, the second pharmaceutical agent is statin. In some embodiments, the second pharmaceutical agent (e.g., statin) is administered in combination with the pharmaceutical agents disclosed herein.

[0195] exosomes

[0196] In one embodiment, the pharmaceutical composition of the agents described herein comprises extracellular vesicles, such as exosomes. Cell-derived exosomes can be collected from a culture medium by any suitable method. Typically, formulations of exosomes can be prepared from cell cultures or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, exosomes can be prepared by differential centrifugation, i.e., at low speeds (< 20,000 x 10⁻⁶), using standard methods. g Centrifuge to precipitate larger particles, then perform high-speed (>100,000 x 10000) centrifugation. gCentrifugation to precipitate exosomes, size filtration using an appropriate filter (e.g., a 0.22-micron filter), gradient ultracentrifugation (e.g., a sucrose gradient), or a combination of these methods. Exosomes are loaded with exogenous inhibitors according to standard methods for systemic delivery to subjects (e.g., humans).

[0197] Liposomes

[0198] In one embodiment, the pharmaceutical composition of the agent described herein comprises liposomes. Liposomes are artificially prepared vesicles that may consist primarily of a lipid bilayer and can be used as delivery media for administering nutrients and pharmaceutical formulations. Liposomes can have different sizes, such as, but not limited to, multilayer vesicles (MLVs) with diameters of several hundred nanometers and containing a series of concentric bilayers separated by narrow aqueous compartments, small single-cell vesicles (SUVs) with diameters less than 50 nm, and large monolayer vesicles (LUVs) with diameters between 50 and 500 nm. To improve the adhesion of liposomes to unhealthy tissues or to activate events such as, but not limited to, endocytosis, liposome design may include, but is not limited to, opsonins or ligands. To improve the delivery of pharmaceutical formulations, liposomes may contain low or high pH. According to standard methods, liposomes are loaded with exogenous inhibitors for systemic delivery to a subject (e.g., a human).

[0199] lipid nanoparticles

[0200] In one embodiment, the pharmaceutical composition of the agent described herein comprises lipid nanoparticles (LNPs). For example, inhibitors, such as 5'-inhibitor-Pro-CGG-1 and / or 5'-inhibitor-Cys-GCA-27, can be formulated in lipid nanoparticles, such as those described in International Publication No. WO 2012170930, which is incorporated herein by reference in its entirety. As a non-limiting example, LNP formulations may contain cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristoyl-propyl-3-amine (PEG-c-DOMG), distearate racemic glycerol (DSG), and / or dimethyl butyrate (DMA). As a non-limiting example, the lipid molar ratio of PEG-c-DOMG is 1%-5% compared to the cationic lipids, DSPC, and cholesterol. In another embodiment, the PEG-c-DOMG can be replaced by a PEG lipid (such as, but not limited to, PEG-DSG (1,2-distearyl-sn-glycerol, methoxy polyethylene glycol) or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxy polyethylene glycol)). The cationic lipid can be selected from any lipid known in the art, such as, but not limited to, 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester (Dlin-MC3-DMA), 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane (Dlin-DMA), C12-200 and N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanolamine (Dlin-KC2-DMA). According to standard methods, LNPs are loaded with exogenous therapeutic agents for systemic delivery to subjects (e.g., humans).

[0201] In some embodiments, the compounds described herein are formulated into lipid-based carriers (or lipid nanoformations). In some embodiments, the lipid-based carrier (or lipid nanoformation) is a liposome or lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is an LNP.

[0202] In some embodiments, the lipid-based carrier (or lipid nanoformation) comprises cationic lipids (e.g., ionizable lipids), non-cationic lipids (e.g., phospholipids), structural lipids (e.g., cholesterol), and PEG-modified lipids. In some embodiments, the lipid-based carrier (or lipid nanoformation) contains one or more compounds described herein or pharmaceutically acceptable salts thereof.

[0203] As described herein, suitable compounds for use in lipid-based carriers (or lipid nanoformations) include all isomers and isotopes of the compounds described above, and include all pharmaceutically acceptable salts, solvates or hydrates thereof, as well as all crystalline forms, mixtures of crystalline forms and anhydrides or hydrates thereof.

[0204] In addition to one or more of the compounds described herein, lipid-based carriers (or lipid nanoformulations) may further comprise a second lipid. In some embodiments, the second lipid is a cationic lipid, a non-cationic lipid (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.

[0205] One or more naturally occurring and / or synthetic lipid compounds can be used in the preparation of lipid-based carriers (or lipid nanoformations).

[0206] Lipid-based carriers (or lipid nanoformations) may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or combinations thereof.

[0207] Cationic lipids (positively charged) and ionizable lipids

[0208] In some embodiments, the lipid-based carrier (or lipid nanoformation) comprises one or more cationic lipids, such as cationic lipids that can exist in a positively charged or neutral form depending on pH, or amine-containing lipids that can be readily kinetically converted. In some embodiments, the cationic lipids are, for example, lipids that can be positively charged under physiological conditions.

[0209] Exemplary cationic lipids include one or more positively charged amine groups. Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-bis(octadecyl)ammonium bromide (DDAB) and ammonium chloride (DDAC), N-(l-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol), 1,2-dioleoyloxy-3-[trimethylammonium]-propane (DOTAP), 1,2-bis(octadecyloxy)-3-[trimethylammonium]-propane (DSTAP), and 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI), N,N-diolenoyl-N,N-dimethylammonium chloride (DOD). AC), N,N-dimethyl-2,3-diolenoyloxy)propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoylcarbamoyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilinoleoyl-3-dimethylammonium-propane (DLINDAP), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-β-oxy)-3′-oxaproloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLin) DMA), N,N-dimethyl-3,4-diolenoyloxybenzylamine (DMOBA), and, for example, Martin et al. Curr Pharm Des. The cationic lipids described in [Current Drug Design] 11(3): 375-394, 2005 are incorporated herein by reference in their entirety. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.

[0210] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa greater than 6.0. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having an effective pKa different from that of the first cationic lipid (e.g., greater than the first effective pKa).

[0211] In some embodiments, cationic lipids that may be used in lipid-based carriers (or lipid nanoformations) include, for example, those described in Table 4 of WO 2019 / 217941 (which is incorporated herein by reference).

[0212] In some embodiments, cationic lipids are ionizable lipids (e.g., lipids that are protonated at low pH but remain neutral at physiological pH). In some embodiments, lipid-based carriers (or lipid nanoformulations) may comprise one or more additional ionizable lipids different from those described herein. Exemplary ionizable lipids include, but are not limited to: (LP01) (SM-086) (SM-102) (ALC-0315) (Lipid 10) (Lipid A9) and (Dlin-MC3-DMA) (See WO 2017 / 004143 A1, which is incorporated herein by reference in its entirety.)

[0213] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described in WO 2021 / 113777 (e.g., lipids having formula (3), such as lipids in Table 3 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0214] In one embodiment, the ionizable lipid is a lipid disclosed in Hou, X. et al. Nat Rev Mater [Nature Review Material] 6: 1078-1094 (2021). https: / / doi.org / 10.1038 / s41578-021-00358-0 (e.g., L319, C12-200 and Dlin-MC3-DMA) (which is incorporated herein by reference in its entirety).

