Methods to stimulate cell proliferation

Abstract

Disclosed is a method for treating or preventing diseases associated with fibrosis, and an agent for use in such a method.The method comprises inhibiting at least one of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.Also disclosed is a method for promoting the regeneration of cells such as hepatocytes.

Description

[Technical Field]

[0001] This application claims priority from SG10202007297P, filed on 30 July 2020, the contents and elements thereof being incorporated herein by reference for all purposes.

[0002] This disclosure relates, in general, to the field of regenerative medicine. In particular, this specification teaches a method for stimulating or increasing cell proliferation and / or regeneration in a subject, comprising the step of administering an inhibitor of a gene associated with organ regeneration or a corresponding gene product to the subject to stimulate or increase cell proliferation in the subject. [Background technology]

[0003] The rising incidence of acute and chronic liver failure, which causes more than 1.3 million deaths worldwide each year (World Health Organization, 2018), is a major global health concern. The main underlying causes of end-stage liver disease include hepatitis virus infections (particularly hepatitis B and C), drug- and alcohol-induced liver injury, and non-alcoholic fatty liver disease (NAFLD; associated with obesity and progressing to non-alcoholic steatohepatitis (NASH)). Asia has a particularly high burden of hepatitis virus infections (WHO) and a high incidence of NAFLD. Despite advances in the prevention and treatment of viral hepatitis (hepatitis B vaccination and hepatitis C combination therapy), the number of people with end-stage liver disease is projected to remain high, mainly exacerbated by the prevalence of obesity and aging societies.

[0004] Currently, the only therapeutic treatment for end-stage liver disease is liver transplantation. However, donor organs are limited, and patients with end-stage liver disease may also develop complications that make them unsuitable for major surgery. Therefore, alternative strategies to slow or reverse end-stage liver disease are being explored. These include cell transplantation, artificial liver devices, and enhancing the endogenous regenerative capacity of organs.

[0005] The liver is the only visceral organ with a remarkable ability to regenerate. It has been shown that it can regenerate to its full size with only 25% of its original mass. Adult hepatocytes have a long lifespan and normally do not undergo cell division (G0). However, when the liver is injured, they enter the cell cycle and gain the ability to proliferate. Once cell proliferation is complete, the newly divided cells undergo reconstruction, and the regeneration process is completed through other regeneration-related processes, such as angiogenesis and the remodeling of the extracellular matrix.

[0006] Despite this remarkable ability, the liver's regenerative capacity appears to be limited, especially under conditions of chronic injury. The liver's regenerative capacity is central to hepatic homeostasis. As the primary site of drug detoxification, the liver is exposed to numerous chemicals throughout the body that are likely to induce cell death and injury. Furthermore, it is exposed to microbiome-related metabolites through enterohepatic circulation. The liver can rapidly regenerate damaged tissue, thereby preventing dysfunction. Liver regeneration is also important for patients who have had part of their liver removed through tumor resection or living-donor organ transplantation.

[0007] Over the past 30 years, scientists have gained a better understanding of the liver regeneration process. For example, cytokines IL-6 and TNFα stimulate hepatocytes to enter the cell cycle, and mitogens such as HGF and EGF are important for driving proliferation. However, the processes that facilitate the regeneration process are not well understood. Importantly, the regeneration response involves not only liver-specific signals but also signals from distal organs.

[0008] Numerous different processes, including those related to nutrients, oxygen levels, and others, are involved in modifying the regeneration reaction. Importantly, the complex structure of the liver, particularly its interactions with other organs, cannot be fully simulated in vitro, and therefore in vivo experiments are essential. The disadvantage of in vivo models lies in the limited potential for high-throughput drug discovery pipelines, especially compound screening.

[0009] Therefore, it is necessary to overcome or at least mitigate one or more of the above problems. [Overview of the project] [Problems that the invention aims to solve]

[0010] This invention relates to the treatment and / or prevention of disease by inhibiting genes and / or proteins identified as being upregulated in the fibrosis-promoting process. Such gene / protein inhibition has protective and regenerative effects. [Means for solving the problem]

[0011] This disclosure provides a method for treating or preventing fibrosis and related diseases, comprising the step of inhibiting at least one of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.

[0012] Also provided is a method for treating or preventing fibrosis-related diseases, comprising the step of administering a therapeutic or prophylactic effective dose of at least one inhibitor of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0013] Also provided are at least one inhibitor of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB for use in methods of treating or preventing fibrosis and related diseases.

[0014] Furthermore, the use of at least one inhibitor of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB is provided in the manufacture of a pharmaceutical product for use in a method of treating or preventing fibrosis and related diseases.

[0015] In some aspects, the disease is a disease or condition of the liver.

[0016] In some embodiments, the disease or condition is selected from acute liver disease, chronic liver disease, metabolic liver disease, fatty liver, hepatic fibrosis, primary sclerosing cholangitis (PSC), cirrhosis, mild hepatic fibrosis, progressive hepatic fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (ALFD), alcohol-related liver disease (ARLD), hepatic ischemia-reperfusion injury, primary biliary cirrhosis (PBC), hepatitis, liver injury, liver damage, liver failure, metabolic syndrome, obesity, diabetes mellitus, end-stage liver disease, inflammation of the liver, inflammation of the lobules, and / or hepatocellular carcinoma (HCC).

[0017] In some embodiments, the inhibitor is selected from nucleic acids, peptides, antibodies, antigen-binding molecules, or small molecule inhibitors. In some embodiments, the inhibitor can bind to polypeptides following one or more of SEQ ID NOs. 7156-7178, or to mRNA following one of SEQ ID NOs. 7179-7195.

[0018] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid having at least 75% sequence identity to any one or a portion thereof of sequence numbers 7179-7195, or having at least 75% sequence identity to the reverse complement or a portion thereof of any one of sequence numbers 7179-7195.

[0019] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid that comprises or comprises a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1 to 7155, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 1 to 7155.

[0020] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 6, 7, 457-1482, 7095, 7096, 7109-7114, 7130-7140, 7144, 7145, 7149, 7150, 7154 and / or 7155, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 6, 7, 457-1482, 7095, 7096, 7109-7114, 7130-7140, 7144, 7145, 7149, 7150, 7154 and / or 7155, wherein the antisense nucleic acid is capable of reducing the gene and / or protein expression of ITFG1.

[0021] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1, 2, 14-347, 7092, 7093, 7097-7102, 7115-7120, 7141, 7142, 7146, 7147, 7151 and / or 7152, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 1, 2, 14-347, 7092, 7093, 7097-7102, 7115-7120, 7141, 7142, 7146, 7147, 7151 and / or 7152, wherein the antisense nucleic acid is capable of reducing the gene and / or protein expression of MFAP4.

[0022] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 3 - 5, 348 - 456, 7094, 7103 - 7108, 7121 - 7129, 7143, 7148, and / or 7153, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 3 - 5, 348 - 456, 7094, 7103 - 7108, 7121 - 7129, 7143, 7148, and / or 7153, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of GRHPR.

[0023] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 1483 - 2208, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 1483 - 2208, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of ABCC4.

[0024] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or encoding an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 2209 - 5060, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 2209 - 5060, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of PAK3.

[0025] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or consisting of an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 5061 - 5389 and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 5061 - 5389, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of TRNP1.

[0026] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or consisting of an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 5390 - 5966 and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 5390 - 5966, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of APLN.

[0027] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or consisting of an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 5967 - 6974 and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 5967 - 6974, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of KIF20A.

[0028] In some embodiments, the inhibitor is an inhibitory nucleic acid comprising or consisting of an antisense nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 6975 - 7091 and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 6975 - 7091, and optionally, the antisense nucleic acid is capable of reducing the gene and / or protein expression of LTB.

[0029] In some embodiments, the inhibitory nucleic acid includes (i) a nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to one of sequence numbers 1-7096 or 7146-7150, or a nucleotide sequence having at least 75% sequence identity to one of sequence numbers 1-7096 or 7146-7150, and (ii) a nucleic acid comprising a nucleotide sequence having the reverse complement of the nucleotide sequence in (i), or having at least 75% sequence identity to the reverse complement of the nucleotide sequence in (i).

[0030] In some embodiments, the inhibitory nucleic acids are 2'-O-methyluridine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 2'- It comprises one or more modified nucleotides selected from ruoraulyzin-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate.

[0031] In some embodiments, the inhibitory nucleic acid comprises (i) a nucleic acid comprising a nucleotide sequence (including modifications thereof) represented in one of sequence numbers 7146 to 7150, and (ii) a nucleic acid comprising a nucleotide sequence (including modifications thereof) represented in one of sequence numbers 7151 to 7155.

[0032] In some embodiments, the inhibitor includes a portion that promotes the uptake of the inhibitory nucleic acid by hepatocytes. In some embodiments, the nucleic acid inhibitor is an antisense nucleic acid, siRNA, or shRNA.

[0033] In some embodiments, the method includes administering an inhibitor to a subject in which the expression and / or activity of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB is upregulated.

[0034] Furthermore, inhibitory nucleic acids are provided for reducing the gene and / or protein expression of ITFG1, the nucleic acids comprising or encoding an antisense nucleic acid having at least 75% sequence identity to SEQ ID NO: 7182 or a portion thereof, or having at least 75% sequence identity to the reverse complement of SEQ ID NO: 7182 or a portion thereof.

[0035] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 6, 7, 457-1482, 7095, 7096, 7109-7114, 7130-7140, 7144, 7145, 7149, 7150, 7154 and / or 7155, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 6, 7, 457-1482, 7095, 7096, 7109-7114, 7130-7140, 7144, 7145, 7149, 7150, 7154 and / or 7155.

[0036] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of MFAP4, wherein the nucleic acids include or encode antisense nucleic acids having at least 75% sequence identity to SEQ ID NO: 7179 or 7180 or a portion thereof, or having at least 75% sequence identity to the reverse complement or a portion thereof of SEQ ID NO: 7179 or 7180.

[0037] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1, 2, 14-347, 7092, 7093, 7097-7102, 7115-7120, 7141, 7142, 7146, 7147, 7151 and / or 7152, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 1, 2, 14-347, 7092, 7093, 7097-7102, 7115-7120, 7141, 7142, 7146, 7147, 7151 and / or 7152.

[0038] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of GRHPR, wherein the nucleic acids include or encode an antisense nucleic acid having at least 75% sequence identity to SEQ ID NO: 7181 or a portion thereof, or having at least 75% sequence identity to the reverse complement of SEQ ID NO: 7181 or a portion thereof.

[0039] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 3-5, 348-456, 7094, 7103-7108, 7121-7129, 7143, 7148 and / or 7153, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 3-5, 348-456, 7094, 7103-7108, 7121-7129, 7143, 7148 and / or 7153.

[0040] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of ABCC4, wherein the nucleic acids contain or encode antisense nucleic acids having at least 75% sequence identity to any one or a portion thereof of SEQ ID NOs. 7183-7186, or having at least 75% sequence identity to the reverse complement or a portion thereof of any one of SEQ ID NOs. 7183-7186.

[0041] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising or consisting of a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1483-2208, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 1483-2208.

[0042] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of PAK3, wherein the nucleic acids include or encode antisense nucleic acids having at least 75% sequence identity to any one or a portion thereof of SEQ ID NOs. 7187-7190, or having at least 75% sequence identity to the reverse complement or a portion thereof of any one of SEQ ID NOs. 7187-7190.

[0043] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs. 2209-5060, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs. 2209-5060.

[0044] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of TRNP1, wherein the nucleic acids include or encode an antisense nucleic acid having at least 75% sequence identity to SEQ ID NO: 7191 or a portion thereof, or having at least 75% sequence identity to the reverse complement of SEQ ID NO: 7191 or a portion thereof.

[0045] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 5061-5389, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs: 5061-5389.

[0046] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of APLN, wherein the nucleic acids include or encode an antisense nucleic acid having at least 75% sequence identity to SEQ ID NO: 7192 or a portion thereof, or having at least 75% sequence identity to the reverse complement of SEQ ID NO: 7192 or a portion thereof.

[0047] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs. 5390-5966, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs. 5390-5966.

[0048] Inhibitory nucleic acids are provided for reducing the gene and / or protein expression of KIF20A, wherein the nucleic acids include or encode an antisense nucleic acid having at least 75% sequence identity to SEQ ID NO: 7193 or a portion thereof, or having at least 75% sequence identity to the reverse complement of SEQ ID NO: 7193 or a portion thereof.

[0049] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs. 5967-6974, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs. 5967-6974.

[0050] Inhibitory nucleic acids are provided for reducing LTB gene and / or protein expression, the nucleic acids comprising or encoding antisense nucleic acids having at least 75% sequence identity to SEQ ID NO: 7194 or 7195 or a portion thereof, or having at least 75% sequence identity to the reverse complement or a portion thereof of SEQ ID NO: 7194 or 7195.

[0051] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising a nucleotide sequence having at least 75% sequence identity to any one of SEQ ID NOs. 6975-7091, and / or having at least 75% sequence identity to the reverse complement of any one of SEQ ID NOs. 6975-7091.

[0052] Furthermore, inhibitory nucleic acids are provided, including (i) a nucleic acid containing the nucleotide sequence shown in one of sequence numbers 7092 to 7096, and (ii) a nucleic acid containing the nucleotide sequence shown in one of sequence numbers 7141 to 7145.

[0053] In some embodiments, the inhibitory nucleic acids are 2'-O-methyluridine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 2'- It comprises one or more modified nucleotides selected from ruoraulyzin-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate.

[0054] Furthermore, inhibitory nucleic acids are provided, which include (i) a nucleic acid containing a nucleotide sequence (including modifications thereof) represented in one of sequence numbers 7146 to 7150, and (ii) a nucleic acid containing a nucleotide sequence (including modifications thereof) represented in one of sequence numbers 7151 to 7155.

[0055] In some embodiments, the inhibitory nucleic acid further includes a portion that promotes the uptake of the inhibitory nucleic acid by hepatocytes. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid, siRNA, or shRNA.

[0056] This disclosure also provides optionally isolated nucleic acids that encode inhibitory nucleic acids according to this disclosure.

[0057] This disclosure also provides an expression vector comprising nucleic acid as described herein.

[0058] This disclosure also provides compositions comprising inhibitory nucleic acids, nucleic acids or expression vectors, and pharmaceutically acceptable carriers, diluents, excipients or adjuvants.

[0059] This disclosure also provides cells comprising inhibitory nucleic acids, nucleic acids, or expression vectors as a result of this disclosure.

[0060] The Disclosure also provides a method for treating or preventing a disease relating to the Disclosure, comprising the step of administering a therapeutic or prophylactic effective amount of an inhibitor, inhibitory nucleic acid, nucleic acid, expression vector, composition or cell relating to the Disclosure to the target.

[0061] The Disclosure also provides inhibitors, inhibitory nucleic acids, nucleic acids, expression vectors, compositions, or cells for use in therapeutics. In some embodiments, inhibitors, inhibitory nucleic acids, nucleic acids, expression vectors, compositions, or cells are provided for use in methods of treating or preventing diseases, such as the diseases described herein.

[0062] This disclosure also provides the use of inhibitors, inhibitory nucleic acids, nucleic acids, expression vectors, compositions or cells according to this disclosure in the manufacture of pharmaceuticals for use in treating or preventing diseases, for example, in methods of treating or preventing diseases according to this disclosure.

[0063] Also disclosed are in vitro or in vivo methods for reducing the expression of one or more genes and / or proteins of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in cells, the methods comprising the step of introducing the inhibitory nucleic acid, nucleic acid, or expression vector according to the present disclosure into cells.

[0064] Also disclosed is a method for regenerating liver tissue in vitro or in vivo, comprising the step of inhibiting at least one of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB in the cells of the tissue.

[0065] Also disclosed is a method for growing / extending hepatocytes in vitro or in vivo, comprising the step of inhibiting at least one of ITFG1, MFAP4, GRHPR, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB in hepatocytes.

[0066] In some embodiments, the methods disclosed herein include the step of introducing the inhibitory nucleic acid, nucleic acid or expression vector according to the disclosure into cells, for example, tissue cells or hepatocytes.

[0067] A method for stimulating or increasing cell proliferation and / or regeneration in a subject is disclosed herein, the method comprising the step of administering an inhibitor of a gene or corresponding gene product associated with organ regeneration to a subject for a period of time and under conditions sufficient to stimulate or increase cell proliferation and / or regeneration in the subject.

[0068] A method for enhancing cellular function in a subject is disclosed herein, the method comprising the step of administering an inhibitor of a gene or corresponding gene product associated with organ regeneration to the subject for a period of time and under conditions sufficient to enhance cellular function in the subject.

[0069] A method for enhancing cell viability in a subject is disclosed herein, the method comprising the step of administering an inhibitor of a gene or corresponding gene product associated with organ regeneration to the subject for a period of time and under conditions sufficient to enhance cell viability in the subject.

[0070] A method for treating a liver condition or disease in a subject is disclosed herein, the method comprising the step of administering an inhibitor of a gene or corresponding gene product associated with organ regeneration to the subject for a period of time and under conditions sufficient to treat the liver condition or disease in the subject.

[0071] A method for protecting a subject from liver injury is disclosed herein, the method comprising the step of administering an inhibitor of a gene or corresponding gene product associated with organ regeneration to the subject for a period of time and under conditions sufficient to protect the subject from liver injury.

[0072] A method for detecting a liver condition or disease in a subject is disclosed herein, the method comprising the step of detecting the level of one or more biomarkers associated with organ regeneration in a sample, wherein a change in the level of one or more biomarkers compared to a reference indicates that the subject has a liver condition or disease.

[0073] Inhibitors of genes or corresponding gene products associated with organ regeneration are disclosed herein for use in the prevention or treatment of liver conditions or diseases in subjects.

[0074] The use of inhibitors of genes or corresponding gene products associated with organ regeneration in the manufacture of pharmaceuticals for the prevention or treatment of liver conditions or diseases in subjects is disclosed herein.

[0075] The methods disclosed herein may use any suitable inhibitor. In some embodiments, the inhibitor is the inhibitor according to this disclosure.

[0076] Disclosed herein are nucleic acid inhibitors comprising, or encoding, an RNAi agent having at least 70%, 80%, 90%, or 95% sequence identity with any of the RNA sequences listed in Tables 1 to 14, or an RNAi agent that hybridizes with a complement of any of the RNA sequences listed in Tables 1 to 14 under stringency conditions.

[0077] A method for screening for inhibitors of genes or corresponding gene products associated with organ regeneration is disclosed herein, comprising the steps of a) contacting a gene or corresponding gene product with a chemical library, and b) identifying chemicals in the library that bind to the gene or corresponding gene product and inhibit the expression or function of the gene or corresponding gene product.

[0078] The present invention includes combinations of the aspects and preferred features described, except where such combinations are clearly unacceptable or explicitly avoided.