[0215] Examples of other ionizable lipids that can be used in lipid-based carriers (or lipid nanoformulations) include, but are not limited to, one or more of the following: X of US 2016 / 0311759; I of US 20150376115 or US 2016 / 0376224; compound 5 or compound 6 of US 2016 / 0376224; I, IA, or II of US 9,867,888; I, II, or III of US 2016 / 0151284; I, IA, II, or IIA of US 2017 / 0210967; Ic of US 2015 / 0140070; A of US 2013 / 0178541; I of US 2013 / 0303587 or US 2013 / 0123338; ​​US US 2015 / 0141678, Part I; US 2015 / 0239926, Parts II, III, IV, or V; US 2017 / 0119904, Part I; WO 2017 / 117528, Part I or II; US 2012 / 0149894, Part A; US 2015 / 0057373, Part A; WO 2013 / 116126, Part A; US 2013 / 0090372, Part A; US 2013 / 0274523, Part A; US 2013 / 0274504, Part A; US 2013 / 0053572, Part A; WO 2013 / 016058, Part A; WO 2012 / 162210, Part A; US 2008 / 042973, Part I; US US 2012 / 01287670, I, II, III, or IV; US 2014 / 0200257, I or II; US 2015 / 0203446, I, II, or III; US 2015 / 0005363, I or III; US 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; US 2013 / 0338210; WO 2009 / 132131, I, II, III, or IV; US 2012 / 01011478, A; US 2012 / 0027796, I or XXXV; US 2012 / 0058144, XIV or XVII; US 2013 / 0323269; US US 2011 / 0117125, Part I; US 2011 / 0256175, Part I, II, or III; US 2012 / 0202871, Part I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII; US 2011 / 0076335, Part I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI; US 2006 / 008378, Part I or II;WO2015 / 074085, Part I (e.g., ATX-002); US 2013 / 0123338, Part I; US 2015 / 0064242, Part I or XAYZ; US 2013 / 0022649, Part XVI, XVII, or XVIII; US 2013 / 0116307, Part I, II, or III; US 2013 / 0116307, Part I, II, or III; US 2010 / 0062967, Part I or II; US 2013 / 0189351, Part IX; US 2014 / 0039032, Part I; US 2018 / 0028664, Part V; US 2016 / 0317458, Part I; US 2013 / 0195920, Part I; US 10,221,127 of 5, 6 or 10; WO 2018 / 081480 of III-3; WO 2020 / 081938 of I-5 or I-8; WO 2015 / 199952 of I (e.g., compound 6 or 22) and Table 1 therein; US 9,867,888 of 18 or 25; US 2019 / 0136231 of A; WO 2020 / 219876 of II; US 2012 / 0027803 of 1; US ​​2019 / 0240349 of OF-02; US 10,086,013 of 23; Miao et al. (2020) of cKK-E12 / A6; WO C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al.; TS-P4C2 of U S9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3) or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, are incorporated herein by reference in their entirety.

[0216] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a biodegradable, ionizable lipid, such as octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester. See, for example, lipids in WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340 and WO 2014 / 136086, which are incorporated herein by reference in their entirety.

[0217] Non-cationic lipids (e.g., phospholipids)

[0218] In some embodiments, the lipid-based carrier (or lipid nanoformation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a phospholipid substitute or replacement. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid.

[0219] Exemplary non-cationic lipids include, but are not limited to, distearyl-sn-glycero-phosphoethanolamine, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidyl... Acylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethylPE), 18-l-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine ( SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disqualylphosphatidylcholine (DEPC), palmitoyl oleoylphosphatidylglycerol (POPG), ditransoleenoyl-phosphatidylethanolamine (DEPE), 1,2-dilaurin Acyl-sn-glycerol-3-phosphate choline (DLPC), sodium 1,2-bis(tetradecanoyl)-sn-glycerol-3-phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebroside, dihexadecanophosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably derived from those having a C 10- C 24 The acyl group of the fatty acid in the carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In some embodiments, other exemplary lipids include, but are not limited to, those of Eygeris et al. Nano Letters. Those described in (20)6: 4543-4549, 2020, are incorporated herein by reference. In some embodiments, such lipids include plant lipids (e.g., DGTS) found to improve liver transfection with mRNA.

[0220] In some embodiments, the lipid-based carrier (or lipid nanoformation) may comprise a combination of distearylphosphatidylcholine / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, 4,5-dimyristoylphosphatidylcholine / cholesterol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) / cholesterol, or lecithin / cholesterol.

[0221] Other examples of suitable noncationic lipids include, but are not limited to, nonphospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfates, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, etc. Other noncationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, which are incorporated herein by reference in their entirety.

[0222] In one embodiment, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids as oleic acid or a compound of formula I, II or IV of US2018 / 0028664 (which is incorporated herein by reference in its entirety).

[0223] The content of non-cationic lipids can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the content of non-cationic lipids is 5%-20% (mol) or 10%-15% (mol) of the total lipid components present.

[0224] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises neutral lipids, and the molar ratio of ionizable lipids to neutral lipids ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1).

[0225] In some embodiments, the lipid-based carrier (or lipid nanoformation) does not contain any phospholipids.

[0226] In some embodiments, the lipid-based carrier (or lipid nanoformation) may further comprise one or more phospholipids, and optionally one or more additional molecules having similar molecular shapes and sizes, which have both hydrophobic and hydrophilic portions (e.g., cholesterol).

[0227] Structural lipids

[0228] The lipid-based carriers (or lipid nanoformulations) described herein may further comprise one or more structured lipids. As used herein, the term "structured lipid" refers to sterols (e.g., cholesterol) and also to lipids containing sterol moieties.

[0229] Incorporating structural lipids into lipid nanoparticles can help reduce the aggregation of other lipids within the particles. Structural lipids can be selected from the group including, but not limited to, cholesterol or cholesterol derivatives, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicosterol, tomatine, tomatine, ursolic acid, α-tocopherol, hornet's salt, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analogue of cholesterol. In some embodiments, the structural lipid is α-tocopherol.

[0230] In some embodiments, structural lipids may be incorporated into lipid-based carriers at a molar ratio (cholesterol phospholipids) ranging from about 0.1 to 1.0.

[0231] In some embodiments, the sterol, when present, may include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or US 2010 / 0130588, which are incorporated herein by reference in their entirety. Other exemplary sterols include phytosterols, including those by Eygeris et al. Nano Lett. Those described in [Nano Communications] 20(6):4543-4549, 2020 are incorporated herein by reference.

[0232] In some embodiments, the structural lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholesterol, 53-cosanosterol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholesterol; nonpolar analogs such as 5α-cholesterol, cholesterone, 5α-cholesterone, 5p-cholesterone, and cholesterol decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in WO 2009 / 127060 and US 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0233] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises sterols in an amount of 0-50 mol% (e.g., 0-10 mol%, 10-20 mol%, 20-50 mol%, 20-30 mol%, 30-40 mol%, or 40-50 mol%) of the total lipid component.

[0234] Polymers and polyethylene glycol (PEG) lipids

[0235] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more polymers or copolymers, such as poly(lactic acid-co-hydroxyacetic acid) (PFAG) nanoparticles.

[0236] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more polyethylene glycol (PEG) lipids. Examples of usable PEG-lipids include, but are not limited to, 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-350] (mPEG 350 PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-550] (mPEG 550 PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-750] (mPEG 750 PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-1000] (mPEG 1000 PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG 2000 PE). PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000] (mPEG3000 PE); 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG 5000 PE); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol) 750] (mPEG 750 ceramide); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol) 2000] (mPEG 2000 ceramide); and N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol) 5000] (mPEG 5000 ceramide). In some embodiments, the PEG lipid is a polyethylene glycol-diacylglycerol (i.e., polyethylene glycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.

[0237] In some embodiments, the lipid-based carrier (or nanoformulation) comprises one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of WO 2019 / 217941, which is incorporated herein by reference in its entirety). In some embodiments, one or more conjugated lipids are formulated with one or more ionic lipids (e.g., non-cationic lipids, such as neutral or anionic or zwitterionic lipids); and one or more sterols (e.g., cholesterol).

[0238] PEG conjugates may include PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14), PEG-dipalmitylglycerol (C16), PEG-distearatelglycerol (C18), PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitylglycamide (C16), and PEG-distearatelglycamide (C18).

[0239] In some embodiments, the conjugated lipids, when present, may include one or more of the following: PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG-dialkoxypropylcarbamate, N-(carbonyl-methoxy-polyethylene glycol 2000)-1,2-distearate-sn-glycerol-3-phosphate ethanolamine sodium salt, and those described in Table 2 of WO 2019 / 051289 (which is incorporated herein by reference in its entirety) and combinations thereof.

[0240] Other exemplary PEG-lipid conjugates are described in, for example, US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, US 2017 / 0119904, US 2018 / 0028664 and WO2017 / 099823, all of which are incorporated herein by reference in their entirety.

[0241] In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of US 2018 / 0028664, which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid has formula II of US 2015 / 0376115 or US 2016 / 0376224, which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dispalmityloxypropyl, or PEG-distearateloxypropyl. In some embodiments, the PEG-lipid comprises one of the following:

[0242] .

[0243] In some embodiments, lipids conjugated to molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates can be used in place of or in combination with PEG-lipids.

[0244] Exemplary conjugated lipids, such as PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are included in Table 2 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0245] In some embodiments, the amount of conjugated lipids (e.g., polyethylene glycol-modified lipids) may be 0-20 mol% of the total lipid component present in the lipid-based carrier (or lipid nanoformulation). In some embodiments, the content of conjugated lipids (e.g., polyethylene glycol-modified lipids) may be 0.5-10 mol% or 2-5 mol% of the total lipid component.

[0246] When needed, the lipid-based carriers (or lipid nanoformations) described herein can be coated with a polymer layer to enhance in vivo stability (e.g., spatially stable LNPs).

[0247] Examples of suitable polymers include, but are not limited to, poly(ethylene glycol) that can form a hydrophilic surface layer that improves the circulating half-life of liposomes and increases the amount of lipid nanoformulations (e.g., liposomes or LNPs) reaching therapeutic targets. See, for example, Working et al. J Pharmacol Exp Ther[Journal of Pharmacology and Experimental Therapeutics], 289: 1128-1133, 1999; Gabizon et al., J Controlled Release [Journal of Controlled Release] 53:275-279, 1998; Adlakha Hutcheon et al. Nat Biotechnol [Nature Biotechnology] 17: 775-779, 1999; and Koning et al., Biochim Biophys Acta [Chinese Journal of Biochemistry and Biophysics] 1420: 153-167, 1999, these references are included in this article in their full text by reference.

[0248] Percentage of lipid nanoform formulation components

[0249] In some embodiments, the lipid-based carrier (or lipid nanoformation) comprises one or more of the compounds described herein, optional non-cationic lipids (e.g., phospholipids), sterols, neutral lipids, and optional conjugated lipids that inhibit particle aggregation (e.g., PEGylated lipids). In some embodiments, the lipid-based carrier (or lipid nanoformation) further comprises a payload (e.g., an inhibitor of the target listed in Table 1; e.g., siRNA listed in Table 4). The amounts of these components may be varied independently to obtain the desired properties. For example, in some embodiments, the amount of ionizable lipids (including the lipid compounds described herein) is from about 20 mol% to about 100 mol% of the total lipid composition (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50-90 mol%); the amount of non-cationic lipids (e.g., phospholipids) is from about 0 mol% to about 50 mol% of the total lipid composition (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%); and the amount of conjugated lipids (e.g., polyethylene glycol-modified lipids) is from about 0.5 mol% to about 20 mol% of the total lipid composition (e.g., 1-10 mol%). (5-10 mol%), and the amount of sterol present is from about 0 mol% to about 60 mol% of the total lipid component (e.g., 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20-40 mol%), provided that the total mol% of the lipid component does not exceed 100%.

[0250] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of ionizable lipids (including lipid compounds described herein), about 0-50 mol% of phospholipids, about 0-50 mol% of sterols, and about 0-10 mol% of polyethylene glycol-modified lipids.

[0251] In some embodiments, the lipid-based load comprises a payload (e.g., an inhibitor of a target listed in Table 1; e.g., siRNA listed in Table 4) formulated in lipid nanoparticles, wherein the lipid nanoparticles comprise about 25-100 mol% ionizable lipids (including lipid compounds described herein), about 0-50 mol% phospholipids, about 0-50 mol% sterols, and about 0-10 mol% polyethylene glycol-modified lipids. In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0252] In one embodiment, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of ionizable lipids (including lipid compounds described herein); about 0-40 mol% of phospholipids (e.g., DSPC), about 0-50 mol% of sterols (e.g., cholesterol), and about 0-10 mol% of polyethylene glycol-modified lipids.

[0253] In some embodiments, the lipid-based load comprises a payload (e.g., inhibitors of targets listed in Table 1; e.g., siRNAs listed in Table 4) formulated in lipid nanoparticles, wherein the lipid nanoparticles comprise about 25-100 mol% ionizable lipids (including lipid compounds described herein); about 0-40 mol% phospholipids (e.g., DSPC), about 0-50 mol% sterols (e.g., cholesterol), and about 0-10 mol% polyethylene glycol-modified lipids. In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0254] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol% (e.g., about 35-55 mol% or about 40-50 mol%) of ionizable lipids (including lipid compounds described herein), about 0-30 mol% (e.g., 5-25 mol% or 10-20 mol%) of phospholipids, about 15-50 mol% (e.g., 18.5-48.5 mol% or 30-40 mol%) of sterols and about 0-10 mol% (e.g., 1-5 mol% or 1.5-2.5 mol%) of polyethylene glycol-modified lipids.

[0255] In some embodiments, the lipid-based load comprises a payload (e.g., an inhibitor of the target listed in Table 1; e.g., siRNA listed in Table 4) formulated in lipid nanoparticles, wherein the lipid nanoparticles comprise about 30-60 mol% (e.g., about 35-55 mol% or about 40-50 mol%) of ionizable lipids (including lipid compounds described herein), about 0-30 mol% (e.g., 5-25 mol% or 10-20 mol%) of phospholipids, about 15-50 mol% (e.g., 18.5-48.5 mol% or 30-40 mol%) of sterols, and about 0-10 mol% (e.g., 1-5 mol% or 1.5-2.5 mol%) of polyethylene glycol-modified lipids. In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0256] In some embodiments, the molar ratio of ionizable lipid / sterol / phospholipid (or another structural lipid) / PEG-lipid / another component varies within the following ranges: ionizable lipid (25%-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).

[0257] In some embodiments, the lipid-based load comprises a payload (e.g., an inhibitor of the target listed in Table 1; e.g., siRNA listed in Table 4) formulated in lipid nanoparticles, wherein the lipid nanoparticles comprise a molar ratio of ionizable lipid / sterol / phospholipid (or another structural lipid) / PEG-lipid / another component in the following ranges: ionizable lipid (25%-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%). In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0258] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises 50%-75% ionizable lipids (including lipid compounds as described herein) by mol% or wt% of total lipid components, 20%-40% sterols (e.g., cholesterol or derivatives), 0-10% noncationic lipids, and 1%-10% conjugated lipids (e.g., polyethylene glycol-modified lipids).

[0259] In some embodiments, the lipid-based load comprises a payload (e.g., inhibitors of targets listed in Table 1; e.g., siRNAs listed in Table 4) formulated in lipid nanoparticles, wherein the lipid nanoparticles comprise 50%-75% by mol% or wt% of total lipid components, including ionizable lipids (including lipid compounds as described herein), 20%-40% by sterols (e.g., cholesterol or its derivatives), 0-10% by non-cationic lipids, and 1%-10% by conjugated lipids (e.g., polyethylene glycol-modified lipids). In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0260] In some embodiments, the lipid-based carrier (or lipid nanoformation) comprises (i) a nucleic acid (e.g., siRNA listed in Table 4); (ii) a cationic lipid comprising from 50 mol% to 65 mol% of the total lipids present in the lipid-based carrier; (iii) a non-cationic lipid comprising a mixture of phospholipids and their cholesterol derivatives, wherein the phospholipids comprise from 3 mol% to 15 mol% of the total lipids present in the lipid-based carrier, and cholesterol or its derivatives comprise from 30 mol% to 40 mol% of the total lipids present in the lipid-based carrier; and (iv) a conjugated lipid comprising from 0.5 mol% to 2 mol% of the total lipids present in the particles.