[0079] Drawing Overview Embodiments and experiments illustrating the principle of the present invention will be discussed below with reference to the accompanying drawings, by an extent not limited to such embodiments. [Brief explanation of the drawing]

[0080] [Figure 1-1]Figure 1 shows an in vivo RNAi screening of functional genes that modulator liver regeneration. A) Screening overview. A library of 250 shRNAs targeting 89 genes was delivered to the liver of five independent mice by hydrodynamic tail vein (HDTV) injection of a transposon-based construct (top panel) combined with a plasmid encoding sleeping beauty 13 (SB13). After stable integration in approximately 5-10% of hepatocytes, thioacetamide (TAA) treatment (3 times per week for 8 weeks) induced chronic liver injury associated with progressive hepatic fibrosis. Changes in shRNA levels were detected by deep sequencing. B) Presentation of reticular changes for each shRNA. The majority of shRNAs are depleted, while a small number are clearly enriched. C) ROMAampl-library (250 shRNA) distribution. Abundances of promising candidates are shown. Heatmap-based presentation of enrichment (dark gray) or depletion (light gray) for each animal. The upper panel shows all shRNAs (each row (raw) represents one animal). The lower panel shows higher magnification of highly enriched, depleted, and neutral shRNAs (each column represents one animal). D) Screening of functional genes identifies highly reliable candidates (enlarged view shown in Figure B). At least two independent shRNAs targeting Mfap4, Grhpr, and Itfg1 were enriched. Furthermore, untargeted control (shNC) shRNAs (Renilla 713 and luciferase 1309) did not show significant enrichment or depletion, and known important liver regeneration genes were depleted, as was the essential receptor for liver regeneration, which is a c-Met-targeting shRNA. These results give confidence to the screening approach. [Figure 1-2]Figure 1 shows an in vivo RNAi screening of functional genes that modulator liver regeneration. A) Screening overview. A library of 250 shRNAs targeting 89 genes was delivered to the liver of five independent mice by hydrodynamic tail vein (HDTV) injection of a transposon-based construct (top panel) combined with a plasmid encoding sleeping beauty 13 (SB13). After stable integration in approximately 5-10% of hepatocytes, thioacetamide (TAA) treatment (3 times per week for 8 weeks) induced chronic liver injury associated with progressive hepatic fibrosis. Changes in shRNA levels were detected by deep sequencing. B) Presentation of reticular changes for each shRNA. The majority of shRNAs are depleted, while a small number are clearly enriched. C) ROMAampl-library (250 shRNA) distribution. Abundances of promising candidates are shown. Heatmap-based presentation of enrichment (dark gray) or depletion (light gray) for each animal. The upper panel shows all shRNAs (each row (raw) represents one animal). The lower panel shows higher magnification of highly enriched, depleted, and neutral shRNAs (each column represents one animal). D) Screening of functional genes identifies highly reliable candidates (enlarged view shown in Figure B). At least two independent shRNAs targeting Mfap4, Grhpr, and Itfg1 were enriched. Furthermore, untargeted control (shNC) shRNAs (Renilla 713 and luciferase 1309) did not show significant enrichment or depletion, and known important liver regeneration genes were depleted, as was the essential receptor for liver regeneration, which is a c-Met-targeting shRNA. These results give confidence to the screening approach. [Figure 2-1]Figure 2 shows in vitro confirmation of Mfap4 targeting for enhanced regeneration - shRNA-mediated knockdown of Mfap4 accelerates growth rate in embryonic liver cell lines. A) Testing of knockdown efficiency of top-scoring shRNAs targeting Mfap4. Top panel, retroviral scaffold for generating stable cell lines. Bottom panel, Western blot showing efficient knockdown of Mfap4 by our shRNA (control: aTub = α-tubulin). B) Schematic overview of stable cell line-based assay. C) Wound healing assay in TIB 73 (BNLCL.2) cell line. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The filling of this “wound” gap was monitored. Representative images of each group are shown in the left panel. Three technical replications were performed. The right panel shows quantifications across different time points (data were analyzed by two-way ANOVA using ImageJ and GraphPad Prism software). Significant differences between shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC are indicated by "*". D) EdU Incorporation Assay. DNA synthesis of TIB 73 cells (BNLCL.2) transfected with shMfap4.1356 (SEQ ID NO: 2) and shNC was evaluated by the EdU assay. Quantifications show significant differences between experiments and controls. Three technical replications were performed. E) Cell Doubling. Doubling time assay results are shown. Cells were seeded at the same seeding density. Doubling time was calculated based on the logarithmic phase of the growth curve. Three technical replications were performed. F) Cell Cycle Analysis by Flow Cytometry using a Guava Muse Cell Analyzer. Percentages of cells at the indicated cell cycle phases are shown. Compared to control NC, the experimental case (cells with Mfap4 stably knocked down by shMfap4.1356 and shMfap4.760) shows a greater number of G2 phase cells. G) Wound healing assay using adult liver mouse cell line AML12. The same effect was observed in the left panel, Figure 2C). Right panel.The quantification in (A) already shows significantly faster wound closure at 14 hours. [Figure 2-2]Figure 2 shows in vitro confirmation of Mfap4 targeting for enhanced regeneration - shRNA-mediated knockdown of Mfap4 accelerates growth rate in embryonic liver cell lines. A) Testing of knockdown efficiency of top-scoring shRNAs targeting Mfap4. Top panel, retroviral scaffold for generating stable cell lines. Bottom panel, Western blot showing efficient knockdown of Mfap4 by our shRNA (control: aTub = α-tubulin). B) Schematic overview of stable cell line-based assay. C) Wound healing assay in TIB 73 (BNLCL.2) cell line. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The filling of this “wound” gap was monitored. Representative images of each group are shown in the left panel. Three technical replications were performed. The right panel shows quantifications across different time points (data were analyzed by two-way ANOVA using ImageJ and GraphPad Prism software). Significant differences between shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC are indicated by "*". D) EdU Incorporation Assay. DNA synthesis of TIB 73 cells (BNLCL.2) transfected with shMfap4.1356 (SEQ ID NO: 2) and shNC was evaluated by the EdU assay. Quantifications show significant differences between experiments and controls. Three technical replications were performed. E) Cell Doubling. Doubling time assay results are shown. Cells were seeded at the same seeding density. Doubling time was calculated based on the logarithmic phase of the growth curve. Three technical replications were performed. F) Cell Cycle Analysis by Flow Cytometry using a Guava Muse Cell Analyzer. Percentages of cells at the indicated cell cycle phases are shown. Compared to control NC, the experimental case (cells with Mfap4 stably knocked down by shMfap4.1356 and shMfap4.760) shows a greater number of G2 phase cells. G) Wound healing assay using adult liver mouse cell line AML12. The same effect was observed in the left panel, Figure 2C). Right panel.The quantification in (A) already shows significantly faster wound closure at 14 hours. [Figure 2-3]Figure 2 shows in vitro confirmation of Mfap4 targeting for enhanced regeneration - shRNA-mediated knockdown of Mfap4 accelerates growth rate in embryonic liver cell lines. A) Testing of knockdown efficiency of top-scoring shRNAs targeting Mfap4. Top panel, retroviral scaffold for generating stable cell lines. Bottom panel, Western blot showing efficient knockdown of Mfap4 by our shRNA (control: aTub = α-tubulin). B) Schematic overview of stable cell line-based assay. C) Wound healing assay in TIB 73 (BNLCL.2) cell line. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The filling of this “wound” gap was monitored. Representative images of each group are shown in the left panel. Three technical replications were performed. The right panel shows quantifications across different time points (data were analyzed by two-way ANOVA using ImageJ and GraphPad Prism software). Significant differences between shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC are indicated by "*". D) EdU Incorporation Assay. DNA synthesis of TIB 73 cells (BNLCL.2) transfected with shMfap4.1356 (SEQ ID NO: 2) and shNC was evaluated by the EdU assay. Quantifications show significant differences between experiments and controls. Three technical replications were performed. E) Cell Doubling. Doubling time assay results are shown. Cells were seeded at the same seeding density. Doubling time was calculated based on the logarithmic phase of the growth curve. Three technical replications were performed. F) Cell Cycle Analysis by Flow Cytometry using a Guava Muse Cell Analyzer. Percentages of cells at the indicated cell cycle phases are shown. Compared to control NC, the experimental case (cells with Mfap4 stably knocked down by shMfap4.1356 and shMfap4.760) shows a greater number of G2 phase cells. G) Wound healing assay using adult liver mouse cell line AML12. The same effect was observed in the left panel, Figure 2C). Right panel.The quantification in (A) already shows significantly faster wound closure at 14 hours. [Figure 2-4]Figure 2 shows in vitro confirmation of Mfap4 targeting for enhanced regeneration - shRNA-mediated knockdown of Mfap4 accelerates growth rate in embryonic liver cell lines. A) Testing of knockdown efficiency of top-scoring shRNAs targeting Mfap4. Top panel, retroviral scaffold for generating stable cell lines. Bottom panel, Western blot showing efficient knockdown of Mfap4 by our shRNA (control: aTub = α-tubulin). B) Schematic overview of stable cell line-based assay. C) Wound healing assay in TIB 73 (BNLCL.2) cell line. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The filling of this “wound” gap was monitored. Representative images of each group are shown in the left panel. Three technical replications were performed. The right panel shows quantifications across different time points (data were analyzed by two-way ANOVA using ImageJ and GraphPad Prism software). Significant differences between shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC are indicated by "*". D) EdU Incorporation Assay. DNA synthesis of TIB 73 cells (BNLCL.2) transfected with shMfap4.1356 (SEQ ID NO: 2) and shNC was evaluated by the EdU assay. Quantifications show significant differences between experiments and controls. Three technical replications were performed. E) Cell Doubling. Doubling time assay results are shown. Cells were seeded at the same seeding density. Doubling time was calculated based on the logarithmic phase of the growth curve. Three technical replications were performed. F) Cell Cycle Analysis by Flow Cytometry using a Guava Muse Cell Analyzer. Percentages of cells at the indicated cell cycle phases are shown. Compared to control NC, the experimental case (cells with Mfap4 stably knocked down by shMfap4.1356 and shMfap4.760) shows a greater number of G2 phase cells. G) Wound healing assay using adult liver mouse cell line AML12. The same effect was observed in the left panel, Figure 2C). Right panel.The quantification in (A) already shows significantly faster wound closure at 14 hours. [Figure 3-1]Figure 3 shows that Mfap4 knockdown accelerates liver regrowth. A) FAH knockout mouse-based liver regrowth assay. The upper panel shows an overview of the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest. The lower panel shows an overview of the assay and rationale. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion compared to control shRNA, starting from stably incorporated hepatocytes. B) GFP imager image. GFP imaging of explanted mouse liver shows enhanced clonal expansion (regrowth) in hepatocytes stably expressing shMfap4.1356 (SEQ ID NO: 1) compared to hepatocytes expressing shNC (18 days after HDTV injection of 25 μg of the plasmid shown). Representative images of each group are shown (n=8 in the group with shMfap4.1356, n=6 in the group with shMfap4.760, and n=6 in the group with shNC). Right, white dots represent macroscopically visible clonal expansion and proliferation of GFP-positive cells. C) Native GFP on tissue sections. Representative GFP fluorescence images of liver sections (200×) of FAH- / - mice 18 days after in vivo delivery of transposon constructs expressing either shMfap4 or the corresponding control shRNA are shown. D) Histological analysis (immunostaining for GFP) of GFP-positive cells in mouse livers stably expressing shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC (representative images are shown, n=8 in the group with shMfap4.1356, n=6 in the group with shMfap4.760, and n=6 in the group with shNC). E) 18 days after HDTV injection of 1.25 μg of the plasmid shown (200x magnification). Enhanced clonal expansion can be seen for shMfap4. E) Quantification of GFP-positive cells (corresponding to Figure D) shows a significant increase in GFP-positive hepatocytes in the case of Mfap4 knockdown compared to the control. Each dot represents one animal.F) Kaplan-Meier survival curves of FAH- / - mice injected with either p / T-FAHIG-shMfap4.1356 (n=5) or p / T-FAHIG-shNC (n=5) and SB13 (p<0.05) in a 1:30 (0.83 μg plasmid and 0.17 mg SB13) dilution. NTBC off indicates NTBC drug clearance time and induces the selection process (1 day after injection). [Figure 3-2]Figure 3 shows that Mfap4 knockdown accelerates liver regrowth. A) FAH knockout mouse-based liver regrowth assay. The upper panel shows an overview of the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest. The lower panel shows an overview of the assay and rationale. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion compared to control shRNA, starting from stably incorporated hepatocytes. B) GFP imager image. GFP imaging of explanted mouse liver shows enhanced clonal expansion (regrowth) in hepatocytes stably expressing shMfap4.1356 (SEQ ID NO: 1) compared to hepatocytes expressing shNC (18 days after HDTV injection of 25 μg of the plasmid shown). Representative images of each group are shown (n=8 in the group with shMfap4.1356, n=6 in the group with shMfap4.760, and n=6 in the group with shNC). Right, white dots represent macroscopically visible clonal expansion and proliferation of GFP-positive cells. C) Native GFP on tissue sections. Representative GFP fluorescence images of liver sections (200×) of FAH- / - mice 18 days after in vivo delivery of transposon constructs expressing either shMfap4 or the corresponding control shRNA are shown. D) Histological analysis (immunostaining for GFP) of GFP-positive cells in mouse livers stably expressing shMfap4.1356 (SEQ ID NO: 1), shMfap4.760 (SEQ ID NO: 2), and shNC (representative images are shown, n=8 in the group with shMfap4.1356, n=6 in the group with shMfap4.760, and n=6 in the group with shNC). E) 18 days after HDTV injection of 1.25 μg of the plasmid shown (200x magnification). Enhanced clonal expansion can be seen for shMfap4. E) Quantification of GFP-positive cells (corresponding to Figure D) shows a significant increase in GFP-positive hepatocytes in the case of Mfap4 knockdown compared to the control. Each dot represents one animal.F) Kaplan-Meier survival curves of FAH- / - mice injected with either p / T-FAHIG-shMfap4.1356 (n=5) or p / T-FAHIG-shNC (n=5) and SB13 (p<0.05) in a 1:30 (0.83 μg plasmid and 0.17 mg SB13) dilution. NTBC off indicates NTBC drug clearance time and induces the selection process (1 day after injection). [Figure 4-1] Figure 4 shows the “Western diet” (WD) mouse fatty liver model A) the WD + fructose diet in practice. The diet used is rich in fat and carbohydrates. 45% of the energy comes from fat, mainly saturated fat, and it contains 0.2% cholesterol. In addition, the animals obtain 60% fructose / water (weight / volume). B) Pathological evaluation. Histological slides of liver tissue from C56Bl6 mice exposed to the “Western diet” or normal solid feed for the indicated time were evaluated and scored by a certified pathologist. Scoring results showing the outcomes of fatty liver and fibrosis are shown. Each point represents one animal. C) “Western diet” mice show progressive weight gain independently of sex. D) The WD model shows progressive fibrosis similar to that of human patients (see Figure E). E) Progressive increase in fibrosis at disease stage, based on human patients, similar to the mouse model (Figures D and B). F) Progressive hepatic fibrosis can be macroscopically detected as early as 24 weeks after WD (representative image). G) After 24 weeks of WD, mouse liver shows a high level of fatty liver (H&E stained liver tissue, representative image). H) Sirius red staining of collagen fibers showing progressive fibrosis after 24 weeks of WD exposure. [Figure 4-2]Figure 4 shows the “Western diet” (WD) mouse fatty liver model A) the WD + fructose diet in practice. The diet used is rich in fat and carbohydrates. 45% of the energy comes from fat, mainly saturated fat, and it contains 0.2% cholesterol. In addition, the animals obtain 60% fructose / water (weight / volume). B) Pathological evaluation. Histological slides of liver tissue from C56Bl6 mice exposed to the “Western diet” or normal solid feed for the indicated time were evaluated and scored by a certified pathologist. Scoring results showing the outcomes of fatty liver and fibrosis are shown. Each point represents one animal. C) “Western diet” mice show progressive weight gain independently of sex. D) The WD model shows progressive fibrosis similar to that of human patients (see Figure E). E) Progressive increase in fibrosis at disease stage, based on human patients, similar to the mouse model (Figures D and B). F) Progressive hepatic fibrosis can be macroscopically detected as early as 24 weeks after WD (representative image). G) After 24 weeks of WD, mouse liver shows a high level of fatty liver (H&E stained liver tissue, representative image). H) Sirius red staining of collagen fibers showing progressive fibrosis after 24 weeks of WD exposure. [Figure 5-1]Figure 5 shows that Mfap4 knockdown attenuates NASH-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct, and then maintained for 3 months for complete regrowth so that all hepatocytes in the liver expressed the target shRNA construct. After complete regrowth was achieved, the mice were exposed to a "Western diet" (high-fat diet and 60% fructose) for 24 weeks. Livers were harvested, processed, and analyzed. B) Representative macrographs of the livers are shown. Macroscopic differences between groups were already observed. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=5 per experimental group and n=7 per control group; representative sections are shown, 50x magnification) of sections of regrowing mouse livers shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. Fibrosis scores were significantly lower in the experimental group compared to the control group. E) Scores for utricle hyperplasia are shown. Scores were given by a blinded certified pathologist for the experimental group. Scores were significantly lower (=0) in the experimental group compared to the control group. Utricle hyperplasia is considered a compensatory mechanism when hepatocyte regeneration is no longer sufficient. F) Representative GFP-scanner macrographs of the liver are shown. Strong GFP signals on the liver surface indicate complete regrowth. [Figure 5-2]Figure 5 shows that Mfap4 knockdown attenuates NASH-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct, and then maintained for 3 months for complete regrowth so that all hepatocytes in the liver expressed the target shRNA construct. After complete regrowth was achieved, the mice were exposed to a "Western diet" (high-fat diet and 60% fructose) for 24 weeks. Livers were harvested, processed, and analyzed. B) Representative macrographs of the livers are shown. Macroscopic differences between groups were already observed. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=5 per experimental group and n=7 per control group; representative sections are shown, 50x magnification) of sections of regrowing mouse livers shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. Fibrosis scores were significantly lower in the experimental group compared to the control group. E) Scores for utricle hyperplasia are shown. Scores were given by a blinded certified pathologist for the experimental group. Scores were significantly lower (=0) in the experimental group compared to the control group. Utricle hyperplasia is considered a compensatory mechanism when hepatocyte regeneration is no longer sufficient. F) Representative GFP-scanner macrographs of the liver are shown. Strong GFP signals on the liver surface indicate complete regrowth. [Figure 6]Figure 6 shows that Mfap4 knockdown attenuates chronic liver injury-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, chronic liver injury was induced by repeated doses of thioacetamide administered intraperitoneally 3 times per week for 8 weeks. Liver tissue was harvested, processed, and analyzed. B) Representative macrographs of the liver are shown. Macroscopic differences between groups were already observed. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=6 per experimental group and n=7 per control group; representative sections are shown, 50x magnification) of sections of regrowing mouse livers shown. D) Fibrosis scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental group. Fibrosis scores were significantly lower in the experimental group compared to the control group. [Figure 7-1]Figure 7 shows the acceleration of liver regeneration after Mfap4 knockdown and partial hepatectomy (PH). A) Experimental overview. FRGN mice were injected with our construct and maintained for 3 months for complete regrowth. FRGN mice were FAH- / -, Rag2- / -, Il2rg- / - in a NOD background and had reduced immune function. After complete regrowth of the mouse liver, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected 48 hours after surgery. B) Representative photographs of liver sections stained with Ki67 immunofluorescence (top row) and DAB Ki67 (bottom row) 48 hours after hepatectomy are shown (200x magnification, n=5 per experimental / control group). C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation after partial hepatectomy in shMfap4-expressing livers compared to shNC livers (individual points represent individual animals, data show mean ± SEM; n=5 per group). D) Western blot analysis of cyclin A (nuclear extracts from regrowing mouse livers at the indicated time) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2). E) Experimental summary. Immunocompatible FAH- / - mice were injected with our construct, and the mice were maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerated liver was harvested 42 and 48 hours after surgery. F) Representative images of DAB Ki67-stained liver sections after hepatectomy are shown at 42 hours (n=5 per experimental group, n=6 per control group) and 48 hours (n=5 per experimental group, n=10 per control group) (200× magnification). G) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in shMfap4-expressing livers compared with shNC livers (individual points represent individual animals, data shows mean ± SEM). H) Western blot analysis of cyclin E (nuclear extracts from regrowing mouse livers at the indicated time points) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2).I) GFP imaging of a completely regrowing FAH- / - liver after partial resection of 2 / 3 of the liver by surgery, corresponding to different time points in PHx (3 months after HDTV injection). Strong GFP signal on the liver surface indicates complete regrowth. J) Representative image of DAB GFP staining showing that complete regrowth in FAH liver is approximately 90-95%. Dark brown areas represent regrowing hepatocytes, and light brown areas represent non-regrowing cells. [Figure 7-2]Figure 7 shows the acceleration of liver regeneration after Mfap4 knockdown and partial hepatectomy (PH). A) Experimental overview. FRGN mice were injected with our construct and maintained for 3 months for complete regrowth. FRGN mice were FAH- / -, Rag2- / -, Il2rg- / - in a NOD background and had reduced immune function. After complete regrowth of the mouse liver, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected 48 hours after surgery. B) Representative photographs of liver sections stained with Ki67 immunofluorescence (top row) and DAB Ki67 (bottom row) 48 hours after hepatectomy are shown (200x magnification, n=5 per experimental / control group). C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation after partial hepatectomy in shMfap4-expressing livers compared to shNC livers (individual points represent individual animals, data show mean ± SEM; n=5 per group). D) Western blot analysis of cyclin A (nuclear extracts from regrowing mouse livers at the indicated time) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2). E) Experimental summary. Immunocompatible FAH- / - mice were injected with our construct, and the mice were maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerated liver was harvested 42 and 48 hours after surgery. F) Representative images of DAB Ki67-stained liver sections after hepatectomy are shown at 42 hours (n=5 per experimental group, n=6 per control group) and 48 hours (n=5 per experimental group, n=10 per control group) (200× magnification). G) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in shMfap4-expressing livers compared with shNC livers (individual points represent individual animals, data shows mean ± SEM). H) Western blot analysis of cyclin E (nuclear extracts from regrowing mouse livers at the indicated time points) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2).I) GFP imaging of a completely regrowing FAH- / - liver after partial resection of 2 / 3 of the liver by surgery, corresponding to different time points in PHx (3 months after HDTV injection). Strong GFP signal on the liver surface indicates complete regrowth. J) Representative image of DAB GFP staining showing that complete regrowth in FAH liver is approximately 90-95%. Dark brown areas represent regrowing hepatocytes, and light brown areas represent non-regrowing cells. [Figure 7-3]Figure 7 shows the acceleration of liver regeneration after Mfap4 knockdown and partial hepatectomy (PH). A) Experimental overview. FRGN mice were injected with our construct and maintained for 3 months for complete regrowth. FRGN mice were FAH- / -, Rag2- / -, Il2rg- / - in a NOD background and had reduced immune function. After complete regrowth of the mouse liver, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected 48 hours after surgery. B) Representative photographs of liver sections stained with Ki67 immunofluorescence (top row) and DAB Ki67 (bottom row) 48 hours after hepatectomy are shown (200x magnification, n=5 per experimental / control group). C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation after partial hepatectomy in shMfap4-expressing livers compared to shNC livers (individual points represent individual animals, data show mean ± SEM; n=5 per group). D) Western blot analysis of cyclin A (nuclear extracts from regrowing mouse livers at the indicated time) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2). E) Experimental summary. Immunocompatible FAH- / - mice were injected with our construct, and the mice were maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerated liver was harvested 42 and 48 hours after surgery. F) Representative images of DAB Ki67-stained liver sections after hepatectomy are shown at 42 hours (n=5 per experimental group, n=6 per control group) and 48 hours (n=5 per experimental group, n=10 per control group) (200× magnification). G) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in shMfap4-expressing livers compared with shNC livers (individual points represent individual animals, data shows mean ± SEM). H) Western blot analysis of cyclin E (nuclear extracts from regrowing mouse livers at the indicated time points) shows earlier cell cycle initiation and faster cell cycle progression in shMfap4-expressing mouse livers (n=2).I) GFP imaging of a completely regrowing FAH- / - liver after partial resection of 2 / 3 of the liver by surgery, corresponding to different time points in PHx (3 months after HDTV injection). Strong GFP signal on the liver surface indicates complete regrowth. J) Representative image of DAB GFP staining showing that complete regrowth in FAH liver is approximately 90-95%. Dark brown areas represent regrowing hepatocytes, and light brown areas represent non-regrowing cells. [Figure 