[0261] In some embodiments, the lipid-based carrier (or lipid nanoformation) comprises (i) a nucleic acid (e.g., siRNA listed in Table 4); (ii) a cationic lipid comprising from 50 mol% to 85 mol% of the total lipids present in the lipid-based carrier; (iii) a non-cationic lipid comprising from 13 mol% to 49.5 mol% of the total lipids present in the lipid-based carrier; and (d) a conjugated lipid comprising from 0.5 mol% to 2 mol% of the total lipids present in the lipid-based carrier.

[0262] In some embodiments, the phospholipid component in the mixture may be present in amounts ranging from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol% (or any portion of these ranges) of the total lipid component. In some embodiments, the lipid-based carrier (or lipid nanoformulation) is phospholipid-free.

[0263] In some embodiments, the sterol component (e.g., cholesterol or its derivatives) in the mixture may account for 25 mol% to 45 mol%, 25 mol% to 40 mol%, 25 mol% to 35 mol%, 25 mol% to 30 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 30 mol% to 35 mol%, 35 mol% to 40 mol%, 27 mol% to 37 mol%, or 27 mol% to 35 mol% (or any portion of these ranges) of the total lipid component.

[0264] In some embodiments, the amount of non-ionizable lipid components in the lipid-based carrier (or lipid nanoformulation) may be from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% of the total lipid components (or any part of these ranges).

[0265] The ratio of total lipid component to payload (e.g., encapsulated agents, such as nucleic acids; e.g., siRNA listed in Table 3 or Table 4) can be varied as needed. For example, the ratio of total lipid component to payload (mass or weight) can be from about 10:1 to about 30:1. In some embodiments, the ratio of total lipid component to payload (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or from about 6:1 to about 9:1. The total lipid component and payload can be adjusted to provide a desired N / P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or higher. Generally, the total lipid content of lipid-based carriers (or lipid nanoformulations) can range from about 5 mg / mL to about 30 mg / mL. The nitrogen:phosphate ratio (N:P ratio) is evaluated at values ​​between 0.1 and 100.

[0266] Encapsulation efficiency of payloads such as proteins and / or nucleic acids describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with lipid nanoformulations (e.g., liposomes or LNPs) after preparation, relative to the initial amount provided. Ideally, encapsulation efficiency is high (e.g., at least 70%, 80%, 90%, 95%, close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing liposomes or LNPs before and after cleavage with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in solution. For the lipid-based carriers (or lipid nanoparticles) described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 70%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0267] Hepatic stellate cell targeting portion

[0268] In some embodiments, the hepatic stellate cell targeting moiety is combined with an inhibitor of the target listed in Table 1 (e.g., an inhibitor listed in Tables 2, 3, or 4) or a carrier thereof. In some embodiments, the hepatic stellate cell targeting moiety is a ligand of one or more receptors expressed or overexpressed in hepatic stellate cells (e.g., activated hepatic stellate cells). In some embodiments, the receptor is selected from type VI collagen receptor, retinol-binding protein (RBP) receptor, platelet-derived growth factor receptor (PDGFR), synaptic growth protein (e.g., major synaptic vesicle protein p38), insulin-like growth factor-II receptor (IGFIIR), low-density lipoprotein receptor (LDLR), TNF-associated apoptosis-inducing ligand receptor (TRAILR), integrin (e.g., αvβ3), and / or differentiation cluster 44 (CD44).

[0269] In some embodiments, one or more ligands of the receptor are selected as the targeting portion for delivering an inhibitor of the targets listed in Table 1. In some embodiments, one or more ligands are conjugated to an inhibitor (e.g., inhibitors listed in Table 2 or Table 3; e.g., siRNA; e.g., siRNA listed in Table 4) via direct conjugation to the inhibitor or by conjugation to a carrier of the inhibitor (e.g., exosomes, liposomes, or lipid nanoparticles). In some embodiments, the ligands are conjugated to the inhibitor in a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ligand is conjugated to the inhibitor in a molar ratio of 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, or greater than 500:1. In some embodiments, one ligand is selected as the targeting moiety. In other embodiments, multiple ligands (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 ligands) are selected as the targeting moiety. In some embodiments, multiple ligands target a single receptor or a mixture of receptors (e.g., 2, 3, 4, 5, or more than 5 different receptors).

[0270] In some embodiments, the ligand is a naturally occurring or synthetic ligand. In other embodiments, the ligand is monovalent or multivalent (e.g., binding to at least two receptors or binding partners). In some embodiments, the ligand is a protein or protein fragment (e.g., a protein domain), a peptide (e.g., an RGD peptide), an antibody or its antigen-binding fragment, a polynucleotide (e.g., an aptamer), a lipid (e.g., cholesterol), a carbohydrate, an antioxidant (e.g., vitamin A), or a combination thereof. In some embodiments, the ligand is linear, branched, or cyclic. In some embodiments, the ligand is a linear peptide. In some embodiments, the ligand is a branched peptide. In some embodiments, the ligand is a cyclic peptide. In some embodiments, the ligand comprises a fusion protein (e.g., an Fc fusion protein or an albumin fusion protein) for increasing stability, half-life, solubility, or a combination thereof. Exemplary targeting portions of hepatic stellate cells can be found elsewhere, for example, Toriyabe et al. J. Pharm. Sci. [Journal of Pharmaceutical Sciences] 106(8):2046-2052, 2017, and Chen et al. J. Pharmacol. Exp. Ther. [Journal of Pharmacology and Experimental Therapy] 370(3):695-702, 2019, each of which is hereby incorporated by reference.

[0271] Carbohydrate Targeting

[0272] In some embodiments, the carbohydrate-targeting portion may form a complex with an inhibitor of the targets listed in Table 1. The carbohydrate-targeting portion may, for example, comprise a sugar, disaccharide, or polysaccharide. In some embodiments, the carbohydrate includes mannose, galactose, or glucose. In some embodiments, the carbohydrate portion described herein comprises one or more monosaccharide portions. In some embodiments, one or more monosaccharide portions comprise at least three carbon atoms (e.g., arranged in a straight chain, branched chain, or cyclic structure) and oxygen, nitrogen, or sulfur atoms, or fragments or variants of a monosaccharide portion comprising at least three carbon atoms (e.g., arranged in a straight chain, branched chain, or cyclic structure) and oxygen, nitrogen, or sulfur atoms. Each monosaccharide portion or fragment or variant thereof may be a tetrasaccharide, pentose, hexose, or heptose. Each monosaccharide portion or fragment or variant thereof may be in the form of an aldose, ketose, sugar alcohol, and, where appropriate, in an L or D form. Exemplary monosaccharide portions may be amino sugars, N-acetylamino sugars, imino sugars, deoxy sugars, or sugar acids. Carbohydrates may comprise individual monosaccharide portions, or may further comprise disaccharides, oligosaccharides (e.g., trisaccharides, tetrasaccharides, pentoses, hexasaccharides, heptasaccharides, octasaccharides), polysaccharides, or combinations thereof. Exemplary carbohydrates include ribose, arabinose, lyseose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, tarose, allose, azulose, allulose, fructose, sorbose, tagatose, rhamnose, pneumose, isorhamnose, fucose, mannoheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine. Glucuronic acid, galacturonic acid, mannulic acid, guluronic acid, iduronic acid, tagatonic acid, fructose, galactosamine, mannulic acid, glucosamine uronic acid, N-acetylglucosamine uronic acid, N-acetylgalactosamine, N-acetylmannulic acid, maltose, lactose, sucrose, trehalose, gentiobiose, cellobiose, chitobiose, kosperidose, aspergillus niger, sophorose, trehalose, isomaltose, xylobiose, starch, cellulose, chitin, and dextran.

[0273] The carbohydrate moiety may comprise one or more monosaccharide moieties linked by glycosidic bonds. In some embodiments, the glycosidic bonds comprise 1→2, 1→3, 1→4, or 1→6 glycosidic bonds. In some embodiments, each glycosidic bond may be in an α or β configuration. In embodiments, one or more monosaccharide moieties are directly linked by glycosidic bonds or separated by linkers.

[0274] As used herein, the term "carbohydrate" refers to a compound comprising one or more monosaccharide moieties containing at least three carbon atoms (e.g., arranged in a straight-chain, branched, or cyclic structure) and oxygen, nitrogen, or sulfur atoms, or a compound comprising fragments or variants of a monosaccharide moieties containing at least three carbon atoms (e.g., arranged in a straight-chain, branched, or cyclic structure) and oxygen, nitrogen, or sulfur atoms. Each monosaccharide moiety or fragment or variant thereof may be a tetrasaccharide, pentose, hexose, or heptose. Each monosaccharide moiety or fragment or variant thereof may be in the form of an aldose, ketose, sugar alcohol, and, where appropriate, in an L or D form. Exemplary monosaccharide moieties may be amino sugars, N-acetylamino sugars, imino sugars, deoxy sugars, or sugar acids. Carbohydrates may comprise individual monosaccharide moieties, or may further comprise disaccharides, oligosaccharides (e.g., trisaccharides, tetrasaccharides, pentoses, hexasaccharides, heptasaccharides, octasaccharides), polysaccharides, or combinations thereof. Exemplary carbohydrates include ribose, arabinose, lysose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, talose, allose, azulose, allulose, fructose, sorbose, tagatose, rhamnose, pneumose, isorhamnose, fucose, mannoheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine. Glucuronic acid, galacturonic acid, mannulic acid, guluronic acid, iduronic acid, tagatonic acid, fructose, galactosamine, mannulic acid, glucosamine uronic acid, N-acetylglucosamine uronic acid, N-acetylgalactosamine, N-acetylmannulic acid, maltose, lactose, sucrose, trehalose, gentiobiose, cellobiose, chitobiose, kosperidose, aspergillus niger, sophorose, trehalose, isomaltose, xylobiose, starch, cellulose, chitin, and dextran.

[0275] Carbohydrates may comprise one or more monosaccharide moieties linked by glycosidic bonds. In some embodiments, the glycosidic bonds comprise 1->2, 1->3, 1->4, or 1->6 glycosidic bonds. In some embodiments, each glycosidic bond may be in an α or β configuration. In embodiments, one or more monosaccharide moieties are directly linked by glycosidic bonds or separated by linkers.

[0276] In some embodiments, the carbohydrate-targeting portion is mannose. For example, the carbohydrate-targeting portion may be α-mannose or high-mannose. In some embodiments, the carbohydrate-targeting portion comprises mannose-6-phosphate (M6P) or an analogue thereof. In embodiments, the carbohydrate-targeting portion comprises a plurality of M6P portions (e.g., M6P), for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more M6P portions. In embodiments, the carbohydrate-targeting portion comprises between 2 and 20 M6P portions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 M6P portions). In some embodiments, the carbohydrate-targeting portion comprises between 2 and 10 M6P portions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P portions). In some embodiments, the carbohydrate-targeting portion comprises between 2 and 5 M6P portions (e.g., 2, 3, 4, or 5 M6P portions). In some embodiments, one or more M6P portions are used to target the inhibitor to the insulin-like growth factor II receptor (IGFIIR) on the surface of hepatic stellate cells (e.g., activated hepatic stellate cells).

[0277] In some embodiments, the carbohydrate targeting portion comprises a galactose (Gal), galactosamine (GalNH2), or N-acetylgalactosamine (GalNAc) moiety, such as Gal, GalNH2, or GalNAc, or the like. In some embodiments, the carbohydrate targeting portion comprises a GalNAc moiety (e.g., GalNAc). In some embodiments, the carbohydrate targeting portion comprises multiple GalNAc moieties (e.g., GalNAc), for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more GalNAc moieties (e.g., GalNAc). In one embodiment, the carbohydrate targeting portion comprises between 2 and 20 GalNAc portions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GalNAc portions). In another embodiment, the carbohydrate targeting portion comprises between 2 and 10 GalNAc portions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 GalNAc portions). In yet another embodiment, the carbohydrate targeting portion comprises between 2 and 5 GalNAc portions (e.g., 2, 3, 4, or 5 GalNAc portions). In yet another embodiment, the carbohydrate targeting portion comprises 2 GalNAc portions. In yet another embodiment, the carbohydrate targeting portion comprises 3 GalNAc portions. In yet another embodiment, the carbohydrate targeting portion comprises 4 GalNAc portions. In yet another embodiment, the carbohydrate targeting portion comprises 5 GalNAc portions. In some embodiments, the carbohydrate targeting portion comprises mono-, di-, tri-, or tetra-GalNAc.

[0278] Any of the aforementioned carbohydrate-targeting portions can also be biotinylated to promote binding to biotin-binding proteins (e.g., avidin, streptavidin, or neutral avidin).

[0279] Treatment

[0280] Treatment with drugs that inhibit the target

[0281] Subjects already identified as having NAFLD (e.g., NAFL or NASH) or at risk of developing NAFLD (e.g., NAFL or NASH) may be treated with the agents described herein (e.g., those listed in Tables 2, 3, or 4). In some embodiments, clinical diagnostic methods known in the art are used to diagnose the subject, such as biopsy of one or more tissues (e.g., from the liver) to detect fibrosis, inflammation, hepatocellular ballooning degeneration, and / or steatosis; magnetic resonance elastography (MRE) to detect or measure fibrosis; magnetic resonance imaging (MRI) to detect or measure steatosis and / or hepatomegaly; ultrasound to detect fibrosis and / or steatosis; blood tests or biomarker tests to detect and / or measure fibrosis, such as the enhanced liver fibrosis (ELF) test, the FibroMeter test, and / or FIBROTEST®, as well as other biomarkers and diagnostic methods known in the art and described herein.

[0282] In addition to the treatments described herein, subjects may be treated with other interventions or therapies such as appropriate diet and exercise, bariatric surgery, and / or medications to induce weight loss. In some embodiments, the additional therapy is a medication to induce weight loss, such as a glucagon-like peptide-1 (GLP-1) receptor agonist, such as duraglutide (TRULICITY®, Eli Lilly and Company), exenatide (BYETTA®, AstraZeneca; and BYDUREON BCise®, AstraZeneca), liraglutide (VICTOZA®, Novo Nordisk A / S; and SAXENDA®, Novo Nordisk), liximaglutide, semaglutide (OZEMPIC®, Novo Nordisk; WEGOVY®, Novo Nordisk; and RYBELSUS®, Novo Nordisk), or telpopotide (MOUNJARO®, Eli Lilly). GLP-1 receptor agonists and their potential for treating NAFLD (e.g., NAFL or NASH) have been described elsewhere, such as by Nevola et al. Int J Mol Sci. [International Journal of Molecular Sciences] 24(2):1703,2023), which is hereby incorporated by reference. In some embodiments, an additional therapy is a thyroid hormone receptor-β agonist, such as resmetiro (CAS No. 920509-32-6), which has recently been approved by the U.S. Food and Drug Administration for the treatment of adults with non-cirrhotic NASH with moderate to advanced liver fibrosis.

[0283] Subjects may also be identified as being at risk of developing NAFLD or progressing from NAFLD to NASH based on a family history of NAFLD or other relevant risk factors described herein (e.g., obesity). Treating subjects who develop NAFLD or are at risk of developing NAFLD can reduce the likelihood of developing NAFL or NASH, suppress the onset of NAFL or NASH, and / or delay the onset of NAFL or NASH. In some embodiments, methods of treating subjects with NAFLD (e.g., NAFL or NASH) can be further used to reduce, slow, suppress, and / or reverse the progression of fibrosis, steatosis, inflammation, and / or hepatocellular ballooning degeneration.

[0284] Subjects diagnosed with NAFL or NASH (e.g., using standard diagnostic methods) are treated with the agents described herein (e.g., those listed in Tables 2, 3, or 4). In some embodiments, the treatment is used to reduce, slow, and / or inhibit the progression of the disease and / or reverse NAFLD (e.g., NAFL or NASH) in subjects diagnosed with NAFLD. In some embodiments, these methods may be further used to reduce, slow, inhibit, and / or reverse the progression of fibrosis in subjects diagnosed with NAFL or NASH. In some embodiments, these methods may be further used to reduce, slow, inhibit, and / or reverse the progression of steatosis in subjects diagnosed with NAFL or NASH. In some embodiments, these methods may be used to stabilize the subject's condition (e.g., stabilize the subject's NAFL or NASH so that it does not progress or worsen). In other embodiments, these methods may be used to alleviate, improve, reduce, or reverse the clinical manifestations of the disease (e.g., alleviate, improve, reduce, or reverse the progression of liver fibrosis, liver inflammation, and / or hepatic steatosis).