8-1]Figure 8 shows that in vivo knockdown of Mfap4 affects mTOR and p38 signaling. A) Schematic overview of the experiment. Whole cell protein extracts were isolated from regrowing mouse livers and analyzed by protein array. B) Heatmap shows the results of the phospho-antibody MAPK pathway protein array. Whole cell protein extracts were analyzed from regrowing mouse livers stably expressing either shMfap4 or shNC (relative spot intensity is shown). C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. D) After performing a broad protein array, a focused Western blot experiment was performed. The results of the Western blot are shown here. Proteins were isolated from fully regrowing livers. P-P70S6k, p-p38, p-mTOR, and p-ERK2 are expressed more in the case of Mfap4 knockdown compared to the control, and therefore show stronger activation in the case of Mfap4 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. E) Schematic diagram of mTOR-mediated regulation. Specific mTOR phosphorylation is upstream of p70S6k activation, leading to forced translation. F) Wound healing under double knockdown conditions. Based on pathway analysis, double knockout experiments were initiated. Our stable cell line was expanded, cells were treated with the respective siRNAs, and the silicone gasket was removed. Wound healing was monitored. Slower growth and migration were observed in the case of double knockdown of Mfap4 and p70S6k and Mfap4 and p38. G) Western blots with proteins isolated from cells in Figure F. Interestingly, p38 knockdown also affects p70S6k. H) Schematic outline of preparation of stable cell line with Mfap4 knocked down for transcriptome analysis. I) Principal component analyses of AML12-shMfap4.1356, AML12-shMfap4.760, AML12-shNC, (Rb88-RMA050 & Ren_RMA061), and AML12 (AML_RMA052) are shown. The inventors observed cluster separation between the experiments and the control.J) Heatmaps of the following samples are shown. Compared to the control, Ptgs2, Areg, Dhrs9, Hmox1, and Nqo1 were upregulated in the experimental samples, and these genes are known to be involved in liver regeneration according to the literature. K) The String database shows the linkages between proteins that are upregulated according to Figures D and J. [Figure 8-2]Figure 8 shows that in vivo knockdown of Mfap4 affects mTOR and p38 signaling. A) Schematic overview of the experiment. Whole cell protein extracts were isolated from regrowing mouse livers and analyzed by protein array. B) Heatmap shows the results of the phospho-antibody MAPK pathway protein array. Whole cell protein extracts were analyzed from regrowing mouse livers stably expressing either shMfap4 or shNC (relative spot intensity is shown). C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. D) After performing a broad protein array, a focused Western blot experiment was performed. The results of the Western blot are shown here. Proteins were isolated from fully regrowing livers. P-P70S6k, p-p38, p-mTOR, and p-ERK2 are expressed more in the case of Mfap4 knockdown compared to the control, and therefore show stronger activation in the case of Mfap4 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. E) Schematic diagram of mTOR-mediated regulation. Specific mTOR phosphorylation is upstream of p70S6k activation, leading to forced translation. F) Wound healing under double knockdown conditions. Based on pathway analysis, double knockout experiments were initiated. Our stable cell line was expanded, cells were treated with the respective siRNAs, and the silicone gasket was removed. Wound healing was monitored. Slower growth and migration were observed in the case of double knockdown of Mfap4 and p70S6k and Mfap4 and p38. G) Western blots with proteins isolated from cells in Figure F. Interestingly, p38 knockdown also affects p70S6k. H) Schematic outline of preparation of stable cell line with Mfap4 knocked down for transcriptome analysis. I) Principal component analyses of AML12-shMfap4.1356, AML12-shMfap4.760, AML12-shNC, (Rb88-RMA050 & Ren_RMA061), and AML12 (AML_RMA052) are shown. The inventors observed cluster separation between the experiments and the control.J) Heatmaps of the following samples are shown. Compared to the control, Ptgs2, Areg, Dhrs9, Hmox1, and Nqo1 were upregulated in the experimental samples, and these genes are known to be involved in liver regeneration according to the literature. K) The String database shows the linkages between proteins that are upregulated according to Figures D and J. [Figure 8-3]Figure 8 shows that in vivo knockdown of Mfap4 affects mTOR and p38 signaling. A) Schematic overview of the experiment. Whole cell protein extracts were isolated from regrowing mouse livers and analyzed by protein array. B) Heatmap shows the results of the phospho-antibody MAPK pathway protein array. Whole cell protein extracts were analyzed from regrowing mouse livers stably expressing either shMfap4 or shNC (relative spot intensity is shown). C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. D) After performing a broad protein array, a focused Western blot experiment was performed. The results of the Western blot are shown here. Proteins were isolated from fully regrowing livers. P-P70S6k, p-p38, p-mTOR, and p-ERK2 are expressed more in the case of Mfap4 knockdown compared to the control, and therefore show stronger activation in the case of Mfap4 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. E) Schematic diagram of mTOR-mediated regulation. Specific mTOR phosphorylation is upstream of p70S6k activation, leading to forced translation. F) Wound healing under double knockdown conditions. Based on pathway analysis, double knockout experiments were initiated. Our stable cell line was expanded, cells were treated with the respective siRNAs, and the silicone gasket was removed. Wound healing was monitored. Slower growth and migration were observed in the case of double knockdown of Mfap4 and p70S6k and Mfap4 and p38. G) Western blots with proteins isolated from cells in Figure F. Interestingly, p38 knockdown also affects p70S6k. H) Schematic outline of preparation of stable cell line with Mfap4 knocked down for transcriptome analysis. I) Principal component analyses of AML12-shMfap4.1356, AML12-shMfap4.760, AML12-shNC, (Rb88-RMA050 & Ren_RMA061), and AML12 (AML_RMA052) are shown. The inventors observed cluster separation between the experiments and the control.J) Heatmaps of the following samples are shown. Compared to the control, Ptgs2, Areg, Dhrs9, Hmox1, and Nqo1 were upregulated in the experimental samples, and these genes are known to be involved in liver regeneration according to the literature. K) The String database shows the linkages between proteins that are upregulated according to Figures D and J. [Figure 8-4]Figure 8 shows that in vivo knockdown of Mfap4 affects mTOR and p38 signaling. A) Schematic overview of the experiment. Whole cell protein extracts were isolated from regrowing mouse livers and analyzed by protein array. B) Heatmap shows the results of the phospho-antibody MAPK pathway protein array. Whole cell protein extracts were analyzed from regrowing mouse livers stably expressing either shMfap4 or shNC (relative spot intensity is shown). C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. D) After performing a broad protein array, a focused Western blot experiment was performed. The results of the Western blot are shown here. Proteins were isolated from fully regrowing livers. P-P70S6k, p-p38, p-mTOR, and p-ERK2 are expressed more in the case of Mfap4 knockdown compared to the control, and therefore show stronger activation in the case of Mfap4 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. E) Schematic diagram of mTOR-mediated regulation. Specific mTOR phosphorylation is upstream of p70S6k activation, leading to forced translation. F) Wound healing under double knockdown conditions. Based on pathway analysis, double knockout experiments were initiated. Our stable cell line was expanded, cells were treated with the respective siRNAs, and the silicone gasket was removed. Wound healing was monitored. Slower growth and migration were observed in the case of double knockdown of Mfap4 and p70S6k and Mfap4 and p38. G) Western blots with proteins isolated from cells in Figure F. Interestingly, p38 knockdown also affects p70S6k. H) Schematic outline of preparation of stable cell line with Mfap4 knocked down for transcriptome analysis. I) Principal component analyses of AML12-shMfap4.1356, AML12-shMfap4.760, AML12-shNC, (Rb88-RMA050 & Ren_RMA061), and AML12 (AML_RMA052) are shown. The inventors observed cluster separation between the experiments and the control.J) Heatmaps of the following samples are shown. Compared to the control, Ptgs2, Areg, Dhrs9, Hmox1, and Nqo1 were upregulated in the experimental samples, and these genes are known to be involved in liver regeneration according to the literature. K) The String database shows the linkages between proteins that are upregulated according to Figures D and J. [Figure 9-1]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 9-2]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 9-3]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 9-4]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 9-5]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 9-6]Figure 9 shows that the Mfap4 effect is conserved in human cells. A) An shRNA that efficiently targets human MFAP4 was identified. Knockdown test by Western blot analysis using whole cell lysates. HepG2 cells stably expressing the identified shRNA that targets human MFAP4 were generated by retroviral infection and selection. Tubulin served as a loading control. B) EdU integration assay. DNA synthesis of HepG2 cells transfected with hushMfap4 and shNC was evaluated by EdU assay. Quantifications indicate significant differences between experiments and controls. C) Transcriptome analysis of liver samples from approximately 150 patients shows increased Mfap4 expression in NAFLD patients with cirrhosis and fibrosis 4 score (Table: Squares indicate disease stages with significant changes but log2 2 digit changes; gray marks indicate significant upregulation of at least log2 2 digits; *p<0.05, **p<0.01, ***p,0.005). D) Human tissue samples from healthy and cirrhotic livers were stained for the Mfap4 protein (Mfap4-specific antibody & DAB staining). On the left, healthy liver tissue was stained without a primary antibody as a control. In the center, staining of the healthy liver shows that hepatocytes are slightly positive for Mfap4. Interestingly, some nuclear staining is also visible. The right panel shows staining of cirrhotic human liver. Human hepatocytes show strong staining in the cytoplasm as well as nuclear staining. On the left side of the cirrhotic liver, fibrous scar tissue can be seen, which is highly positive for Mfap4. E) Knockdown test of the human MFAP4 siRNA pool. Western blot analysis of protein extracts from immortalized human hepatocytes (Creative Bioarray CSC-I9016L) treated with either si huMFAP4 or siNC, α-tubulin served as a loading control (n=3). F) The EdU-incorporated assay shows a greater number of EdU-positive cells in the experiment compared to the control. G) EdU-incorporated assay (3 technical replicas). EdU-positive cell percentages ± SEM values ​​are shown.Immortalized human hepatocytes were treated with either siRNA targeting human MFAP4 or siNC as a control (*p<0.05). H) Retroviral scaffold scheme for generating stable cell lines. I) Representative GFP image of immortalized human hepatocytes (Creative Bioarray CSC-I9016L) with stably incorporated shRNA targeting human Mfap4. J) qPCR analysis showing efficient knockdown of huMfap4 by two shRNAs - hu shMfap4.1812 (SEQ ID NO: 7100) and hu shMfap4.1602 (7097) compared to an untargeted control. K) Western blot showing efficient knockdown of human MFAP4 by two independent shRNAs in immortalized human hepatocytes - SV40. L) Mfap4 knockdown in human immortalized hepatocytes accelerates wound healing. Wound healing assay using immortalized human hepatocytes stably expressing shhuMFAP4.1602 or shNC, respectively. Cells were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free area (0 hours). The closure of this "wound" gap was monitored (48 hours; n=3 for each state). M)L) quantification of wound healing area (n=3; *p<0.05, ns=not significant). [Figure 10]Figure 10 shows in vitro confirmation of targeting Grhpr for enhanced regeneration. A) Outline of the retroviral scaffold for generating stable cell lines. B) Test of knockdown efficiency of top-scoring shRNAs targeting Grhpr. Western blot showing efficient knockdown of Grhpr by our shRNA (alpha-tubulin, αTub serving as a loading control). C) Wound healing assay. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The filling of this “wound” gap was monitored. Representative images of each group are shown. D) Quantification of the wound healing assay at various time points is shown (data were analyzed by two-way ANOVA using ImageJ and GraphPad Prism software. Significant differences between shGrhpr361 (SEQ ID NO: 3) and shNC are indicated by “*”). [Figure 11-1]Figure 11 shows that Grhpr knockdown accelerates liver regrowth. A) The outline shows the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest. B) FAH knockout mouse-based liver regrowth assay. The outline shows the rationale for the assay. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion compared to control shRNA, starting from stably incorporated hepatocytes. C) GFP imager image. GFP imaging of explanted mouse liver shows enhanced clonal expansion (regrowth) in hepatocytes stably expressing shGrhpr.361 (SEQ ID NO: 3) compared to hepatocytes expressing shNC (18 days after HDTV injection of 25 μg of the plasmid shown). Representative images of each group are shown (n=5). Right, white dots represent macroscopically visible clonal expansion of GFP-positive cells. D) Native GFP on tissue sections. Representative GFP fluorescence images of liver sections (200×) of FAH- / - mice 18 days after in vivo delivery of a transposon construct expressing either shGrhpr or the corresponding control shRNA (C) are shown. (E) Histological analysis (immunostaining for GFP) of GFP-positive cells in mouse livers stably expressing shGrhpr.361 (SEQ ID NO: 3) and shNC (representative images are shown, n=5 in the group with shGrhpr.361, n=5 in the group with shNC). 18 days after HDTV injection of 1.25 μg of the plasmid shown (200x magnification). Increased clonal expansion can be seen for shMfap4. (F) Quantification of GFP-positive cells (corresponding to E) shows a significant increase in GFP-positive hepatocytes in the case of Mfap4 knockdown compared to the control. Each dot represents one animal. (G) Survival curves using construct dilution (1:30) as 0.83 μg plasmid and 0.17 mg SB13. All experimental mice (n=5) with the shGrhpr construct survived, while the control mice (n=5) died. [Figure 11-2]Figure 11 shows that Grhpr knockdown accelerates liver regrowth. A) The outline shows the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest. B) FAH knockout mouse-based liver regrowth assay. The outline shows the rationale for the assay. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion compared to control shRNA, starting from stably incorporated hepatocytes. C) GFP imager image. GFP imaging of explanted mouse liver shows enhanced clonal expansion (regrowth) in hepatocytes stably expressing shGrhpr.361 (SEQ ID NO: 3) compared to hepatocytes expressing shNC (18 days after HDTV injection of 25 μg of the plasmid shown). Representative images of each group are shown (n=5). Right, white dots represent macroscopically visible clonal expansion of GFP-positive cells. D) Native GFP on tissue sections. Representative GFP fluorescence images of liver sections (200×) of FAH- / - mice 18 days after in vivo delivery of a transposon construct expressing either shGrhpr or the corresponding control shRNA (C) are shown. (E) Histological analysis (immunostaining for GFP) of GFP-positive cells in mouse livers stably expressing shGrhpr.361 (SEQ ID NO: 3) and shNC (representative images are shown, n=5 in the group with shGrhpr.361, n=5 in the group with shNC). 18 days after HDTV injection of 1.25 μg of the plasmid shown (200x magnification). Increased clonal expansion can be seen for shMfap4. (F) Quantification of GFP-positive cells (corresponding to E) shows a significant increase in GFP-positive hepatocytes in the case of Mfap4 knockdown compared to the control. Each dot represents one animal. (G) Survival curves using construct dilution (1:30) as 0.83 μg plasmid and 0.17 mg SB13. All experimental mice (n=5) with the shGrhpr construct survived, while the control mice (n=5) died. [Figure 12]Figure 12 shows that Grhpr knockdown accelerates liver regeneration after partial hepatectomy. A) Experimental overview. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected at various postoperative time points. B) Representative photographs (200x magnification) of Ki67 DAB-stained liver sections at 24 hours (n=5 per group), 38 hours (n=6 per group), and 48 hours (n=9 per group) after partial hepatectomy. Faster and increased hepatocyte proliferation after partial hepatectomy can be seen in shGrhpr-expressing livers compared to shNC livers. C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to B) shows faster and increased hepatocyte proliferation after partial hepatectomy in shGrhpr-expressing livers compared to shNC livers (individual points represent individual animals, data shows mean ± SEM). D) Schematic diagram of the peak shift of the mitotic period in the case of Grhpr knockdown compared to control shNC (C). [Figure 13] Figure 13 shows that Grhpr knockdown attenuates chronic liver injury-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, chronic liver injury was induced by repeated doses of thioacetamide administered intraperitoneally three times a week for 8 weeks. Livers were harvested, processed, and analyzed. B) Representative macrographs of the livers are shown. Macroscopic differences between groups were already observed. C) Sirius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=5 per group, 50× magnification) of sections of regrowing mouse livers shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. [Figure 14]Figure 14 shows that Grhpr knockdown does not protect against NASH-associated liver fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. After complete regrowth was achieved, the mice were exposed to a "Western diet" (high-fat diet and 60% fructose) for 24 weeks. Livers were harvested, processed, and analyzed. B) Representative macrographs of the liver are shown. C) Sirius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=6 per experimental group and n=7 per control group; representative sections are shown, 50× magnification) of sections of regrowing mouse liver shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. [Figure 15-1] Figure 15 shows changes in Grhpr expression in human NAFLD. A) Transcriptome analysis of liver samples from approximately 150 patients shows a slight but significant decrease in Grhpr expression in NASH patients with progressive fibrosis and cirrhosis. Consistent with this, we detected a significant reduction in patients with fibrosis scores 3 and 4 (*p<0.05, **p<0.01, ***p<0.005). [Figure 15-2] Figure 15 shows changes in Grhpr expression in human NAFLD. A) Transcriptome analysis of liver samples from approximately 150 patients shows a slight but significant decrease in Grhpr expression in NASH patients with progressive fibrosis and cirrhosis. Consistent with this, we detected a significant reduction in patients with fibrosis scores 3 and 4 (*p<0.05, **p<0.01, ***p<0.005). [Figure 16-1]Figure 16 shows that Itfg1 knockdown accelerates wound healing and liver regrowth. A) Outline of the retroviral scaffold for generating stable cell lines. B) Testing of the knockdown efficiency of top-scoring shRNAs targeting Itfg1. qPCR and Western blot analysis demonstrate efficient knockdown of Itfg1 by our shRNAs. C) Itfg1 knockdown accelerates wound healing in vitro. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The closure of this “wound” gap was monitored. Representative images are shown at the top. Quantifications of the wound healing assay at various time points are shown (bottom; data were analyzed by two-way ANOVA using ImageJ and GraphPrizm software. Significant differences between shItfg1.698 (SEQ ID NO: 6), shItfg1.680 (SEQ ID NO: 7), and shNC are indicated by “*”). D) The summary shows the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest (upper panel). The lower panel shows the FAH knockout mouse-based liver regrowth assay. The summary shows the rationale for the assay. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion and proliferation, starting from stably incorporated hepatocytes, compared to control shRNA. E) GFP imager images. GFP imaging of explanted mouse liver shows enhanced clonal expansion and proliferation (regrowth) of hepatocytes stably expressing shItfg1 compared to hepatocytes expressing shNC (18 days after HDTV injection of 1.25 μg of the plasmid shown; representative images are shown; n=8 per experimental group with knockdown by shItfg1.698, n=6 per experimental group with knockdown by shItfg1.680, and n=6 per control group). On the right, the white dots represent macroscopic clonal expansion and proliferation of GFP-positive cells.F) Histological analysis (immunostaining for GFP) of GFP-positive cells from mouse livers stably expressing shItfg1.698, shItfg1.680, and shNC (representative photographs are shown). 18 days after HDTV injection of 1.25 μg of the plasmids shown (200x magnification). Increased clonal expansion can be seen for shItfg1. G) Quantification of GFP-positive cells (corresponding to F) shows a significant increase in GFP-positive hepatocytes in the case of Itfg1 knockdown compared to the control. Each dot represents one animal. H) Re-expansion survival assay. The panel on the right shows a summary of the experiment. The inventors further diluted the amount of plasmid delivered to the liver. At certain dilutions, the amount of hepatocytes with stable integration becomes insufficient to expand and compensate for the loss of FAH- / - hepatocytes. However, knockdown by the inventors' candidates may be sufficient to compensate and enable survival if it accelerates regrowth. The left panel shows the survival curves after a 1:30 dilution. All animals injected with our construct expressing the control shRNA died, while all mice injected with our construct expressing shItfg1 survived. There is a statistically significant difference between the experiment and the control. Statistical significance was calculated using the log-rank test (n=5 per group). [Figure 16-2]Figure 16 shows that Itfg1 knockdown accelerates wound healing and liver regrowth. A) Outline of the retroviral scaffold for generating stable cell lines. B) Testing of the knockdown efficiency of top-scoring shRNAs targeting Itfg1. qPCR and Western blot analysis demonstrate efficient knockdown of Itfg1 by our shRNAs. C) Itfg1 knockdown accelerates wound healing in vitro. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The closure of this “wound” gap was monitored. Representative images are shown at the top. Quantifications of the wound healing assay at various time points are shown (bottom; data were analyzed by two-way ANOVA using ImageJ and GraphPrizm software. Significant differences between shItfg1.698 (SEQ ID NO: 6), shItfg1.680 (SEQ ID NO: 7), and shNC are indicated by “*”). D) The summary shows the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest (upper panel). The lower panel shows the FAH knockout mouse-based liver regrowth assay. The summary shows the rationale for the assay. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion and proliferation, starting from stably incorporated hepatocytes, compared to control shRNA. E) GFP imager images. GFP imaging of explanted mouse liver shows enhanced clonal expansion and proliferation (regrowth) of hepatocytes stably expressing shItfg1 compared to hepatocytes expressing shNC (18 days after HDTV injection of 1.25 μg of the plasmid shown; representative images are shown; n=8 per experimental group with knockdown by shItfg1.698, n=6 per experimental group with knockdown by shItfg1.680, and n=6 per control group). On the right, the white dots represent macroscopic clonal expansion and proliferation of GFP-positive cells.F) Histological analysis (immunostaining for GFP) of GFP-positive cells from mouse livers stably expressing shItfg1.698, shItfg1.680, and shNC (representative photographs are shown). 18 days after HDTV injection of 1.25 μg of the plasmids shown (200x magnification). Increased clonal expansion can be seen for shItfg1. G) Quantification of GFP-positive cells (corresponding to F) shows a significant increase in GFP-positive hepatocytes in the case of Itfg1 knockdown compared to the control. Each dot represents one animal. H) Re-expansion survival assay. The panel on the right shows a summary of the experiment. The inventors further diluted the amount of plasmid delivered to the liver. At certain dilutions, the amount of hepatocytes with stable integration becomes insufficient to expand and compensate for the loss of FAH- / - hepatocytes. However, knockdown by the inventors' candidates may be sufficient to compensate and enable survival if it accelerates regrowth. The left panel shows the survival curves after a 1:30 dilution. All animals injected with our construct expressing the control shRNA died, while all mice injected with our construct expressing shItfg1 survived. There is a statistically significant difference between the experiment and the control. Statistical significance was calculated using the log-rank test (n=5 per group). [Figure 16-3]Figure 16 shows that Itfg1 knockdown accelerates wound healing and liver regrowth. A) Outline of the retroviral scaffold for generating stable cell lines. B) Testing of the knockdown efficiency of top-scoring shRNAs targeting Itfg1. qPCR and Western blot analysis demonstrate efficient knockdown of Itfg1 by our shRNAs. C) Itfg1 knockdown accelerates wound healing in vitro. Stable cell lines were grown to full confluence, then the silicone gasket was removed, leaving a defined cell-free region. The closure of this “wound” gap was monitored. Representative images are shown at the top. Quantifications of the wound healing assay at various time points are shown (bottom; data were analyzed by two-way ANOVA using ImageJ and GraphPrizm software. Significant differences between shItfg1.698 (SEQ ID NO: 6), shItfg1.680 (SEQ ID NO: 7), and shNC are indicated by “*”). D) The summary shows the transposon-based vector for the expression of the enzyme FAH, the marker GFP, and the shRNA of interest (upper panel). The lower panel shows the FAH knockout mouse-based liver regrowth assay. The summary shows the rationale for the assay. If knockdown of a particular shRNA can enhance regrowth and accelerate hepatocyte proliferation, we should be able to observe more rapid clonal expansion and proliferation, starting from stably incorporated hepatocytes, compared to control shRNA. E) GFP imager images. GFP imaging of explanted mouse liver shows enhanced clonal expansion and proliferation (regrowth) of hepatocytes stably expressing shItfg1 compared to hepatocytes expressing shNC (18 days after HDTV injection of 1.25 μg of the plasmid shown; representative images are shown; n=8 per experimental group with knockdown by shItfg1.698, n=6 per experimental group with knockdown by shItfg1.680, and n=6 per control group). On the right, the white dots represent macroscopic clonal expansion and proliferation of GFP-positive cells.F) Histological analysis (immunostaining for GFP) of GFP-positive cells from mouse livers stably expressing shItfg1.698, shItfg1.680, and shNC (representative photographs are shown). 18 days after HDTV injection of 1.25 μg of the plasmids shown (200x magnification). Increased clonal expansion can be seen for shItfg1. G) Quantification of GFP-positive cells (corresponding to F) shows a significant increase in GFP-positive hepatocytes in the case of Itfg1 knockdown compared to the control. Each dot represents one animal. H) Re-expansion survival assay. The panel on the right shows a summary of the experiment. The inventors further diluted the amount of plasmid delivered to the liver. At certain dilutions, the amount of hepatocytes with stable integration becomes insufficient to expand and compensate for the loss of FAH- / - hepatocytes. However, knockdown by the inventors' candidates may be sufficient to compensate and enable survival if it accelerates regrowth. The left panel shows the survival curves after a 1:30 dilution. All animals injected with our construct expressing the control shRNA died, while all mice injected with our construct expressing shItfg1 survived. There is a statistically significant difference between the experiment and the control. Statistical significance was calculated using the log-rank test (n=5 per group). [Figure 17-1]Figure 17 shows that Itfg1 knockdown attenuates chronic liver injury-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, chronic liver injury was induced by repeated doses of thioacetamide administered intraperitoneally 3 times per week for 8 weeks. Liver tissue was harvested, processed, and analyzed. B) Representative macrographs of the livers are shown. Macroscopic differences between groups were already observed. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=6 for shItfg1.698 and n=7 for the control group, 50× magnification) of sections of regrowing mouse livers shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. E) Representative macrographs of the livers using a GFP imaging system are shown. All livers are green and therefore completely regrowing. [Figure 17-2] Figure 17 shows that Itfg1 knockdown attenuates chronic liver injury-associated hepatic fibrosis. A) Experimental summary. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, chronic liver injury was induced by repeated doses of thioacetamide administered intraperitoneally 3 times per week for 8 weeks. Liver tissue was harvested, processed, and analyzed. B) Representative macrographs of the livers are shown. Macroscopic differences between groups were already observed. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining (n=6 for shItfg1.698 and n=7 for the control group, 50× magnification) of sections of regrowing mouse livers shown. D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. E) Representative macrographs of the livers using a GFP imaging system are shown. All livers are green and therefore completely regrowing. [Figure 18-1]Figure 18 shows that ITFG1 expression knockdown in human liver tissue protects against NASH-associated fibrosis (see also Figure 35). A) Macroscopic photograph of mice with regrowthed livers exposed to a Western diet. shItfg1 indicates that the liver regrows so that every hepatocyte expresses shRNA targeting Itfg1, while shNC indicates regrowth so that every hepatocyte expresses untargeted control shRNA. Livers with already macroscopically present ITFG1 knockdown show reduced fibrosis. B) Transcriptome analysis of liver samples from approximately 150 patients shows no significant changes in ITFG1 expression. C) ITFG1 is expressed in healthy liver tissue in NASH cirrhosis. D) ITFG1 expression in human tissue is shown. Data taken from The Human Protein Atlas. E) Low ITFG1 expression is associated with long-term survival in the case of liver cancer. Data taken from The Human Protein Atlas. F) Retroviral scaffold scheme for generating stable cell lines. G) ShRNAs that efficiently target human ITFG1 are identified. Knockdown analysis by Western blot using whole cell lysates. HepG2 cells stably expressing the target shRNA were generated by retroviral infection and selection. GAPDH serves as a loading control. [Figure 18-2]Figure 18 shows that ITFG1 expression knockdown in human liver tissue protects against NASH-associated fibrosis (see also Figure 35). A) Macroscopic photograph of mice with regrowthed livers exposed to a Western diet. shItfg1 indicates that the liver regrows so that every hepatocyte expresses shRNA targeting Itfg1, while shNC indicates regrowth so that every hepatocyte expresses untargeted control shRNA. Livers with already macroscopically present ITFG1 knockdown show reduced fibrosis. B) Transcriptome analysis of liver samples from approximately 150 patients shows no significant changes in ITFG1 expression. C) ITFG1 is expressed in healthy liver tissue in NASH cirrhosis. D) ITFG1 expression in human tissue is shown. Data taken from The Human Protein Atlas. E) Low ITFG1 expression is associated with long-term survival in the case of liver cancer. Data taken from The Human Protein Atlas. F) Retroviral scaffold scheme for generating stable cell lines. G) ShRNAs that efficiently target human ITFG1 are identified. Knockdown analysis by Western blot using whole cell lysates. HepG2 cells stably expressing the target shRNA were generated by retroviral infection and selection. GAPDH serves as a loading control. [Figure 18-3]Figure 18 shows that ITFG1 expression knockdown in human liver tissue protects against NASH-associated fibrosis (see also Figure 35). A) Macroscopic photograph of mice with regrowthed livers exposed to a Western diet. shItfg1 indicates that the liver regrows so that every hepatocyte expresses shRNA targeting Itfg1, while shNC indicates regrowth so that every hepatocyte expresses untargeted control shRNA. Livers with already macroscopically present ITFG1 knockdown show reduced fibrosis. B) Transcriptome analysis of liver samples from approximately 150 patients shows no significant changes in ITFG1 expression. C) ITFG1 is expressed in healthy liver tissue in NASH cirrhosis. D) ITFG1 expression in human tissue is shown. Data taken from The Human Protein Atlas. E) Low ITFG1 expression is associated with long-term survival in the case of liver cancer. Data taken from The Human Protein Atlas. F) Retroviral scaffold scheme for generating stable cell lines. G) ShRNAs that efficiently target human ITFG1 are identified. Knockdown analysis by Western blot using whole cell lysates. HepG2 cells stably expressing the target shRNA were generated by retroviral infection and selection. GAPDH serves as a loading control. [Figure 18-4]Figure 18 shows that ITFG1 expression knockdown in human liver tissue protects against NASH-associated fibrosis (see also Figure 35). A) Macroscopic photograph of mice with regrowthed livers exposed to a Western diet. shItfg1 indicates that the liver regrows so that every hepatocyte expresses shRNA targeting Itfg1, while shNC indicates regrowth so that every hepatocyte expresses untargeted control shRNA. Livers with already macroscopically present ITFG1 knockdown show reduced fibrosis. B) Transcriptome analysis of liver samples from approximately 150 patients shows no significant changes in ITFG1 expression. C) ITFG1 is expressed in healthy liver tissue in NASH cirrhosis. D) ITFG1 expression in human tissue is shown. Data taken from The Human Protein Atlas. E) Low ITFG1 expression is associated with long-term survival in the case of liver cancer. Data taken from The Human Protein Atlas. F) Retroviral scaffold scheme for generating stable cell lines. G) ShRNAs that efficiently target human ITFG1 are identified. Knockdown analysis by Western blot using whole cell lysates. HepG2 cells stably expressing the target shRNA were generated by retroviral infection and selection. GAPDH serves as a loading control. [Figure 18-5]Figure 18 shows that ITFG1 expression knockdown in human liver tissue protects against NASH-associated fibrosis (see also Figure 35). A) Macroscopic photograph of mice with regrowthed livers exposed to a Western diet. shItfg1 indicates that the liver regrows so that every hepatocyte expresses shRNA targeting Itfg1, while shNC indicates regrowth so that every hepatocyte expresses untargeted control shRNA. Livers with already macroscopically present ITFG1 knockdown show reduced fibrosis. B) Transcriptome analysis of liver samples from approximately 150 patients shows no significant changes in ITFG1 expression. C) ITFG1 is expressed in healthy liver tissue in NASH cirrhosis. D) ITFG1 expression in human tissue is shown. Data taken from The Human Protein Atlas. E) Low ITFG1 expression is associated with long-term survival in the case of liver cancer. Data taken from The Human Protein Atlas. F) Retroviral scaffold scheme for generating stable cell lines. G) ShRNAs that efficiently target human ITFG1 are identified. Knockdown analysis by Western blot using whole cell lysates. HepG2 cells stably expressing the target shRNA were generated by retroviral infection and selection. GAPDH serves as a loading control. [Figure 19-1]Figure 19 shows the emulsion + 500 in vivo functional gene screening. A) Schematic overview of the screening. A pooled shRNA library screening is set up targeting 467 dysregulated genes in human NAFLD patients. The screening is performed in mice of both sexes using two diet-based NAFLD models. B) Presentation of the pluripotency change of each shRNA passing through a p-value of 0.1 from male mice exposed to a choline-deficient L-amino acid-restricted high-fat diet for 8 weeks. The majority of shRNAs are depleted, but a small number are clearly enriched. C) Principal component analysis based on normalized shRNA abundance levels. We can see clear separation based on dietary exposure. D) Heatmap-based enrichment / depletion for each animal for top enriched and depleted shRNAs. Based on our analysis, six highly reliable targets were identified. [Figure 19-2] Figure 19 shows the emulsion + 500 in vivo functional gene screening. A) Schematic overview of the screening. A pooled shRNA library screening is set up targeting 467 dysregulated genes in human NAFLD patients. The screening is performed in mice of both sexes using two diet-based NAFLD models. B) Presentation of the pluripotency change of each shRNA passing through a p-value of 0.1 from male mice exposed to a choline-deficient L-amino acid-restricted high-fat diet for 8 weeks. The majority of shRNAs are depleted, but a small number are clearly enriched. C) Principal component analysis based on normalized shRNA abundance levels. We can see clear separation based on dietary exposure. D) Heatmap-based enrichment / depletion for each animal for top enriched and depleted shRNAs. Based on our analysis, six highly reliable targets were identified. [Figure 20]Figure 20 shows a CDHFD mouse fatty liver model. A) A choline-deficient L-amino acid-modified high-fat diet (CDHFD) leads to rapid, progressive fatty liver disease in mice. After as little as 8 weeks of dietary exposure, mice exhibit NASH with progressive fibrosis. B) Pathological evaluation. Histological slides of liver tissue from C56Bl6 mice exposed to CDHFD or normal solid feed for the indicated time were evaluated and scored by a certified pathologist. Scoring results for fatty liver and fibrosis are shown. Each point represents one animal. [Figure 21-1] Figure 21 shows that Abcc4 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Abcc4 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Abcc4. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Abcc4 gene expression during the late fibrotic and cirrhotic stages of NASH. Furthermore, increased expression can be detected based on ballooning and fibrotic stages (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 21-2]Figure 21 shows that Abcc4 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Abcc4 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Abcc4. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Abcc4 gene expression during the late fibrotic and cirrhotic stages of NASH. Furthermore, increased expression can be detected based on ballooning and fibrotic stages (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 21-3] Figure 21 shows that Abcc4 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Abcc4 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Abcc4. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Abcc4 gene expression during the late fibrotic and cirrhotic stages of NASH. Furthermore, increased expression can be detected based on ballooning and fibrotic stages (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 22-1]Figure 22 shows that Pak3 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Pak3 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Pak3. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Pak3 gene expression during the NASH cirrhosis stage. Furthermore, increased expression can be detected based on the fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 22-2] Figure 22 shows that Pak3 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Pak3 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Pak3. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Pak3 gene expression during the NASH cirrhosis stage. Furthermore, increased expression can be detected based on the fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 22-3]Figure 22 shows that Pak3 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Pak3 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Pak3. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Pak3 gene expression during the NASH cirrhosis stage. Furthermore, increased expression can be detected based on the fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 23-1] Figure 23 shows that Trnp1 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Trnp1 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Trnp1. Mean relative reads for each group, comparison of CDHFD mice with NC mice, metric changes and log2 metric changes are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Trnp1 gene expression during the NASH cirrhosis stage. Interestingly, expression appears to be downregulated as fatty liver and inflammation increase (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 23-2]Figure 23 shows that Trnp1 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Trnp1 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Trnp1. Mean relative reads for each group, comparison of CDHFD mice with NC mice, metric changes and log2 metric changes are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Trnp1 gene expression during the NASH cirrhosis stage. Interestingly, expression appears to be downregulated as fatty liver and inflammation increase (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 23-3] Figure 23 shows that Trnp1 is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Trnp1 are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Trnp1. Mean relative reads for each group, comparison of CDHFD mice with NC mice, metric changes and log2 metric changes are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Trnp1 gene expression during the NASH cirrhosis stage. Interestingly, expression appears to be downregulated as fatty liver and inflammation increase (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 24-1]Figure 24 shows that Apln is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Apln are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Apln. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Apln gene expression during the NASH cirrhosis stage. Interestingly, as inflammation increases, expression appears to be downregulated (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 24-2] Figure 24 shows that Apln is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Apln are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Apln. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Apln gene expression during the NASH cirrhosis stage. Interestingly, as inflammation increases, expression appears to be downregulated (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 24-3]Figure 24 shows that Apln is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Apln are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Apln. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a significant increase in Apln gene expression during the NASH cirrhosis stage. Interestingly, as inflammation increases, expression appears to be downregulated (Table: Gray marks indicate significant upregulation of at least log2 2 metric; Gray marks enclosed in squares indicate significant downregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 25-1] Figure 25 shows that Kif20a is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Kif20a are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Kif20a. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Kif20a gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 25-2]Figure 25 shows that Kif20a is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Kif20a are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Kif20a. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Kif20a gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 25-3] Figure 25 shows that Kif20a is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Kif20a are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Kif20a. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Kif20a gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 26-1]Figure 26 shows that Ltb is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Ltb are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Ltb. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Ltb gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 26-2] Figure 26 shows that Ltb is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Ltb are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Ltb. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Ltb gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 26-3]Figure 26 shows that Ltb is a promising therapeutic target for NAFLD. A) Relative reads of shRNA expression cassettes targeting Ltb are shown for each animal (NC = normal solid feed, CD = CDHFD). B) Summary of screening results for shRNA expression cassettes targeting Ltb. Mean relative reads for each group, metric changes and log2 metric changes comparing CDHFD mice to NC mice are shown. C) Transcriptome analysis of liver samples from approximately 150 patients shows a progressive increase in Ltb gene expression up to the NASH progressive fibrosis stage (Table: Gray marks indicate significant upregulation of at least log2 2 metric; *p<0.05, **p<0.01, ***p,0.005). [Figure 27] Figure 27 shows the configuration for in vivo functional gene screening to block NASH disease. A) Genome-wide in vivo functional gene screening for disease blockade. Screening of approximately 80,000 shRNAs divided into 32 subpools. ShRNA expression is inducible and activated only after the liver shows signs of fatty liver, but before NASH progression. [Figure 28]Figure 28 shows that one year of Mfap4 knockdown does not lead to liver cancer. A) Schematic diagram of the experiment. FAH- / - mice were injected with a p / T-FAHIG-shRNA & SB13 expression construct via HDTV, and the mice were then maintained for one year to observe any tumorigenesis or abnormal liver histology. B) Bright-field imaging. Representative images are shown (both surfaces of the liver) (n=5 mice per experimental group, n=5 mice per control group). C) GFP imaging. Representative images are shown (both surfaces of the liver). No GFP-positive tumors were observed. The liver is completely regrowthed (strong GFP-positive signal). D) Hematoxylin & eosin staining. Representative images are shown. Both: No malignant disease was observed in the experimental and control groups. Pathological evaluation was performed by a certified pathologist. The pathologist found no malignant lesions in the liver. E) GFP (DAB) staining. Representative images are shown. Approximately 95% of the liver cells were GFP-positive, which means that the liver had completely regrowthed. [Figure 29] Figure 29 shows GalNAC conjugates with siRNA against Mfap4 (BNL CL.2 cell line; 72 hours post-transfection). A) Structure of the GalNAC-siRNA conjugate used in the study. Precise skeletal modifications can be found in the sequence appendix (SEQ ID NOs. 7092 and 7093). The siRNA target sequences were based on shRNA guide sequences. B) Western blot analysis using a concentration of 6 μM shows efficient knockdown of Mfap4 by two different conjugates, GalNAC-si Mfap4.1356 (SEQ ID NO: 7092) and GalNAC-si Mfap4.760 (SEQ ID NO: 7093), compared to a control. C) Western blot analysis using a concentration of 11 μM shows efficient knockdown of Mfap4 by two different conjugates, GalNAC-si Mfap4.1356 and GalNAC-si Mfap4.760, compared to a control. [Figure 30]Figure 30 shows that one year of Grhpr knockdown does not lead to liver cancer. A) Schematic diagram of the experiment. FAH- / - mice were injected with a p / T-FAHIG-shRNA&SB13 expression construct via HDTV, and the mice were then maintained for one year to observe any tumorigenesis or abnormal liver histology. Liver tissue was harvested one year after injection. B) Bright-field imaging. Representative images are shown (both surfaces of the liver) (n=3 mice per experimental group, n=5 mice per control group). C) GFP imaging. Representative images are shown (both surfaces of the liver). No GFP-positive tumors were observed. The liver was completely regrowthed (strong GFP-positive signal). [Figure 31] Figure 31 shows Grhpr expression in human hepatocytes (HpG2 cell line). A) Retroviral scaffold scheme for generating stable cell lines. B) Identification of shRNAs that efficiently target human Grhpr. Knockdown test by qPCR using whole cell lysates. HepG2 cells were co-transfected with the pMSCV vector. C) Knockdown test by Western blotting using whole cell lysates. Retroviral infection and selection generated HepG2 cells that stably express the indicated shRNAs. Tubulin serves as a loading control. [Figure 32] Figure 32 shows the GalNAC conjugate with siRNA against Grhpr (BNL CL.2 cell line; 72 hours post-transfection). A) Structure of the GalNAC-siRNA conjugate used in the study. The exact skeletal modifications can be found in the sequence appendix (SEQ ID NO: 7094). The siRNA target sequence was based on the shRNA guide sequence. B) Western blot analysis using a concentration of 6 μM shows efficient knockdown of Grhpr by the conjugated GalNAC-si Grhpr.361 (SEQ ID NO: 7094) compared to a scrambled control. Western blot analysis using a concentration of 11 μM shows efficient knockdown of Grhpr by the conjugated GalNAC-si Grhpr.361 compared to a scrambled control. [Figure 33-1]Figure 33 shows that Itfg1 knockdown accelerates liver regeneration after partial hepatectomy (PH). A) Experimental overview. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected 42 and 48 hours after surgery. B) Representative photographs of DAB Ki67-stained liver sections after hepatectomy are shown at 42 hours (n=4 per experimental group, n=6 per control group) and 48 hours (n=5 per experimental group, n=10 per control group) (200× magnification). C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation after partial hepatectomy in shItfg1-expressing livers compared to shNC livers (individual points represent individual animals, data shows mean ± SEM). [Figure 33-2] Figure 33 shows that Itfg1 knockdown accelerates liver regeneration after partial hepatectomy (PH). A) Experimental overview. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. Subsequently, 2 / 3 of the liver was surgically removed. The remaining regenerative liver was collected 42 and 48 hours after surgery. B) Representative photographs of DAB Ki67-stained liver sections after hepatectomy are shown at 42 hours (n=4 per experimental group, n=6 per control group) and 48 hours (n=5 per experimental group, n=10 per control group) (200× magnification). C) Quantification of Ki67-positive cells in DAB-stained liver sections (corresponding to Figure B) shows increased hepatocyte proliferation after partial hepatectomy in shItfg1-expressing livers compared to shNC livers (individual points represent individual animals, data shows mean ± SEM). [Figure 34-1]Figure 34 shows that one year of Itfg1 knockdown does not lead to liver cancer. A) Schematic diagram of the experiment. FAH- / - mice were injected with a p / T-FAHIG-shRNA & SB13 expression construct via HDTV, and the mice were then maintained for one year to observe any tumor formation or abnormal liver histology. Liver tissue was harvested one year after injection. B) Bright-field imaging. Representative images are shown (both surfaces of the liver). No tumors were observed (n=5 mice per experimental group, n=5 mice per control group). C) GFP imaging. Representative images are shown (both surfaces of the liver). No GFP-positive tumors were observed. The liver was completely regrowthed (GFP-positive). D) Hematoxylin & eosin staining. Representative images are shown. Both: No malignant disease was observed in the experimental and control groups. Pathological evaluation is performed by a certified pathologist. E) GFP (DAB) staining. Representative images are shown. Approximately 95% of the liver cells were GFP-positive, which means that the liver had completely regrowthed. [Figure 34-2] Figure 34 shows that one year of Itfg1 knockdown does not lead to liver cancer. A) Schematic diagram of the experiment. FAH- / - mice were injected with a p / T-FAHIG-shRNA & SB13 expression construct via HDTV, and the mice were then maintained for one year to observe any tumor formation or abnormal liver histology. Liver tissue was harvested one year after injection. B) Bright-field imaging. Representative images are shown (both surfaces of the liver). No tumors were observed (n=5 mice per experimental group, n=5 mice per control group). C) GFP imaging. Representative images are shown (both surfaces of the liver). No GFP-positive tumors were observed. The liver was completely regrowthed (GFP-positive). D) Hematoxylin & eosin staining. Representative images are shown. Both: No malignant disease was observed in the experimental and control groups. Pathological evaluation is performed by a certified pathologist. E) GFP (DAB) staining. Representative images are shown. Approximately 95% of the liver cells were GFP-positive, which means that the liver had completely regrowthed. [Figure 35]Figure 35 shows that Itfg1 knockdown attenuates chronic liver injury-associated hepatic fibrosis in a NASH model. A) Experimental overview. FAH- / - mice were injected with our construct and maintained for 3 months for complete regrowth. After complete regrowth was achieved, the mice were exposed to a "Western diet" (high-fat diet and 60% fructose) for 24 weeks. Livers were harvested, processed, and analyzed. B) Representative macroscopic images of the livers are shown. Macroscopic differences were already observed between the groups. C) Picrosilius red staining (staining of fibrous scar tissue) and hematoxylin & eosin staining of sections of regrowing mouse livers shown (representative images are shown; n=6 for shItfg1.698 and n=7 for the control group, 50× magnification). D) Fibrousity scores for each animal are shown. Scores were given by certified pathologists blinded to the experimental groups. E) Objective, AI-based analysis of fatty liver performed by HistoIndex. Representative photographs are shown. F) Quantitative analysis shows that the experimental group (n=7 mice per group) has a significantly lower fatty liver score compared to the control group (n=7 mice per group). [Figure 36-1] Figure 36 shows that knockdown of Itfg1 affects MKK6, JNK, and RPS6 signaling. A) Schematic outline of isolating proteins from a fully regrowing liver for broader protein array analysis. B) After performing a broad protein array, focused Western blot experiments were performed. The results of the Western blots are shown here. Proteins were isolated from a fully regrowing liver. In particular, P-MKK6 / P-MKK3 is more activated in the case of Itfg1 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. [Figure 36-2]Figure 36 shows that knockdown of Itfg1 affects MKK6, JNK, and RPS6 signaling. A) Schematic outline of isolating proteins from a fully regrowing liver for broader protein array analysis. B) After performing a broad protein array, focused Western blot experiments were performed. The results of the Western blots are shown here. Proteins were isolated from a fully regrowing liver. In particular, P-MKK6 / P-MKK3 is more activated in the case of Itfg1 knockdown compared to the control. There are 3 biological replicas in the experiment and 3 biological replicas in the control. C) According to the STRING database, all shown proteins interact and are associated with cell growth and proliferation. [Figure 37] Figure 37 shows GalNAC conjugates with siRNA against Itfg1 (BNL CL.2 cell line; 72 hours post-transfection). A) Structure of the GalNAC-siRNA conjugates used in the study. Precise skeletal modifications can be found in the sequence appendix (SEQ ID NOs. 7095 and 7096). The siRNA target sequences were based on shRNA guide sequences. B) Western blot analysis using a concentration of 6 μM shows efficient knockdown of Itfg1 by two different conjugates, GalNAC-si Itfg1.698 (SEQ ID NO: 7095) and GalNAC-si Itfg1.680 (SEQ ID NO: 7096), compared to a control. Western blot analysis using a concentration of 11 μM shows efficient knockdown of Itfg1 by two different conjugates, GalNAC-si Itfg1.698 and GalNAC-si Itfg1.680, compared to a control. [Figure 38]Figure 38 shows that knockdown of Mfap4 and Itfg1 enhances proliferation and regeneration beyond the liver. A) Overview of the wound healing assay. Stable cell lines expressing each shRNA were generated. B) Knockdown of Mfap4 and Itfg1 accelerates wound healing in mouse lung cells (cell line CCL206). C) Knockdown of Mfap4 and Itfg1 accelerates wound healing in mouse myoblasts (myoblast cell line CRL1772). [Figure 39] Figure 39 shows that Pak3 knockdown accelerates wound healing in vitro. Stable knockdown of Pak3 in the AML12 adult hepatocyte cell line accelerates wound healing (representative images are shown). [Modes for carrying out the invention]