[0285] This method may include delivering the disclosed pharmaceutical agent or pharmaceutical composition thereof to the liver, muscle, blood, or any affected tissue of a subject (e.g., a human) via any suitable route of administration (e.g., subcutaneous, intravenous, intramuscular, etc.). This method may include delivering the disclosed pharmaceutical agent or pharmaceutical composition thereof to hepatic stellate cells via any suitable route of administration. Exemplary routes of administration are oral administration, intravenous injection, subcutaneous injection, or intramuscular injection. The pharmaceutical agent may be administered at any suitable dose. The actual dose of the disclosed composition administered to a patient may be determined by physical and physiological factors such as weight, severity of illness, prior or concurrent treatment interventions, the patient's idiopathic condition, and the route of administration. Depending on the dose and route of administration, the preferred dose and / or the frequency of administration of the effective amount may vary depending on the subject's response. In any case, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and one or more appropriate doses for the individual subject. Administration may be performed daily, weekly, monthly, or annually at any suitable frequency and whenever necessary. The subject may be an adult or pediatric human, with or without comorbidities.

[0286] Combination therapy

[0287] This method may include administering medications in combination with one or more other therapies (e.g., 1, 2, or 3 additional agents). Two or more agents may be administered simultaneously (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes, or less). These agents may also be administered simultaneously via co-prepared formulations. Two or more agents may also be administered sequentially, such that the effects of the two or more agents overlap, and their combined effect results in a decrease in symptoms or other parameters related to the disorder greater than would be observed with the delivery of a single agent or treatment alone or without the other. The effects of the two or more treatments may be partially additive, completely additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each agent may be achieved via, but is not limited to, oral, intravenous, intramuscular, local, and any suitable route of direct absorption through mucosal tissue. These agents may be administered via the same or different routes. For example, the first agent of the combination may be administered intravenously, while the second agent of the combination may be administered locally. The first dose can be applied immediately before or after the second dose, for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7, 1-14, 1-21, or 1-30 days.

[0288] For use in the treatment of NAFL or NASH (e.g., in subjects diagnosed with NAFL or NASH or at risk of developing either of these conditions), the second inhibitor may be one or more agents listed in Tables 2, 3, or 4. In some embodiments, two or more inhibitors are inhibitors of different targets listed in Table 1. In some embodiments, two or more inhibitors are inhibitors of two or more of EPHA, ITGAV, and NAV2. In some embodiments, two or more inhibitors are inhibitors of EPHA3 and ITGAV. In some embodiments, two or more inhibitors are inhibitors of EPHA3 and NAV2. In some embodiments, two or more inhibitors are inhibitors of ITGAV and NAV2. In some embodiments, three or more inhibitors are inhibitors of all three of EPHA3, ITGAV, and NAV2. In some embodiments, the same targets listed in Table 1 are inhibited by two or more therapeutic platforms listed in Table 2. In other embodiments, the same targets listed in Table 1 are inhibited by two or more inhibitory nucleic acid molecules (e.g., two or more siRNAs as listed in Tables 3 or 4). One or more inhibitors targeting one or more of the targets listed in Table 1 may be used in combination with other therapies (e.g., thyroid hormone receptor-β agonists, such as resimeltiro) to treat NAFL or NASH in subjects in need.

[0289] Evaluate efficacy

[0290] Determining the efficacy of treatments for subjects with NAFLD (e.g., NAFL or NASH) or at risk of developing it may require evaluating the subject's response to treatment. The subject's response can be evaluated in an in-patient treatment setting or an out-of-patient treatment setting. The subject's response can be evaluated once or multiple times after administration of one or more inhibitors (e.g., one or more inhibitors of the targets listed in Table 1, or one or more inhibitors as described herein). The subject's response can be evaluated continuously, occasionally, or at specified time points after administration of one or more inhibitors. The subject's response can be evaluated, for example, 1–10 days after administration of one or more inhibitors (e.g., 7–10 days, 6–8 days, 5–7 days, 3–5 days, 2–4 days, 1–3 days, or 1 day after administration of one or more inhibitors; e.g., 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less than one day after administration of one or more inhibitors). Subject responses can be evaluated 1–12 weeks or longer after administration of one or more inhibitors (e.g., 10–12 weeks, 8–12 weeks, 6–12 weeks, 8–10 weeks, 6–10 weeks, 4–10 weeks, 6–8 weeks, 4–8 weeks, 2–8 weeks, 4–6 weeks, 2–6 weeks, 3–6 weeks, 2–4 weeks, 1–3 weeks, or 1–2 weeks after administration of one or more inhibitors; e.g., later than 12 weeks, 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more inhibitors). Subject response can be evaluated 1–12 months after administration of one or more inhibitors (e.g., 10–12 months, 8–10 months, 6–8 months, 4–6 months, 3–5 months, 2–4 months, or 1–3 months after administration of one or more inhibitors; e.g., 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month after administration of one or more inhibitors). Subject response can be evaluated 1–5 years or longer after administration of one or more inhibitors (e.g., 4–5 years, 3–5 years, 2–3 years, or 1–3 years after administration of one or more inhibitors; e.g., later than 5 years, 5 years, 4 years, 3 years, 2 years, or 1 year after administration of one or more inhibitors).

[0291] The efficacy of a treatment method can be evaluated by comparing one or more measures of a subject to be treated with a relevant control or reference. In some embodiments, the efficacy of the treatment method is evaluated by comparing one or more measures of a subject undergoing treatment with one or more measures of the same subject prior to treatment. In some embodiments, the efficacy of the treatment method is evaluated by comparing one or more measures of a subject undergoing treatment with one or more measures of different subjects who have NAFLD or are at risk of developing NAFLD and have not yet undergone treatment. In other embodiments, the efficacy of the treatment method is evaluated by comparing one or more measures of a subject undergoing treatment with one or more measures of healthy subjects (e.g., subjects who do not have NAFLD or are currently not at risk of developing NAFLD).

[0292] In some embodiments, the efficacy of a treatment for a subject is evaluated by histopathological analysis of liver tissue in a subject with NAFLD or at risk of developing NAFLD, using one or more methods known in the art, to assess fibrosis, inflammation, hepatocellular ballooning degeneration, and / or steatosis. In some embodiments, histopathological analysis of liver tissue or other tissues is performed via one or more biopsies during treatment to evaluate liver fibrosis, inflammation, and / or steatosis. In some embodiments, treatment efficacy is evaluated by tissue staining of samples (e.g., liver samples) using trichrome staining or Sirius red staining to measure the levels of one or more extracellular matrix components (e.g., collagen (e.g., type I, III, and IV collagen), hyaluronic acid, laminin, fibronectin, crude fibroregulatory agents, elastin) during treatment. In some embodiments, one or more magnetic resonance elastography (MRE) devices are used to detect or measure fibrosis during treatment to evaluate treatment efficacy. In other embodiments, treatment efficacy is evaluated by one or more ultrasound methods (e.g., FIBROSCAN®) to detect or measure fibrosis and / or steatosis during treatment.

[0293] In some embodiments, after administration of one or more inhibitors described herein, the level of liver fibrosis in subjects with NAFLD or at risk of developing NAFLD is reduced by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the subject's pre-treatment level of liver fibrosis. In some embodiments, after administration of one or more inhibitors described herein, the level of liver fibrosis in subjects with NAFLD or at risk of developing NAFLD is reduced by one or more levels or stages (e.g., F3 to F2, F2 to F1, or F1 to F0) compared to the subject's pre-treatment level of fibrosis. In some embodiments, after administration of one or more inhibitors described herein, the level of hepatic steatosis in subjects with NAFLD or at risk of developing NAFLD is reduced by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the subject's pre-treatment level of hepatic steatosis. In some embodiments, after administration of one or more inhibitors described herein, the level of hepatic steatosis in subjects with NAFLD or at risk of developing NAFLD is reduced such that the steatosis stage of the subject is reduced by one or more levels or stages compared to the subject's steatosis level before treatment (e.g., as measured by NAS; e.g., 3 to 2, 2 to 1, or 1 to 0).

[0294] In some embodiments, after administration of one or more inhibitors described herein, the level of liver inflammation in subjects with NAFLD or at risk of developing NAFLD is reduced by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the subject's pre-treatment level of liver inflammation. In some embodiments, after administration of one or more inhibitors described herein, the level of liver inflammation in subjects with NAFLD or at risk of developing NAFLD is reduced by one or more levels or stages (e.g., as measured by NAS; e.g., 3 to 2, 2 to 1, or 1 to 0) compared to the subject's pre-treatment level of liver inflammation.

[0295] In some embodiments, after administration of one or more inhibitors described herein, the level of hepatocyte ballooning in a subject with NAFLD or at risk of developing NAFLD is reduced such that the level of hepatocyte ballooning is reduced by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the subject's pre-treatment level of hepatocyte ballooning. In some embodiments, after administration of one or more inhibitors described herein, the level of hepatocyte ballooning in a subject with NAFLD or at risk of developing NAFLD is reduced such that the subject's hepatocyte ballooning stage is reduced by one or more levels or stages (e.g., as measured by NAS; e.g., 2 to 1 or 1 to 0) compared to the subject's pre-treatment level of hepatocyte ballooning.

[0296] In some embodiments, the efficacy of a treatment for a subject with NAFLD or at risk of developing NAFLD is evaluated by measuring the expression or activity of one or more targets listed in Table 1 during treatment. In some embodiments, after administration of one or more inhibitors targeting one or more targets listed in Table 1 (e.g., inhibitors listed in Tables 2, 3, or 4) to a subject, the expression or activity of one or more targets listed in Table 1 is reduced by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the expression or activity of one or more targets listed in Table 1 is evaluated by obtaining one or more biological samples (e.g., blood samples or components thereof or liver biopsies) from the subject and comparing the results of earlier measurements from the subject with / or results from the subject measured prior to administration of one or more inhibitors. In some embodiments, the expression or activity of one or more targets listed in Table 1 is detected by biological or analytical methods known in the art (e.g., mass spectrometry, Western blotting, ELISA, RT-PCR, flow cytometry, immunofluorescence, microscopy, in situ hybridization, colorimetric assay, arrays of targeting antibodies or hybrid nucleotides (e.g., aptamers), and other methods known in the art).

[0297] The additional biomarkers described herein can be used to evaluate the efficacy of treatment administered to subjects in need. Efficacy can be evaluated using methods known in the art, such as the ELF test, the FibroMeter test, and / or FIBROTEST®. Efficacy can be assessed by measuring blood or serum levels of one or more biomarkers, such as type III procollagen peptide (PICP), hyaluronic acid, tissue inhibitor of metalloproteinases 1 (TIMP-1), metalloproteinases (e.g., matrix metallopeptidase-1 (MMP1), MMP2, and MMP3), retinol-binding protein 4 (RBP4), adiponectin (ADIPOQ), leptin (LEP), resistin (RETN), or gastroglycemic index (GHRL); liver transaminase levels, such as the AST / ALT ratio; cholesterol levels, such as low-density lipoprotein (LDL) cholesterol or reduced high-density lipoprotein (HDL) cholesterol levels; triglyceride levels; uric acid levels; and other biomarkers described herein or known in the art.

[0298] Depending on the efficacy of the treatment administered to subjects with NAFLD or at risk of developing NAFLD, the treatment regimen may be modified during treatment. The treatment regimen and its efficacy can be determined by those skilled in the art (e.g., physicians, clinicians, or medical specialists). In some embodiments, the dosage of one or more inhibitors of the targets listed in Table 1 may be adjusted (e.g., decreased or increased) during treatment. In some embodiments, the dosage of one or more inhibitors may be increased by about 10%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, or more during treatment. In some embodiments, the dosage of one or more inhibitors may be decreased by about 10%, 25%, 50%, 75%, or more than 75% during treatment. In some embodiments, the frequency of administration of one or more inhibitors may be increased or decreased during treatment. In other embodiments, the duration of treatment may be increased or decreased. In some embodiments, different combinations of inhibitors may be administered, such that new combinations of different types of inhibitors listed in Tables 2, 3, or 4 are administered to subjects in need during treatment. In other embodiments, different combinations of the same inhibitor agents (e.g., two or more siRNAs listed in Table 4) may be administered to subjects in need during treatment.

[0299] Example

[0300] Example 1. NAFLD targets inhibited by siRNA

[0301] This example describes siRNA knockdown of several representative targets identified by the inventors for the treatment of NAFLD, steatosis, liver inflammation, hepatocellular ballooning degeneration and / or liver fibrosis, or for reducing fibrosis-related proteins in the liver cells of subjects.

[0302] Immortalized human stellate cells (e.g., LX-2) or primary human hepatic stellate cells were seeded in 12-well plates at a density of 75,000 cells / well. One day later, lipid-based transfection with siRNA (e.g., Lipofectamine) was performed at a final concentration of 100 nM in serum and antibiotic-free Duchenne Modified Eagle's Medium (DMEM, LX-2) or serum-free stellate cell medium (SteCM, ScienCell, primary human hepatic stellate cells). TM RNAiMAX (Thermo Fisher Scientific) transfected cells with a mixture of siRNAs targeting identified targets. As a negative control, some stellate cells were transfected with disordered siRNA (siNEG) or a mediator-only formulation. As a positive control, some stellate cells were transfected with siRNA that inhibits COL1A1 expression. The next day, the medium in the 12-well plates was replaced with fresh serum-free basal medium with 1 ng / mL transforming growth factor-β (TGF-β) to induce stellate cell activation.

[0303] Changes in gene expression were analyzed 24 hours after TGF-β administration. The efficiency of siRNA knockdown was assessed by qPCR. Changes in gene expression of representative query targets were observed. Figure 1 As shown in Table 5, siRNA effectively reduces gene expression of the targets listed in Table 5.

[0304] Table 5. Knockdown efficiency

[0305] EPHA3 = type A liver ligand receptor 3; ITGAV = integrin subunit αV; NAV2 = neuronal navigator 2; + = Compared with the siNEG control, gene expression was reduced by 30% or more.

[0306] Example 2. Reduction in the secretion of activation-related genes and proteins in astrocytes treated with siRNA targeting the identified target.

[0307] The cell supernatant of TGF-β-induced, siRNA-treated, and control human stellate cells described in Example 1 was harvested and lysed in RLT buffer (Qiagen) with 1% β-mercaptoethanol. Secreted procollagen 1α1 was measured in conditioned medium using the R&D Systems Human Procollagen Iα1 DuoSet ELISA Kit (catalog number DY6220-05). COL1A1 and ACTA2 mRNA expression levels were assessed by qPCR. The reduction in stellate cell activation was assessed by comparing cell populations treated with disordered siRNA as a control (siNEG). Figure 2 As illustrated in the examples and Table 6, siRNA inhibition of representative identified targets reduced stellate cell activation in human hepatic stellate cells.

[0308] Table 6. Activation of hepatic stellate cells

[0309] EPHA3 = type A liver ligand receptor 3; ITGAV = integrin subunit αV; NAV2 = neuronal navigator 2

[0310] +Reduced by 10%-39.9% compared to the siNEG control.

[0311] ++Reduced by 40%-69.9% compared to the siNEG control.

[0312] +++ Reduction of 70%-89.9% compared to the siNEG control.

[0313] ++++Reduced by >90% compared to siNEG control

[0314] Example 3. Reduction in the secretion of activation-related proteins in astrocytes treated with compounds targeting the identified targets.

[0315] This example describes treatment with small molecules targeting EPHA3 for the treatment of NAFLD, steatosis, liver inflammation, hepatocellular ballooning degeneration and / or liver fibrosis, or for reducing fibrosis-related proteins in the liver cells of a subject.