[0081] The present invention relates to the identification of proteins involved in the development of liver disease and / or detrimental to liver regeneration after injury, and to targeting such proteins to treat liver disease.

[0082] Without intending to be constrained by theory, the inventors used unbiased in vivo functional gene screening to identify novel therapeutic targets upregulated in liver disease and fibrosis-associated conditions. Enrichment of target shRNAs indicates that knockdown / inhibition of these targets provides a viable advantage to hepatocytes under chronic liver injury conditions. Since enrichment shows relative increased proliferation compared to controls, knockdown or inhibition of the identified genes supports increased hepatocyte proliferation, growth, and robustness. This is therapeutically beneficial for blocking liver disease, accelerating liver regeneration, protecting from liver injury, promoting cell proliferation, halting and recovering from liver fibrosis, and increasing survival. target This disclosure relates to the inhibition of the expression of one or more genes and / or proteins of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. Any one or combination of these genes (i.e., any one, two, three, four, five, six, seven, eight, or nine) may be inhibited in the manner provided herein. Any one or combination of these genes may be referred herein to as a target gene(s), target mRNA(s), or target protein(s). Any one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be referred herein to as “genes or corresponding gene products associated with organ regeneration.”

[0083] MFAP4, GRHPR, and ITFG1 are found in recurrent amplification in hepatocellular carcinoma (Nat Med. 2014 Oct; 20(10):1138-1146). ABCC4, PAK3, TRNP1, APLN, KIF20A, and LTB were all found by the inventors to be dysregulated in a localized patient cohort with non-alcoholic fatty liver disease (NAFLD).

[0084] Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-associated domain (FReD) superfamily. Human MFAP4 has been identified by UniProtKB P55083.

[0085] The structure and function of MFAP4 are described, for example, in Pilecki B. et al., J. Biol. Chem. 291: pp. 1103-1114 (2016), which are incorporated herein by reference in their entirety.

[0086] MFAP4 is an extracellular glycoprotein found in elastic fibers and is essential for proper elastic fiber organization. It specifically binds to tropoelastin, fibrillin-1 and -2, and the elastin-crosslinking amino acid desmosine, and co-localizes with fibrillin-1-positive fibers in vivo. Human MFAP4 is localized to elastic fibers in a variety of elastic tissues, including the aorta, skin, and lungs.

[0087] MFAP4 is closely associated with remodeling-related diseases, including hepatic fibrosis, atherosclerosis, arterial injury-induced remodeling, and asthma (Wang HB et al., J Am Heart Assoc. 2020; 9(17):e015307). Pan Z et al., FASEB J. 2020, 34(11):14250~14263, reported that MFAP4 deficiency alleviates renal fibrosis by inhibiting the activation of NF-κB and TGF-β / Smad signaling pathways and downregulating the expression of fibrosis-related proteins. MFAP4 is produced by activated myofibroblasts and may be a predictive biomarker for the severity of hepatic fibrosis (Madsen BS et al., Liver Int. 2020; 40(7): pp. 1701-1712; Saekmose SG et al., PLoS One. 2015; 10(10):e0140418). Example 2 of this application shows that genes known to be involved in liver regeneration, such as Ptgs2, Areg, Dhrs9, Hmox1, and Nqo1, are upregulated after Mfap4 knockdown.

[0088] Alternative splicing of mRNA transcribed from the human MFAP4 gene results in two isoforms: isoform 1 (UniProtKB:P55083-1, v2; SEQ ID NO: 7156) and isoform 2 (UniProtKB:P55083-2; SEQ ID NO: 7157), in which the amino acid sequences corresponding to positions 1-2 of SEQ ID NO: 7156 are replaced with the sequence "MGELSPLQRPLATEGTMKAQGVLLKL".

[0089] The 255-amino acid sequence of human MFP4 isoform 1 includes an N-terminal signal peptide at positions 1-21 of SEQ ID NO: 7156 and a mature protein region at positions 22-255. Positions 26-28 of SEQ ID NO: 7156 constitute the cell attachment site, and positions 32-255 constitute the fibrinogen C-terminal domain.

[0090] In this specification, references to “MFAP4” include human MFAP4, isoforms of human MFAP4, homologs of human MFAP4 (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, MFAP4 according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7156, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0091] Glyoxylate reductase / hydroxypyruvate reductase (GRHPR) is an NADPH / NADH-dependent enzyme possessing hydroxypyruvate reductase, glyoxylate reductase, and D-glycerate dehydrogenase enzymatic activity. It reduces the toxic intermediate glyoxylate to readily excreted glycolic acid and reduces hydroxypyruvate to D-glyceric acid for use in glucose synthesis. GRHPR deficiency is the underlying cause of primary hyperoxaluria type 2 (PH2), leading to increased urinary oxalate levels, kidney stone formation, and renal failure (Cregeen DP et al., Hum Mol Genet. 1999;8(11):2063-9). Human GRHPR has been identified by UniProtKB Q9UBQ7.

[0092] The structure and function of GRHPR are described, for example, in Rumsby G. and Cregeen DP Biochim. Biophys. Acta 1446:383-388 (1999), which are incorporated herein by reference in their entirety, and Booth et al. J Mol Biol, 2006; 360(1):178-89.

[0093] Alternative splicing of mRNA transcribed from the human GRHPR gene results in two isoforms: isoform 1 (UniProtKB:Q9UBQ7-1, v1; SEQ ID NO: 7158) and isoform 2 (UniProtKB:Q9UBQ7-2; SEQ ID NO: 7159), in which the amino acid sequence corresponding to positions 1-21 of SEQ ID NO: 7158 is replaced with the sequence "MLGGVPTLCGTGNETWTLLAL", positions 22-164 of SEQ ID NO: 7158 are lost, and positions 246-328 of SEQ ID NO: 7158 are replaced with the sequence "YPRATLPSKPGEEPSPLLPSGDFLPRGLLVRPQAELAGFHKPNNQLRNSWEYTRPPYREEEPSEWAWPVCFSAVAPTRRGLAHSSVASGSVPREPLQAHYPPPQRAGLEDLKGPLEAASHTAEPGFVWLWFSDTLNLMLLGGQTLKLTWS".

[0094] The 328-amino acid sequence of human GRHPR isoform 1 contains NADP binding sites at positions 217, 243, 162-164, 185-188 and 295 of SEQ ID NO: 7158, and substrate (glyoxylic acid / hydroxypyruvic acid) binding sites at positions 83-84, 245, 269 and 293-296 of SEQ ID NO: 7158.

[0095] In this specification, references to “GRHPR” include human GRHPR, isoforms of human GRHPR, homologs of human GRHPR (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, the GRHPRs according to this disclosure include or consist of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7158, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0096] T cell immunomodulatory proteins (ITFG1; also known as protein TIP, integrin-alpha-FG-GAP repeat-containing protein 1, or Linkin / LNKN-1) are modulators of T cell function. Human ITFG1 has been identified by UniProtKB Q8TB96.