[0316] Immortalized human stellate cells (e.g., LX-2) or primary human hepatic stellate cells were plated as described in Example 1. After 24 hours, the cells were treated with different doses (typically ranging from 0.01 μM to 10 μM, but within the in vitro pharmacodynamic parameters of a given molecule) of a compound designed to inhibit a given target protein. As a control, some cells were treated with a mediator-only formulation (e.g., DMSO only). As an additional control, some stellate cells were treated with an inhibitor of the TGF-β receptor. 1 ng / mL of TGF-β was administered to the cells concurrently with the compound administration. All compound / TGF-β formulations were prepared in serum-free basal medium. The stellate cells were treated for 24 hours prior to harvest. Following treatment, hepatic stellate cell activation was assessed by measuring secreted COL1A1 in the medium using ELISA, as described in Example 2. Treatment of human hepatic stellate cells with the small molecule AWL-II-38.3, which inhibits EPHA3, resulted in a dose-dependent reduction in collagen secretion, indicating reduced stellate cell activation, as shown in Example 2. Figure 3 As shown.

[0317] Example 4. Treatment of subjects at risk of developing NAFLD

[0318] Due to poor dietary and exercise habits, large waist circumference (e.g., approximately ≥80 cm for men and approximately ≥78 cm for women), and age (e.g., ≥45 years), clinicians identified subjects as being at risk of developing NAFLD and further developing liver fibrosis. Subjects were evaluated for steatosis by magnetic resonance imaging (MRI) and for fibrosis by magnetic resonance elastography (MRE), and were classified as having a fibrosis score of F0 or no detectable fibrosis. To reduce the likelihood of developing F1, F2, F3, or F4 liver fibrosis, subjects were then administered (e.g., intravenously or subcutaneously) a therapeutically effective amount of a pharmaceutical composition containing one or more inhibitory nucleic acid molecules (e.g., one or more siRNA molecules having a nucleotide sequence of any one or more of SEQ ID NO: 9-32, e.g., the siRNA molecules in Table 4). An inhibitory nucleic acid molecule is formulated in an LNP conjugated to a hepatic stellate cell-targeting moiety (e.g., vitamin A or a cyclic RGD peptide) that binds to a hepatic stellate cell receptor (e.g., an RBP receptor or a type VI collagen receptor). Following treatment (e.g., administration of the drug composition 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times over a period of 3, 6, 9, 12, 15, or 18 months), the levels of hepatic steatosis and fibrosis are measured again and compared to the fibrosis levels measured before treatment.

[0319] Example 5. Treatment of fibrosis in subjects with NAFLD

[0320] Subjects previously diagnosed with NAFLD (e.g., NAFL or NASH) have an F2 fibrosis score, indicating moderate liver fibrosis, as determined by ultrasound (e.g., FIBROSCAN®). The subjects are then treated with a therapeutically effective amount of siRNA formulated as a pharmaceutical composition with LNP (e.g., one or more siRNA molecules from SEQ ID NO: 9-32, e.g., siRNA molecules from Table 4). Following siRNA treatment (e.g., 1, 2, 3, or 4 doses over a one-year period), additional ultrasound is performed to determine changes in fibrosis in the liver. No changes indicate that the treatment is effective and reducing progression. The subject continues treatment, and their NAFLD does not progress to cirrhosis. Ultrasound is performed approximately 18 to 24 months after treatment. Ultrasound determines that the subject has an F1 liver fibrosis score or mild fibrosis, compared to an F2 fibrosis score measured before treatment, indicating that the treatment can reverse fibrosis.

[0321] Example 6. Treatment of subjects with NASH

[0322] Subjects previously diagnosed with NASH had approximately 16% hepatic steatosis (i.e., 16% of hepatocytes in the liver exhibiting lipid accumulation), as determined by magnetic resonance imaging (MRI), and evidence of liver inflammation and hepatocyte ballooning, as determined by liver biopsy. Magnetic resonance elastography (MRE) and ultrasound (e.g., FIBROSCAN®) also determined that the subject's liver had no measurable fibrosis. The subject was then treated with a therapeutically effective amount of siRNA formulated as a pharmaceutical composition with LNP (e.g., any one or more siRNA molecules in SEQ ID NO: 9-32, e.g., siRNA molecules in Table 4) to reduce the likelihood of the subject developing liver fibrosis. After treatment with siRNA (e.g., 1, 2, 3, or 4 doses of siRNA over a one-year period), MRI was performed again to determine the subject's hepatic steatosis. The subject may be further treated and re-evaluated with MRE at 2, 3, 5 years, or longer to determine the progression of fibrosis.

[0323] Other embodiments

[0324] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0325] While the invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention that generally follow the principles and include known or conventional practices within the field to which the invention pertains, and may be applied to the essential features set forth above and in accordance with the scope of the claims.

[0326] Other embodiments are defined in the claims.

Claims

1. A method for treating a human subject identified as having non-alcoholic fatty liver disease (NAFLD), the method comprising administering to the subject an inhibitor of type A liver ligand receptor 3 (EPHA3), integrin subunit αV (ITGAV), or neuronal navigator 2 (NAV2).

2. A method for treating a human subject identified as being at risk of developing NAFLD, the method comprising administering an inhibitor of EPHA3, ITGAV, or NAV2 to the subject.

3. The method of claim 1 or 2, wherein the subject has liver fibrosis.

4. The method of claim 3, wherein the liver fibrosis is stage 0 fibrosis (F0), stage 1 fibrosis (F1), stage 2 fibrosis (F2), stage 3 fibrosis (F3), or stage 4 fibrosis (F4).

5. The method of any one of claims 1-4, wherein the NAFLD is non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH).

6. The method of any one of claims 1-5, wherein the inhibitor is delivered to the hepatic stellate cells of the subject.

7. A method for reducing the deposition of one or more extracellular matrix components in the liver of a human subject, the method comprising administering an inhibitor of EPHA3, ITGAV, or NAV2 to the subject.

8. The method of claim 7, wherein the one or more extracellular matrix components are selected from the group consisting of: Collagen, hyaluronic acid, laminin, fibronectin, crude fiber regulator and elastin.

9. A method for treating liver fibrosis in a human subject identified as having NAFLD or at risk of having NAFLD, the method comprising administering an inhibitor of EPHA3, ITGAV, or NAV2 to the subject.

10. The method of claim 9, wherein the subject suffers from NASH or NAFL.

11. The method of claim 9 or 10, wherein the subject suffers from liver fibrosis.

12. The method of claim 9 or 10, wherein the subject does not have liver fibrosis.

13. The method of claim 11, wherein the liver fibrosis is F0, F1, F2, F3 or F4.

14. The method of any one of claims 1-13, wherein the inhibitor is a pharmaceutical agent listed in Table 2.

15. The method of any one of claims 1-14, wherein the inhibitor is selected from the group consisting of: (i) Small interfering RNA (siRNA); (ii) Antisense oligonucleotides (ASO); (iii) The small molecules listed in Table 3; and (iv) Antibodies listed in Table 3.

16. The method of claim 15, wherein the siRNA is one of the siRNAs listed in Table 4.

17. The method of any one of claims 1-16, wherein the method further comprises the step of administering an additional therapeutic agent to the subject.

18. The method of claim 17, wherein the additional therapeutic agent is selected from the group consisting of: (i) siRNAs listed in Table 4; (ii) ASO; (iii) Small molecules listed in Table 3; and (iv) Antibodies listed in Table 3.

19. The method of any one of claims 1-18, wherein the inhibitor is administered parenterally.

20. The method of any one of claims 1-19, wherein the inhibitor is administered in an amount sufficient to reduce liver fibrosis in the subject.

21. The method of claim 20, wherein the inhibitor further reduces hepatic steatosis and / or liver inflammation in the subject.

22. The method of any one of claims 1-21, wherein the inhibitor comprises a delivery medium.

23. The method of claim 22, wherein the delivery medium is lipid nanoparticles.

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