[0097] The structure and function of ITFG1 are described, for example, in Fiscella M. et al., Nat. Biotechnol. 21:302-307 (2003), the entire text of which is incorporated herein by reference. Treatment of primary human and mouse T cells with ITFG1 in vitro results in the secretion of IFN-gamma, TNF-alpha, and IL-10, while in vivo, ITFG1 has been reported to have a protective effect in a mouse acute graft-versus-host disease (GVHD) model. The interaction between ITFG1 and the ATPase RUVBL1 has been reported to be necessary for the invasion and progression of breast cancer cells (Fan W. et al., Biochim Biophys Acta Gen Subj. 2017; 1861(7):1788-1800).

[0098] The 612-amino acid sequence of human ITFG1 is shown in SEQ ID NO: 7160 (UniprotKB:Q8TB96-1, v1). This sequence contains an N-terminal signal peptide at positions 1-33 of SEQ ID NO: 7160, an FG-GAP repeat at positions 258-293, and a transmembrane domain at positions 567-587.

[0099] In this specification, references to “ITFG1” include human ITFG1, isoforms of human ITFG1, homologs of human ITFG1 (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, ITFG1 according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7160, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0100] ATP-binding cassette subfamily C member 4 (ABCC4; also known as multidrug resistance protein 4 (MRP4)) is an ATP-dependent transporter of the ATP-binding cassette (ABC) family that actively pushes physiological compounds and xenobiotics out of cells. It transports a wide range of endogenous molecules that play important roles in cellular signal transduction and communication, including cyclic nucleotides such as cyclic AMP (cAMP) and cyclic GMP (cGMP), bile acids, steroid conjugates, uric acid, and prostaglandins. It is expressed in several tissues, including hepatocytes, and is most highly expressed in the kidney and choroidal plexus (Maher JM et al., Drug Metab. Dispos., 33 (2005), pp. 947-955). Human ABCC4 has been identified by UniProtKB O15439.

[0101] The structure and function of ABCC4 are described, for example, in its entirety by reference in Russel et al., Trends Pharmacol Sci. 2008, 29(4):200-7. ABCC4 is an inducible gene in the liver after exposure to toxic acetaminophen in both humans and rodents. In mice, ABCC4 deficiency is associated with an increased risk of liver injury, altered intestinal epithelial function, and altered drug pharmacokinetics, while protein expression has been reported to be increased in human livers with fatty liver, alcoholic cirrhosis, and diabetic cirrhosis (More VR et al., Drug Metab Dispos. 2013; 41(5):1148-1155).

[0102] Alternative splicing of mRNA transcribed from the human ABCC4 gene results in four isoforms: isoform 1 (UniProtKB:O15439-1, v3; SEQ ID NO: 7161), isoform 2 (O15439-2, SEQ ID NO: 7162), and the amino acid sequence corresponding to positions 846-859 of SEQ ID NO: 7161 is lost, replaced with the sequence "RWDLAVLSWLVSNS", and SEQ ID NO: 7 Isoform 3 (O15439-3, SEQ ID NO: 7163) has the amino acid sequence corresponding to positions 860-1325 of 161 missing, and isoform 4 (O15439-4, SEQ ID NO: 7164) has the amino acid sequence corresponding to positions 103-177 of SEQ ID NO: 7161 missing, and the amino acid sequence corresponding to positions 846-859 of SEQ ID NO: 7161 replaced with the sequence "RWDLAVLSWLVSNS", resulting in the loss of the amino acid sequence corresponding to positions 860-1325 of SEQ ID NO: 7161.

[0103] The 1325 amino acid sequence of human ABCC4 isoform 1 includes the ABC transmembrane-11 domain at positions 92-377, the ABC transporter 1 domain at positions 410-633, the ABC transmembrane-12 domain at positions 714-1005, the ABC transporter 2 domain at positions 1041-1274, and the ATP binding domains at positions 445-452 and 1075-1082.

[0104] In this specification, references to “ABCC4” include human ABCC4, isoforms of human ABCC4, homologs of human ABCC4 (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, ABCC4 according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7161, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0105] p21-activated kinase 3 (PAK3; also known as serine / threonine-protein kinase PAK3, Beta-PAK, or Oligophrenin-3) is a serine / threonine protein kinase that plays a role in various distinct signaling pathways, including cytoskeletal regulation, cell migration, or cell cycle regulation. Activation by binding of active CDC42 and RAC1 results in conformational changes and subsequent autophosphorylation at several serine and / or threonine residues. It phosphorylates MAPK4 and MAPK6, activating the downstream target MAPK5, a regulator of F-actin polymerization and cell migration. PAK3 is also a core mediator of integrin beta-1 signaling (a key mediator of HSC activation and fibrous disease progression). Human PAK3 has been identified by UniProtKB O75914.

[0106] The structure and function of PAK3 are described, for example, in Deleris P. et al., J. Biol. Chem. 286:6470-6478 (2011) and Chong C. et al., J. Biol. Chem. 276:17347-17353 (2001), both of which are incorporated herein by reference in their entirety.

[0107] Alternative splicing of mRNA transcribed from the human PAK3 gene results in four isoforms: isoform 1 (UniProtKB:O75914-1, v2; SEQ ID NO: 7165), in which the amino acid sequence corresponding to positions 93-107 of SEQ ID NO: 7165 is lost; isoform 2 (O75914-2, SEQ ID NO: 7166); isoform 3 (O75914-3, SEQ ID NO: 7167), in which the amino acid at position 92 of SEQ ID NO: 7165 is replaced with the sequence "TNSPFQTSRPVTVASSQSEGKM"; and isoform 4 (O75914-4, SEQ ID NO: 7168), in which the amino acid sequence corresponding to positions 92-107 of SEQ ID NO: 7165 is replaced with the sequence "TNSPFQTSRPVTVASSQSEGKM".

[0108] The 559-amino acid sequence of human PAK3 isoform 1 contains a CRIB domain at positions 70-83 and a protein kinase domain at positions 283-534 of sequence number 7165.

[0109] In this specification, references to “PAK3” include human PAK3, isoforms of human PAK3, homologs of human PAK3 (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, PAK3 according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7165, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0110] TMF regulatory nucleoprotein 1 (TRNP1) is a DNA-binding factor that regulates the expression of a subset of genes and plays a crucial role in the tangential, radial, and lateral expansion and growth of the neocortex. Human TRNP1 has been identified by UniProtKB Q6NT89.

[0111] The TRNP1 structure and function are described, for example, in Stahl R. et al., Cell 153: pp. 535-549 (2013), which are incorporated herein by reference in their entirety.

[0112] The 227-amino acid sequence of human TRNP1 is shown in SEQ ID NO: 7169 (UniprotKB:Q6NT89-1, v2).

[0113] In this specification, references to "TRNP1" include human TRNP1, isoforms of human TRNP1, homologs of human TRNP1 (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, TRNP1 according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7169, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0114] Apelin (APLN) is a peptide ligand for the G protein-coupled apelin receptor (APLNR). The APLN system plays a variety of crucial roles in the physiology and pathophysiology of numerous organs, including the regulation of blood pressure, myocardial contractility, angiogenesis, metabolic balance and cell proliferation, and apoptosis or inflammation. Apelin is expressed in the heart, endothelium, vascular smooth muscle cells (VSMCs), brain, kidney, testes, ovaries, liver, and adipose tissue, with the highest expression levels in the lungs and mammary glands. Human APLN has been identified by UniProtKB Q9ULZ1.

[0115] The structure and function of APLN are described, for example, in Tatemoto K. et al., Biochem. Biophys. Res. Commun. 251:471-476 (1998) and Lee DK et al., J. Neurochem. 74:34-41 (2000), both of which are incorporated herein by reference in their entirety.

[0116] The 77-amino acid sequence of human APLN is shown in SEQ ID NO: 7170 (UniprotKB: Q9ULZ1-1, v1). SEQ ID NO: 7170 contains a signal peptide at positions 1-22 and a propeptide at positions 23-41. SEQ ID NO: 7170 is cleaved into one or more active peptides by proteolytic processing: Apelin-36 at positions 42-77 of SEQ ID NO: 7170 (SEQ ID NO: 7171), Apelin-31 at positions 47-77 of SEQ ID NO: 7170 (SEQ ID NO: 7172), Apelin-28 at positions 50-77 of SEQ ID NO: 7170 (SEQ ID NO: 7173), or Apelin-13 at positions 65-77 of SEQ ID NO: 7170 (SEQ ID NO: 7174).

[0117] In this specification, references to “APLN” include human APLN, isoforms of human APLN, homologs of human APLN (i.e., encoded by the genomes of non-human animals), proteolytic peptides derived from human APLN, and their variants. In some embodiments, the APLN according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7170, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0118] Kinesin-like protein KIF20A (GG10_2, also known as mitotic kinesin-like protein 2 (MKlp2), Rab6-interacting kinesin-like protein (RAB6KIFL), and Rab kinesin-6) is a mitotic kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis. KIF20A is a target of polo-like kinase 1 (Plk1), and phosphorylated KIF20A binds to the polo box domain of Plk1. Phosphorylation of KIF20A by Plk1 is required for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis. Human KIF20A has been identified by UniProtKB O95235.

[0119] The structure and function of KIF20A are described, for example, in Neef R. et al., J Cell Biol. 2003;162(5):863-865, which are incorporated herein by reference in their entirety.

[0120] Alternative splicing of mRNA transcribed from the human KIF20A gene results in two isoforms: isoform 1 (UniProtKB:O95235-1, v1; SEQ ID NO: 7175) and isoform 2 (UniProtKB:O95235-2; SEQ ID NO: 7176), in which the amino acid sequence corresponding to positions 65-82 of SEQ ID NO: 7175 is lost.

[0121] The 890-amino acid sequence of human KIF20A isoform 1 includes a kinesin motor domain at positions 64-507 and a coiled-coil domain at positions 611-762 of sequence number 7175.

[0122] In this specification, references to “KIF20A” include human KIF20A, isoforms of human KIF20A, homologs of human KIF20A (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, KIF20A according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7175, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0123] Lymphotoxin-beta (LTB, also known as tumor necrosis factor C (TNF-C) or tumor necrosis factor ligand superfamily member 3) is a pro-inflammatory cytokine belonging to the TNF family that binds to the receptor LTBR / TNFRSF3. It participates in the regulation of immune and inflammatory responses and, together with other LT-related cytokines, such as LT-alpha, TNFα, and LIGHT (TNFSF14) and their receptors, plays a role in the development and homeostasis of secondary lymphoid organs. Human LTB has been identified by UniProtKB Q06643.

[0124] The LTB structure and function are described, for example, in Sudhamsu J. et al., Proc Natl Acad Sci USA 110:19896-19901 (2013), all of which are incorporated herein by reference in their entirety; Browning JL et al., Cell 72:847-856 (1993); Neville MJ & Campbell RDJ Immunol. 162:4745-4754 (1999); Crowe PD et al., Science. 1994; 264(5159):707-710; and Bjordahl RL et al., Curr Opin Immunol. 2013, 25(2):222-229.

[0125] Alternative splicing of mRNA transcribed from the human LTB gene results in two isoforms: isoform 1 (UniProtKB:Q06643-1, v1; SEQ ID NO: 7177) and isoform 2 (UniProtKB:Q06643-2; SEQ ID NO: 7178), in which the amino acid sequence corresponding to positions 53-77 of SEQ ID NO: 7177 is replaced with the sequence "GLGFRSCQRRSQKQISAPGSQLPTS", and positions 78-244 of SEQ ID NO: 7177 are lost.

[0126] The 244-amino acid sequence of human LTB isoform 1 includes a cytoplasmic domain at positions 1-18, a transmembrane domain at positions 19-48, and an extracellular domain at positions 49-244 of sequence number 7177.

[0127] In this specification, references to “LTB” include human LTB, isoforms of human LTB, homologs of human LTB (i.e., encoded by the genomes of non-human animals) and their variants. In some embodiments, the LTB according to this disclosure comprises or consists of an amino acid sequence having 70% or greater amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 7177, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0128] As used herein, a protein “fragment,” “variant,” or “homologous” may optionally be characterized to have at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, a fragment, variant, isoform, or homolog of a reference protein may be characterized by its ability to perform a function performed by the reference protein.

[0129] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein that has an amino acid sequence containing one or more amino acid substitutions, insertions, deletions, or other modifications to the amino acid sequence of a reference protein, but retains a considerable degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homologous" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologous compounds include orthologues.

[0130] The “fragment” may be of any length (in terms of amino acid count), but may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment originates), and may have one maximum length of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0131] In some embodiments, the target gene / protein (i.e., MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB) is a target gene / protein derived from a mammal (any species in the class Mammalia, e.g., primates (rhesus macaques, crab-eating macaques, non-human primates, or humans) and / or rodents (e.g., rats or mice).

[0132] An isoform, fragment, variant, or homolog of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be optionally characterized to have at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of an immature or mature MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB isoform of a given species, e.g., human.

[0133] Homogenetics of human genes described herein may originate from any animal. In some embodiments, homologetics of human genes described herein may originate from mammals. In some embodiments, mammals may be non-human mammals, such as primates (e.g., non-human primates, e.g., animals of the genus Macaca (e.g., crab-eating macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta)), e.g., non-hominin animals (e.g., chimpanzees (Pan troglodytes)). In some embodiments, mammals may be rabbits, guinea pigs, rats, mice or rodents (Rodentia), cats, dogs, pigs, sheep, goats, animals of the order Artiodactyla (Bos) (e.g., cattle), animals of the family Equidae (e.g., horses), or donkeys.

[0134] Homogenetics of human proteins described herein may be optionally characterized to have 70% or greater amino acid sequence identity with respect to the amino acid sequences of SEQ ID NOs. 7156-7178, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. Variants of human proteins described herein may be optionally characterized to have 70% or greater amino acid sequence identity with respect to the amino acid sequences of SEQ ID NOs. 7156-7178, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity.

[0135] The isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs having the functional properties / activities of reference MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, as determined, for example, by analysis by an assay suitable for the functional properties / activities. Target inhibition The present invention relates to the inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB (i.e., target genes / proteins described herein). In other words, the present invention relates to the inhibition of the expression and / or activity of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB, and the downstream functional consequences thereof.

[0136] Inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB includes a decrease / reduction in the expression (gene and / or protein expression) of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, and / or a decrease / reduction in the activity of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, relative to the expression / activity levels observed in the absence of inhibition. "Inhibition" may also be referred to as "antagonism" herein. Any one, two, three, four, five, six, seven, eight, or nine of the genes / proteins may be inhibited by the methods of this disclosure.

[0137] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be characterized by one or more of the following (compared to the uninhibited state): • Reduces the expression (e.g., gene and / or protein expression) of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces levels of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces / inhibits the transcription of nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Increases the degradation of RNA (e.g., mRNA) encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces levels of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB proteins. • Reduces / hinders post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Promotes / increases the degradation of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB proteins. • Reduces / hinders the level of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB functions, and / or Reduce / hinder the interaction between MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB and their interaction partners.

[0138] Gene expression can be determined by means well known to those skilled in the art. The level of RNA encoding one or more target proteins can be determined by techniques such as RT-qPCR and Northern blotting. As an example, qRT-PCR can be used to determine the level of RNA encoding a target protein.

[0139] A reduction in the level of RNA encoding the target protein may result, for example, from a decrease in the transcription of nucleic acids encoding the target protein or an increase in the degradation of RNA encoding the target protein.

[0140] A reduction in the transcription of nucleic acids encoding a target protein may result from the inhibition of the assembly and / or activity of factors necessary for the transcription of DNA encoding the target protein. An increase in the degradation of RNA encoding a target protein may result from increased enzymatic degradation of RNA encoding the target protein, for example, as a result of RNA interference (RNAi) and / or reduced stability of RNA encoding the target protein.

[0141] Protein expression can be determined by means well known to those skilled in the art. The level of protein encoding the target protein can be determined, for example, by antibody-based methods including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, ELISA, and ELISPOT, or by reporter-based methods.

[0142] A reduction in the target protein level may result from, for example, a reduction in the level of RNA encoding the target protein, a reduction in post-transcriptional processing of RNA encoding the target protein, or an increase in the degradation of the target protein.

[0143] Reducing post-transcriptional processing of a target protein may involve, for example, reduced splicing of pre-mRNA encoding the target protein to mature mRNA encoding the target protein, reduced translation of mRNA encoding the target protein, or reduced post-translational processing of the target protein.

[0144] Reduced splicing of pre-mRNA encoding the target protein to mature mRNA encoding the target protein may result from the inhibition of the assembly and / or activity of factors required for splicing. Reduced translation of mRNA encoding the target protein may result from the inhibition of the assembly and / or activity of factors required for translation. Reduced post-translational processing of the target protein (e.g., enzymatic processing, folding) may result from the inhibition of the assembly and / or activity of factors required for post-translational processing of the target protein. Increased degradation of the target protein may result from increased enzymatic (e.g., protease-mediated) degradation of the target protein.

[0145] In some embodiments, inhibition of a target gene / protein may be characterized by a reduction in the level of function of the target protein. The function of the target protein can be any functional characteristic of the target protein.

[0146] Interaction partners may be any nucleic acids or proteins that interact with or jointly contribute to functions shared with any one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0147] In some embodiments, the interaction partners of MFAP4 are integrin αvβ3, tropoelastin, fibrillin-1, fibrillin-2, desmosine, LOX, MFAP2, FBLN1, FBLN2, MFAP5, EFEMP2, EFEMP1, SFTPD, or elastin.

[0148] In some embodiments, the interaction partners of GRHPR are glyoxylic acid, hydroxypyruvic acid, D-glyceric acid, AGXT, HYI, GLYCTK, PGP, GLO1, HAO1, HAO2, DAO, NADPH, or NADH.

[0149] In some embodiments, the interaction partners of ITFG1 are RUVBL1, RUVBL2, alpha-tubulin, TIPIN, ATP9A, ASCC2, RFX7, or TM7SF3.

[0150] In some embodiments, the interaction partners of ABCC4 are ATP, ABCG4, SNX27, ABCA3, ABCE1, MRPS7, SLC22A8, SLCO1B1, NR1H4, or SLC22A6.

[0151] In some embodiments, the interaction partners of PAK3 are PAK1, CDC42, NCK1, MAPK14, RAC1, PXN, GIT1, GIT2, ARHGEF7, or ARHGEF6.

[0152] In some embodiments, the interaction partners of TRNP1 are TMF1, FAM18A, CNIH3, SMARCC2, FAM19A3, TBC1D3A, TBC1D3D, ARHGAP11B, or GPR56.

[0153] In some embodiments, the interaction partners of APLN are APLNR, AGTR1, AGT, CXCR4, CCR5, KNG1, NPY, PDYN, NMU, or POMC.

[0154] In some embodiments, the interaction partners of KIF20A are MAD2L1, AURKB, RACGAP1, KIF11, PLK1, CDCA8, KIF4A, CENPE, PRC1, or INCENP.

[0155] In some embodiments, the interaction partners of LTB are LTBR, ​​LTA, TNF, TNFSF14, TNFRSF1B, TNFSF13B, TNFRSF11A, CD40LG, MAP3K14, and TNFSF11.

[0156] The functional characteristics of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB can be analyzed using appropriate assays, such as in vitro assays.

[0157] In some embodiments, MFAP4 inhibition increases the expression and / or activation of one or more of Ptgs2, Areg, Dhrs9, Hmox1, Nqo1, P70S6k, p38, mTOR, and / or ERK2. In some embodiments, inhibitors of MFAP4 activate the mTOR, p70S6K, ERK, and p38 signaling pathways.

[0158] Inhibition of the interaction between a target protein and its interaction partner can be identified, for example, by detecting a reduction in the level of interaction between the target protein and its interaction partner compared to a control state where the interaction is not inhibited. The ability of proteins to interact can be analyzed by methods well known to those skilled in the art, such as immunoprecipitation and resonance energy transfer (RET) assays.

[0159] Inhibition of target protein function can also be assessed by analyzing one or more correlates of target protein function. That is, target protein function can be assessed by analyzing the downstream functional consequences of target protein function. For example, inhibition of target protein function can be identified by detecting a reduction in the expression (gene and / or protein expression) and / or activity of one or more proteins whose expression is directly or indirectly upregulated as a result of target protein function. Inhibition of target protein function can also be identified by detecting an increase in the expression (gene and / or protein expression) and / or activity of one or more proteins whose expression is directly or indirectly downregulated as a result of target protein function. inhibitors Inhibitors targeting one or more genes / proteins selected from MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and LTB are provided herein.

[0160] "Inhibitors of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB" means any agonist capable of inhibiting one or more of the expression and / or function of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. Such agonists may be effectors (i.e., directly or indirectly) of the inhibition of one or more of the above-described MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0161] Agents capable of inhibiting one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be referred to herein as MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB inhibitors. MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB inhibitors may also be referred herein as MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB antagonists.

[0162] The “inhibitors” of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may refer to any agonist capable of inhibiting any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB. Furthermore, “inhibitors of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB” may refer to two or more agonists capable of inhibiting two, three, four, five, six, seven, eight, or nine target genes / proteins selected from the group consisting of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and LTB. Multiple inhibitors can be used in the methods of this disclosure to target two or more of the target genes / proteins.

[0163] In some embodiments, inhibitors of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB (i.e., target proteins) may be: • Reduces / inhibits the expression (e.g., gene and / or protein expression) of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces levels of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces / inhibits the transcription of nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Increases the degradation of RNA (e.g., mRNA) encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Reduces levels of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB proteins. • Reduces / hinders post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB. • Promotes / increases the degradation of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB proteins. • Reduces / hinders the level of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB functions, and / or Reduce / hinder the interaction between MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB and their interaction partners.

[0164] It will be acknowledged that a given inhibitor may exhibit more than one of the properties listed in the preceding paragraph. A given inhibitor can be evaluated for the properties listed in the preceding paragraph using a suitable assay. The assay may be, for example, an in vitro assay, optionally a cell-based assay, or a cell-free assay. The assay may also be, for example, an in vivo assay, i.e., one performed in a non-human animal.

[0165] If the assay is a cell-based assay, it may include a step of treating cells with an inhibitor (e.g., nucleic acid) to determine whether the inhibitor exhibits one or more of the listed properties. The assay may utilize species labeled with detectable entities to facilitate detection. The assay may include a step of evaluating the listed properties after treating cells with a wide range of amounts / concentrations of a given inhibitor (e.g., a diluted series) individually. It will be acknowledged that the cells are preferably cells expressing the target protein to be inhibited, e.g., liver cells (e.g., HepG2 cells or HuH7 cells).

[0166] Analysis of the results of such assays may include a step of determining the concentration at which 50% of the maximum level of the relevant activity is obtained. The nucleic acid concentration at which 50% of the maximum level of the relevant activity is obtained may be called the “maximum half-dose effective concentration” of the inhibitor in relation to the relevant activity, or “EC 50 It is sometimes called "EC." As an example, consider the EC of a given inhibitor (e.g., inhibitory nucleic acid) for increased degradation of RNA encoding a target protein. 50 This concentration may be such that 50% of the maximum level of degradation of the RNA encoding the target protein is achieved.

[0167] Depending on the characteristics, EC 50 Also, "maximum half dose inhibitory concentration" or "IC 50 This is sometimes called the IC50, which is the concentration of the inhibitor at which 50% of the maximum level of inhibition of a given characteristic is observed. As an example, consider the IC50 of a given inhibitor (e.g., inhibitory nucleic acid) to reduce the expression of a gene encoding a target protein. 50 This concentration may be such that 50% of the maximum level of inhibition of gene expression is achieved.

[0168] Agitators capable of reducing / inhibiting the expression of one or more of the following genes (e.g., reducing the level of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB); reducing / inhibiting the transcription of nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB; and / or increasing the degradation of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB) can be identified, for example, by RT-qPCR (a technique well known to those skilled in the art), using an assay that includes a step of detecting the level of RNA encoding the target protein. The method utilizes primers and / or probes for the detection and / or quantification of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0169] Such assays may include the steps of: (i) introducing a putative inhibitor (e.g., an inhibitory nucleic acid) or (ii) a control agonist (e.g., a control nucleic acid, such as a nucleic acid known not to affect the level of RNA encoding the target protein) into cells expressing the target protein in in vitro culture (e.g., by transfection); then measuring the level of RNA encoding the target protein in the cells following (i) and (ii) after a suitable period of time, i.e., a period of time sufficient to observe a reduction in gene expression of the target protein / transcription level of the nucleic acid encoding the target protein / level of RNA encoding the target protein or an increase in the level of degradation of RNA encoding the target protein; and (iii) comparing the detected level of RNA encoding the target protein to determine whether the putative inhibitor reduces / hinders gene expression of the target protein, reduces / hinders transcription of the nucleic acid encoding the target protein, reduces the level of RNA encoding the target protein, and / or increases the degradation of RNA encoding the target protein.

[0170] Agitators capable of reducing the expression of one or more of the following proteins (e.g., reducing the levels of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins, or increasing the degradation of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins) can be identified using an assay that includes a step of detecting the level of the target protein, for example, using an antibody / reporter-based method (e.g., Western blotting, ELISA, immunohistochemistry, etc.). Such assays may include the steps of treating cells / tissues with the agonist and then comparing the level of the target protein in such cells / tissues to the level of the target protein in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues).

[0171] The method can utilize antibodies specific to the target protein. Such an assay may include the steps of: (i) introducing a putative inhibitor (e.g., an inhibitory nucleic acid) or (ii) a control agonist (e.g., a nucleic acid known not to affect the level of the target protein) into cells expressing the target protein in in vitro culture (e.g., by transfection); subsequently (e.g., after a suitable period, i.e., a period long enough for a reduction in the level of the target protein to be observed) measuring the level of the target protein in the cells following (i) and (ii); and (iii) comparing the detected level of the target protein to determine whether the putative inhibitor reduces the level of the target protein and / or reduces / hinders the translation of the mRNA encoding the target protein.

[0172] Agents capable of reducing the level of one or more functions of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB (e.g., the functions of the target proteins described herein) can be identified using an assay that includes the step of detecting the level of the relevant function. Such an assay may include the steps of: introducing (i) a putative inhibitor (e.g., an inhibitory nucleic acid) or (ii) a control agonist (e.g., a nucleic acid known not to affect the function of the target protein) into cells expressing the target protein in in vitro culture (e.g., by transfection); subsequently (e.g., after a suitable period, i.e., a period sufficient to observe a reduction in the level of the function of the target protein) in the cells following (i) and (ii); and (iii) comparing the detected level of the function of the target protein to determine whether the putative inhibitor reduces the level of the function of the target protein.

[0173] In this specification, references to “target protein function” may refer to any functional properties and / or activities mediated by them of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins.

[0174] Agitators capable of reducing / inhibiting the normal splicing of premRNAs encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB can be identified using assays that include a step of detecting and / or quantifying the level of RNA (e.g., mature mRNA) encoding one or more isoforms of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. Such assays may include a step of quantifying RNA (e.g., mature mRNA) encoding one or more isoforms of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB by RT-qPCR. The method utilizes primers and / or probes for the detection and / or quantification of mature mRNA produced by canonical splicing of premRNA transcribed from genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB, as well as primers and / or probes for the detection and / or quantification of mature mRNA produced by alternative splicing of premRNA transcribed from genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.

[0175] Mature mRNA produced by canonical splicing of premRNA transcribed from genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be mature mRNA encoding the major isoform produced by the expression of genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. The major isoform may be the most commonly produced / detected isoform. For example, mature mRNA produced by canonical splicing of premRNA transcribed from human MFAP4 may be mature mRNA encoding human MFAP4 isoform 1 (i.e., having the amino acid sequence shown in SEQ ID NO: 7156). Mature mRNA produced by alternative splicing of premRNA transcribed from genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may be mature mRNA encoding an isoform other than the major isoform produced by the expression of the said gene. For example, mature mRNA produced by alternative splicing of premRNA transcribed from human MFAP4 may be mature mRNA encoding a human MFAP4 isoform other than isoform 1 (i.e., having an amino acid sequence not identical to SEQ ID NO: 7156), such as mature mRNA encoding human MFAP4 isoform 2 (i.e., having the amino acid sequence shown in SEQ ID NO: 7157).

[0176] Such assays may include the steps of: (i) introducing a putative inhibitor (e.g., an inhibitory nucleic acid) or (ii) a control agonist (e.g., a nucleic acid known not to affect the splicing of premRNA encoding the target gene) into cells expressing MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB in in vitro culture (e.g., by transfection); subsequently (e.g., after a suitable period, i.e., a period sufficient to observe the effect on the splicing of premRNA encoding the target gene), measuring the level of mature mRNA encoding one or more isoforms of the target gene in the cells following (i) and (ii); and (iii) comparing the levels of mature mRNA encoding the isoform(s) to determine whether the putative inhibitor reduces / hinders the normal splicing of premRNA encoding the target gene.

[0177] Agents capable of reducing the interaction between a target protein and its interaction partner, as described herein, can be identified using an assay that includes a step of detecting the level of interaction between the target protein and its interaction partner, for example, using an antibody / reporter-based method. The level of interaction between the target protein and its interaction partner can be analyzed using, for example, resonance energy transfer techniques (e.g., FRET, BRET) or a method that analyzes the correlation of the interaction between the target protein and its interaction partner. The assay may include the steps of treating cells / tissues with the agent and then comparing the level of interaction between the target protein and its interaction partner in such cells / tissues to the level of interaction between the target protein and its interaction partner in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). The level of interaction between the target protein and its interaction partner can also be analyzed using techniques such as ELISA, surface plasmon resonance, or biolayer interference analysis. The assay may include the step of comparing the level of interaction between the target protein and its interaction partner in the presence of the agent to the level of interaction between the target protein and its interaction partner under appropriate control conditions (e.g., in the absence of the agent).

[0178] In some embodiments, the inhibitors of the present disclosure may reduce the expression of one or more genes encoding any of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than one-fold the level of expression observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to inhibit the expression of the relevant genes, for example, ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or ≤0.01. In some embodiments, inhibitors according to the present disclosure may reduce the expression of one or more genes encoding any of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than 100% of the level of expression observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to inhibit the expression of the relevant genes, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0179] In some embodiments, the inhibitors of this disclosure reduce the levels of RNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in a given assay, either in the absence of the inhibitor or by reducing the levels of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. It may be possible to reduce the level to less than 1x the level observed in the presence of the same amount of control agent known not to have the same effect, for example, to one of ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the inhibitors of this disclosure may reduce the level of RNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the level of RNA encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, for example, ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0180] In some embodiments, the inhibitors of this disclosure reduce the level of transcription of one or more nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in a given assay, either in the absence of the inhibitor or by reducing the transcription of nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. It may be possible to reduce the level to less than 1x the level observed in the presence of the same amount of control agent known not to have the same effect, for example, to one of ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the inhibitors of this disclosure reduce the level of transcription of one or more nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB in a given assay, in the absence of the inhibitor, or MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or It may be possible to reduce the level to less than 100% of the level observed in the presence of the same amount of control agonist known not to reduce the transcription of nucleic acids encoding LTB, for example, one of the following: ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0181] In some embodiments, the inhibitors according to this disclosure reduce the levels of one or more of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins in a given assay in the absence of the inhibitor or without reducing the levels of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins. It may be possible to reduce the level to less than 1x the level observed in the presence of a known equal amount of control agent, for example, to one of the following: ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the inhibitors according to the present disclosure may reduce the level of one or more of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the levels of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB proteins, for example, ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0182] In some embodiments, the inhibitors according to this disclosure are found not to reduce the level of function of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in a given assay, either in the absence of the inhibitor or by other means. It may be possible to reduce the level to less than 1x the level observed in the presence of the same amount of control agent, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the inhibitors according to the present disclosure may reduce the level of function of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the level of function of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, for example, ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0183] In some embodiments, the inhibitors according to the present disclosure may reduce the level of binding to one or more interaction partners, MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, to less than 1 times the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the level of binding, for example, ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or ≤0.01. In some embodiments, the inhibitors according to the present disclosure may reduce the level of binding to one or more interaction partners, MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the level of relevant binding, for example, ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0184] In some embodiments, the inhibitors according to this disclosure are known not to reduce the normal splicing of premRNAs encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in a given assay, either in the absence of the inhibitor or in the absence of the inhibitor or in the absence of the premRNAs encoding the associated target protein(s). It may be possible to reduce the level to less than one times the level observed in the presence of the same amount of control agent, for example, to one of the following: ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or ≤0.01. In some embodiments, the inhibitors of the present disclosure may reduce the level of normal splicing of premRNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce normal splicing of premRNA encoding the relevant target protein(s), e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0185] In some embodiments, the inhibitors of this disclosure reduce the translation of mRNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB in a given assay, either in the absence of the inhibitor or in the presence of an equal amount of a control agent known not to reduce the translation of mRNA encoding the associated target protein(s). It may be possible to reduce the observed level to less than 1x, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the inhibitors of the present disclosure may reduce the translation of mRNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB to less than 100% of the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to reduce the translation of mRNA encoding the relevant target protein(s), e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%.

[0186] The preferred level of reduction according to the eight paragraphs mentioned above is a reduction to 0.5x / ≤50%, for example, to one of 0.4x / ≤40%, 0.3x / ≤30%, 0.2x / ≤20%, 0.15x / ≤15%, or 0.1x / ≤10%.

[0187] In some embodiments, the inhibitors of the present disclosure may be able to increase the degradation of RNA encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB by more than 1x the level observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to increase the degradation of RNA encoding the relevant target protein(s), for example, by ≥1.01x, ≥1.02x, ≥1.03x, ≥1.04x, ≥1.05x, ≥1.1x, ≥1.2x, ≥1.3x, ≥1.4x, ≥1.5x, ≥1.6x, ≥1.7x, ≥1.8x, ≥1.9x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, or ≥10x.

[0188] In some embodiments, the inhibitors of this disclosure interfere with or silence the expression of one or more genes encoding any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. In some embodiments, the inhibitors of this disclosure interfere with or silence the expression of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB at the protein level. As used herein, the expression of a given gene / protein may be considered "interfered" or "silenced" if the level of expression is reduced to 0.1 times / ≤10% of the level observed in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to be an inhibitor of the expression of the relevant gene / protein(s)(s).

[0189] In a preferred embodiment, the inhibitors according to this disclosure (e.g., inhibitory nucleic acids such as siRNA or shRNA) inhibit the gene and / or protein expression of one or more of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB genes and / or proteins observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to inhibit the gene and / or protein expression of the relevant gene(s) / protein(s). Inhibits more than 50% of the following, for example, ≥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%.

[0190] In a preferred embodiment, the inhibitor according to this disclosure (e.g., an inhibitory nucleic acid such as siRNA or shRNA) is used to inhibit the gene and / or protein expression of one or more of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB genes (determined, e.g., by qRT-PCR) observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to inhibit the gene and / or protein expression of the relevant gene(s) / protein(s)(s). Inhibits more than 50% of (as shown), for example, ≥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 one of 100%.

[0191] In a preferred embodiment, the inhibitor according to this disclosure (e.g., an inhibitory nucleic acid such as siRNA or shRNA) is used to inhibit the expression of one or more of the following proteins (e.g., determined by ELISA) observed in a given assay in the absence of the inhibitor or in the presence of an equal amount of a control agonist known not to inhibit the gene and / or protein expression of the relevant gene(s) / protein(s) (e.g., determined by ELISA). Inhibits more than 50% of (as shown), for example, ≥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 one of 100%.

[0192] In some embodiments, the inhibitors of this disclosure (e.g., inhibitory nucleic acids such as siRNA or shRNA) can inhibit the expression of one or more of the following genes and / or proteins: MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, with a range of ≤1 μM, e.g., ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM One IC with a capacitance of ≤3nM, ≤2nM, ≤1nM, ≤900pM, ≤800pM, ≤700pM, ≤600pM, ≤500pM, ≤400pM, ≤300pM, ≤200pM, ≤100pM, ≤50pM, ≤40pM, ≤30pM, ≤20pM, ≤10pM, or ≤1pM. 50 It holds.

[0193] In some embodiments, the inhibitors according to this disclosure can inhibit the expression of one or more of the following genes (as determined, for example, by qRT-PCR): MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, with ICs of ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, or ≤1 pM. 50 It holds.

[0194] In some embodiments, the inhibitors according to this disclosure can inhibit the expression of one or more of the following proteins (as determined, for example, by ELISA): MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, with IC20 50 It holds. Types of inhibitors The inhibitors described herein may be any type of agonist having appropriate inhibitory activity.

[0195] When used herein, the term "inhibitor" refers to an agent that reduces or inhibits at least one function or biological activity of a target molecule, such as those described herein.

[0196] The inhibitors described herein may be molecules capable of binding to one or more of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB mRNA or protein, molecules capable of binding to one or more interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, or molecules capable of reducing the expression of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0197] In some embodiments, the inhibitor can bind to polypeptides following one or more of sequence numbers 7156-7178 or mRNA following one or more of sequence numbers 7179-7195.

[0198] In some embodiments, the inhibitor can target, for example, one or more functional domains or regions of SEQ ID NOs. 7156-7178, or bind to them. In some embodiments, the inhibitor targets the region of SEQ ID NOs. 7156 including positions 22-255, 26-28, or 32-255. In some embodiments, the inhibitor targets the region of SEQ ID NOs. 7158 including one or more positions 83-84, 162-164, 185-188, 217, 243, 245, 269, and 293-296. In some embodiments, the inhibitor targets the region of SEQ ID NOs. 7160 including positions 258-293. In some embodiments, the inhibitor targets the region of SEQ ID NOs. 7161 including positions 92-377, 410-633, 714-1005, 1041-1274, 445-452, or 1075-1082. In some embodiments, the inhibitor targets the region including positions 70-83 or 283-534 of SEQ ID NO: 7165. In some embodiments, the inhibitor targets the region including positions 64-507 or 611-762 of SEQ ID NO: 7175. In some embodiments, the inhibitor targets the region including positions 1-18, 19-48 or 49-244 of SEQ ID NO: 7177.

[0199] In some embodiments, the inhibitor may bind to one or more interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, such as those described herein.

[0200] Such binding molecules can be identified using any suitable assay for detecting the binding of the molecule to a relevant factor (i.e., a target gene / protein as described herein, or an interaction partner of the protein(s) described herein). Such assays may include a step of detecting the formation of a complex between the relevant factor and the molecule.

[0201] In some embodiments, the inhibitor is a nucleic acid, peptide, antibody, antigen-binding molecule, or small molecule inhibitor.

[0202] Small molecule inhibitors that bind to target mRNA / proteins or their binding partners as described herein can be identified by screening a small molecule library. As used herein, “small molecule” refers to an organic compound with a low molecular weight (<1000 daltons, typically between approximately 300 and 700 daltons). Small molecule inhibitors that bind to target mRNA / proteins as described herein can be identified, for example, using the method described by Horswill AR et al., PNAS, 2004, 101 (44) pp. 15591–15596, which is incorporated herein by reference in its entirety.

[0203] A possible inhibitor of GRHPR is 4-hydroxy-2-oxoglutaric acid.

[0204] ABCC4 inhibitors may include methotrexate, mercaptopurine, zidovudine, dipyridamole, probenecid, sulfinpyrazone, fluorouracil, lucaparib, adefovir dipivoxil, cefazolin, tyrofostine AG1478, dantrolene, graphenin, nalidixic acid, or prazosin.

[0205] A possible inhibitor of PAK3 is FRAX597.

[0206] Inhibitors of APLN may include ML221, an apelin receptor (APJ) antagonist.

[0207] Inhibitors of KIF20A may be BKS0349 or Paprotrain.

[0208] The inhibitors provided herein include, for example, peptides / polypeptides, such as peptide aptamers, thioredoxins, monobodies, antikalin, Knitz domains, avimers, Nottin, fynomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), obodies, and fibronectins (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48). Inhibitors include peptides / polypeptides that can be identified by screening a library of relevant peptides / polypeptides. Peptide / polypeptide inhibitors are sometimes called inhibitory peptides / polypeptides.

[0209] Inhibitory peptides / polypeptides may also include, for example, peptide / polypeptide interaction partners of the target gene / mRNA / protein of interest (i.e., MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB).

[0210] Peptide / polypeptide interaction partners may be based on interaction partners of the target gene / mRNA / protein of interest, and may include, for example, fragments of the interaction partner of the target(s). Peptide / polypeptide interaction partners may be based on one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB, and may include, for example, fragments of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB that bind to the mRNA / protein interaction partner. Such activators may act as "decoy" molecules, preferably exhibiting competitive inhibition of interactions between MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB and their corresponding interaction partners.

[0211] MFAP4 inhibitors may be peptides / polypeptides capable of blocking the interaction between MFAP4 and integrin receptors, integrin αvβ3, tropoelastin, fibrillin-1, fibrillin-2, desmosine, LOX, MFAP2, FBLN1, FBLN2, MFAP5, EFEMP2, EFEMP1, SFTPD, or elastin.

[0212] GRHPR inhibitors may be peptides / polypeptides capable of blocking the interaction between GRHPR and glyoxylic acid, hydroxypyruvic acid, D-glyceric acid, AGXT, HYI, GLYCTK, PGP, GLO1, HAO1, HAO2, DAO, NADPH, or NADH.

[0213] ITFG1 inhibitors may be peptides / polypeptides capable of blocking the interaction between ITFG1 and RUVBL1, RUVBL2, alpha-tubulin, TIPIN, ATP9A, ASCC2, RFX7, or TM7SF3.

[0214] ABCC4 inhibitors may be peptides / polypeptides capable of blocking the interaction between ABCC4 and ATP, ABCG4, SNX27, ABCA3, ABCE1, MRPS7, SLC22A8, SLCO1B1, NR1H4, or SLC22A6.

[0215] PAK3 inhibitors may be peptides / polypeptides capable of blocking the interaction between PAK3 and PAK1, CDC42, NCK1, MAPK14, RAC1, PXN, GIT1, GIT2, ARHGEF7, or ARHGEF6.

[0216] TRNP1 inhibitors may be peptides / polypeptides capable of blocking the interaction between TRNP1 and TMF1, FAM18A, CNIH3, SMARCC2, FAM19A3, TBC1D3A, TBC1D3D, ARHGAP11B, or GPR56.

[0217] APLN inhibitors may be peptides / polypeptides capable of blocking the interaction between APLN and APLNR, AGTR1, AGT, CXCR4, CCR5, KNG1, NPY, PDYN, NMU, or POMC.

[0218] KIF20A inhibitors may be peptides / polypeptides capable of blocking the interaction between KIF20A and MAD2L1, AURKB, RACGAP1, KIF11, PLK1, CDCA8, KIF4A, CENPE, PRC1, or INCENP.

[0219] LTB inhibitors may be peptides / polypeptides capable of blocking the interaction between LTB and LTBR, ​​LTA, TNF, TNFSF14, TNFRSF1B, TNFSF13B, TNFRSF11A, CD40LG, MAP3K14, and TNFSF11.

[0220] In some embodiments, the inhibitory peptide / polypeptide may contain or consist of an amino acid sequence having at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence or fragment of one or more interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0221] In some embodiments, the inhibitory peptide / polypeptide may contain or consist of an amino acid sequence having at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to one or more amino acid sequences or fragments of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. In such embodiments, it will be observed that the inhibitory peptide / polypeptide will lack normal activity and / or have reduced activity compared to the wild-type version of the protein. For example, in some embodiments, the inhibitory peptide / polypeptide may be a variant (e.g., mutant) version of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB, having reduced function compared to wild-type MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.

[0222] Inhibitory peptides / polypeptides include aptamers. Nucleic acid aptamers are outlined, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202, and can be identified and / or generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968):505-510, and WO91 / 19813. Nucleic acid aptamers may contain DNA and / or RNA, and may be single-stranded or double-stranded. These may include chemically modified nucleic acids, for example, those with chemically modified sugars and / or phosphates and / or bases. Such modifications may improve the stability of the aptamer or make it more resistant to degradation, and may include modifications at the 2' position of ribose. Nucleic acid aptamers can be chemically synthesized, for example, on a solid-phase support. Solid-phase synthesis may involve the use of phosphoramidite chemistry. Briefly, a solid-phase supported nucleotide is detritylated and then coupled with an appropriately activated nucleoside phosphoramidite to form a phosphite triester bond. Capping may then occur, followed by oxidation of the phosphite triester using an oxidized product, usually iodine. Next, this cycle is repeated to assemble the aptamer (see, for example, Sinha, ND; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, SL; Lyer, RP (1992). Tetrahedron 48 (12): 2223).Peptide aptamers and methods for their generation and identification are outlined in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101, which are incorporated herein by reference in their entirety.

[0223] Inhibitory peptides / polypeptides also include antibodies (immunoglobulins), such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and their fragments and derivatives (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single-domain antibodies (e.g., VhH), etc.).

[0224] In some embodiments, the inhibitors described herein are antibodies capable of binding to one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0225] Inhibitors of MFAP4 may be antibodies with catalog numbers PA5-42013 (ThermoFisher) or ab169757 (abcam). Inhibitors of GRHPR may be antibodies with catalog numbers PA5-54652 (ThermoFisher) or ab155604 (abcam). Inhibitors of ITFG1 may be antibodies with catalog numbers PA5-54067 (ThermoFisher) or TA339563 (ORIGENE). Inhibitors of ABCC4 may be antibodies with catalog numbers PA5-82019 (ThermoFisher) or ab15602 (abcam). Inhibitors of PAK3 may be antibodies with catalog numbers PA5-79781 (ThermoFisher) or ab40808 (abcam). Inhibitors of TRNP1 may be antibodies with catalog numbers PA5-71277 (ThermoFisher) or ab174303 (abcam). Inhibitors of APLN may be APLN blocking antibodies. Inhibitors of APLN may be antibodies with catalog numbers PA5-114860 (ThermoFisher) or ab125213 (abcam). Inhibitors of KIF20A may be antibodies with catalog number PA5-38648 (ThermoFisher). Inhibitors of LTB may be antibodies (e.g., recombinant mouse anti-LTA and LTB antibodies (CBL543)).

[0226] Inhibitory molecules that bind to any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB, or to their interaction partners, may exhibit specific binding to the relevant factor (i.e., the relevant mRNA / protein or its interaction partner). As used herein, “specific binding” refers to a binding that is selective and can be distinguished from nonspecific binding to a non-target molecule.

[0227] Inhibitors or binding molecules that specifically bind to one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB preferably bind to one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB with greater affinity and / or for a longer duration than they bind to other non-target molecules. Such binding molecules may be described as "specific to" one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. Inhibitors or binding molecules that specifically bind to one of the interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB preferably bind to one of the interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB with greater affinity and / or for a longer duration than they bind to other non-target molecules; such binding molecules may be described as “specific to” one of the interaction partners of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0228] In some embodiments, the inhibitors / binding molecules described herein inhibit the ability to bind to the corresponding interaction partner of any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB (i.e., the interaction partner of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB, respectively). In some embodiments, the inhibitor / binding molecule acts as a competitive inhibitor of the interaction between any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB and its corresponding interaction partner. The binding molecule may occupy the region of the protein required for binding to the corresponding interaction partner or otherwise reduce access thereto, or may occupy the region of the interaction partner required for binding to the corresponding protein or otherwise reduce access thereto.

[0229] The ability of an inhibitor, e.g., a binding molecule, to inhibit the interaction between a protein of interest and its corresponding interaction partner can be evaluated, for example, by analysis of the interaction in the presence of the inhibitor or after incubation of one or both of the interaction partners with the inhibitor. One example of a suitable assay for determining whether a given binder is capable of inhibiting the interaction between a protein of interest and its corresponding interaction partner is a competitive ELISA.

[0230] The inhibitors described herein can be molecules capable of reducing the expression of any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. The "molecule capable of reducing the expression of any one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB" refers to a molecule capable of reducing the expression of any one of the genes, mRNAs, and / or proteins of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. In some embodiments, the molecule reduces or inhibits the expression of the polypeptide according to SEQ ID NOs: 7156-7178. In some embodiments, the molecule reduces or inhibits the expression of the polypeptide from the sequence according to SEQ ID NOs: 7179-7195.

[0231] Suppression of the expression of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB or their isoforms will preferably result in a decrease in the amount of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB expressed by the cell / tissue / organ / organ system / subject. For example, in a given cell, suppression of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB by administration of a suitable nucleic acid will result in a decrease in the level of expression compared to an untreated cell. The suppression may be partial. Preferred levels of suppression are at least 50%, more preferably at least one of 60%, 70%, 80%, 85%, or 90%. A level of suppression between 90% and 100% is considered "silencing" of expression or function. Gene and protein expression can be determined as described herein or by methods well known in the art to those skilled in the art.

[0232] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may involve modifying cells to reduce or inhibit the expression of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB may involve modifying nucleic acids encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. The modification causes cells to have reduced levels of gene and / or protein expression for MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB compared to unmodified cells.

[0233] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB may include the step of modifying the genes encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.

[0234] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of introducing an insertion, substitution or deletion into the nucleic acid sequence encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB.

[0235] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of introducing a modification that reduces or interferes with the expression of a polypeptide following any one of SEQ ID NOs. 7156-7178 from a modified nucleic acid sequence. In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of modifying a cell to contain alleles of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB that do not encode an amino acid sequence following any one of SEQ ID NOs. 7156-7178. In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of modifying cells to lack a nucleic acid encoding a polypeptide according to any one of SEQ ID NOs. 7156-7178.

[0236] In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of modifying the relevant gene(s) to introduce an immature stop codon into the sequence transcribed from the gene(s). In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of modifying the relevant gene(s) to encode a truncated and / or non-functional polypeptide(s). In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB includes the step of modifying the relevant gene(s) to encode a misfolded and / or degraded polypeptide(s).

[0237] Methods for modifying nucleic acids encoding a target protein and activators for achieving this are well known in the art, and include, for example, modification of the target nucleic acid by homologous recombination and targeted nucleic acid editing using site-specific nucleases (SSNs).

[0238] Suitable methods include, for example, targeting by homologous recombination, outlined in Mortensen Curr Protoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98(15): pp. 8403-8410, both of which are incorporated herein by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequences through crossover events induced by homologous sequences.

[0239] In some embodiments, the method utilizes targeted nucleic acid editing using SSNs. Gene editing using SSNs is outlined, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of producing site-directed double-strand breaks (DSBs) can be manipulated to introduce DSBs into the desired target nucleic acid sequence(s). DSBs may be repaired by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, often resulting in nucleotide insertion or deletion. Alternatively, DSBs may be repaired by advanced homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the DSB site.

[0240] SSNs that can be manipulated to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems.

[0241] The ZFN system is outlined, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636–46, which is incorporated herein by reference in its entirety. A ZFN comprises a programmable zinc finger DNA-binding domain and a DNA-cleaving domain (e.g., a FokI endonuclease domain). The DNA-binding domain can be identified by screening zinc finger arrays capable of binding to a target nucleic acid sequence.

[0242] The TALEN system is outlined, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, the entire system of which is incorporated herein by reference. TALEN comprises a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g., a FokI endonuclease domain). TALE contains a repeat domain consisting of 33-39 amino acid repeats, each repeat being identical except for two residues at positions 12 and 13, which are repeat variable two residues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501).

[0243] CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are outlined, for example, in Nakade et al., Bioengineered (2017) 8(3):265–273, which are incorporated herein by reference in their entirety. These systems include endonucleases (e.g., Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. sgRNAs can be manipulated to target endonuclease activity to a desired nucleic acid sequence.

[0244] In some embodiments, inhibition of one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB utilizes site-directed nuclease (SSN) systems that target the relevant nucleic acid sequence(s). Thus, in some embodiments, the inhibitor comprises or consists of an SSN system that targets one or more nucleic acids encoding MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB. In some embodiments, inhibition of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB utilizes nucleic acids encoding an SSN system that targets the relevant nucleic acid sequence(s).

[0245] In some embodiments, the SSN system targets a region of nucleic acid that encodes a domain of the MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB protein, which is necessary for protein function, such as the domains described herein.

[0246] In some embodiments, the SSN system is a ZFN system, TALEN system, CRISPR / Cas9 system, CRISPR / Cpf1 system, CRISPR / C2c1 system, CRISPR / C2c2 system, or CRISPR / C2c3 system.

[0247] In some embodiments, the SSN system is a CRISPR / Cas9 system. In such embodiments, inhibition can utilize CRISPR RNA (crRNA) that targets nucleic acids encoding one or more of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A and / or LTB, as well as nucleic acids (possibly multiple) that encode transactivating crRNA (tracrRNA) for processing the crRNA into its mature form. Nucleic acid inhibitors In some embodiments, the inhibitor is a nucleic acid inhibitor. Nucleic acid inhibitors may also be referred to herein as inhibitory nucleic acids.

[0248] The nucleic acid inhibitors described herein include or consist of DNA and / or RNA. Nucleic acid inhibitors may be single-stranded (e.g., in the case of antisense oligonucleotides (e.g., gapmers)). Nucleic acid inhibitors may be double-stranded or contain double-stranded regions (e.g., in the case of siRNA, shRNA, etc.). Inhibitory nucleic acids may contain both double-stranded and single-stranded regions (e.g., in the case of shRNA and pre-miRNA molecules, where the stem region of the hairpin structure is double-stranded and the loop region of the hairpin structure is single-stranded).

[0249] In some embodiments, the nucleic acid inhibitors provided herein may be antisense nucleic acids as described herein. In some embodiments, the nucleic acid inhibitors may comprise antisense nucleic acids as described herein. In some embodiments, the nucleic acid inhibitors may encode antisense nucleic acids as described herein.

[0250] As used herein, "antisense nucleic acid" refers to a nucleic acid (e.g., DNA or RNA) that is complementary to at least a portion of a target nucleotide sequence (e.g., of RNA encoding a target gene described herein). Antisense nucleic acids according to the present disclosure are preferably single-stranded nucleic acids that bind by complementary Watson-Crick base pairing to the target nucleotide sequence. Complementary base pairing may involve hydrogen bonding between complementary bases. Antisense nucleic acids may be provided as single-stranded molecules, as in the case of antisense oligonucleotides, or may be composed of double-stranded molecular species, as in the case of siRNA, shRNA, and pre-miRNA molecules.

[0251] Complementary base pairing between an antisense nucleic acid and its target nucleotide sequence may be complete. In such an embodiment, the antisense nucleic acid comprises or consists of the reverse complement of its target nucleotide sequence, and complementary base pairing occurs between each nucleotide of the target nucleotide sequence and the complementary nucleotide in the antisense nucleic acid. Alternatively, complementary base pairing between an antisense nucleic acid and its target nucleotide sequence may be incomplete / partial. In such an embodiment, complementary base pairing occurs between the target nucleotide sequence and some, but not all, of the nucleotides of the complementary nucleotide in the antisense nucleic acid.

[0252] Such binding between nucleic acids by complementary base pairing may be referred to as "hybridization." By binding to its target nucleotide sequence, an antisense nucleic acid may form a nucleic acid complex comprising (i) the antisense nucleic acid and (ii) a target nucleic acid comprising the target nucleotide sequence.

[0253] The nucleotide sequence of an antisense nucleic acid is sufficiently complementary to the target nucleotide sequence so as to bind to or hybridize with the target nucleotide sequence. It will be acknowledged that the antisense nucleic acid preferably has a high degree of sequence identity with respect to the reverse complement of the target nucleotide sequence. In some embodiments, the antisense nucleic acid comprises or consists of a nucleotide sequence having at least 75% sequence identity with respect to the reverse complement of the target nucleotide sequence (e.g., at least 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 greater sequence identity).

[0254] In some embodiments, the antisense nucleic acids according to the present disclosure include a nucleotide sequence that is the reverse complement of the target nucleotide sequence, or a nucleotide sequence comprising 1 to 10 substitutions (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) to the reverse complement of the target nucleotide sequence.

[0255] In some embodiments, the target nucleotide sequence of the antisense nucleic acid according to the Disclosure comprises or consists of 5 to 100 nucleotides, e.g., 10 to 80, 12 to 50, or 15 to 30 nucleotides (e.g., 20 to 27, e.g., about 21 to 23). In some embodiments, the target nucleotide sequence of the antisense nucleic acid according to the Disclosure comprises or consists of DNA and / or RNA. In some embodiments, the target nucleotide sequence of the antisense nucleic acid according to the Disclosure comprises or consists of RNA.

[0256] In some embodiments, antisense nucleic acids reduce / hinder the transcription of nucleic acids containing their target nucleotide sequence. In some embodiments, antisense nucleic acids reduce / hinder the association of factors necessary for normal transcription (e.g., enhancers, RNA polymerases) with nucleic acids containing their target nucleotide sequence.

[0257] In some embodiments, antisense nucleic acids increase / enhance the degradation of nucleic acids containing their target nucleotide sequence, for example, by RNA interference. In some embodiments, antisense nucleic acids reduce / hinder the translation of nucleic acids containing their target nucleotide sequence, for example, by RNA interference or antisense degradation via RNase H.

[0258] RNA interference is described, for example, in Agrawal et al., Microbiol. Mol. Bio. Rev. (2003) 67(4):657-685 and Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), both of which are incorporated herein in their entirety by reference. Briefly, double-stranded RNA molecules are recognized by the Argonaut components of the RNA-induced silencing complex (RISC). The double-stranded RNA is separated into single strands and incorporated into the active RISC by the RISC loading complex (RLC). The strand incorporated into the RISC binds to its target RNA by complementary base pairing, depending on the identity of the RNA incorporated into the RISC and the degree of complementarity to the target RNA. The RISC then either cleaves the target RNA, resulting in its degradation, or otherwise blocks ribosome access, thereby preventing its translation. RNAi-based therapies have been approved for several indications (Kim, Chonnam Med J. (2020) 56(2):87-93).

[0259] In some embodiments, antisense nucleic acids reduce / hinder the normal post-transcriptional processing (e.g., splicing and / or translation) of nucleic acids containing their target nucleotide sequence. In some embodiments, antisense nucleic acids reduce or alter the splicing of pre-mRNA containing their target nucleotide sequence to mature mRNA. In some embodiments, antisense nucleic acids reduce the translation of mRNA containing their target nucleotide sequence into protein.

[0260] In some embodiments, antisense nucleic acids reduce or prevent the association of factors necessary for normal post-transcriptional processing (e.g., components of spliceosomes) with nucleic acids containing their target nucleotide sequences. In such cases, antisense nucleic acids are sometimes referred to as “splice-switching” nucleic acids.

[0261] Splice-switching nucleic acids are outlined, for example, in Haves and Hastings, Nucleic Acids Res. (2016) 44(14):6549–6563, which is incorporated herein by reference in its entirety. Splice-switching nucleic acids include, for example, splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of target RNA transcripts by blocking RNA:RNA base pairing and / or protein:RNA binding interactions that occur between the components of the splicing mechanism and premRNA. Splice-switching nucleic acids can be used to alter the number / proportion of mature mRNA transcripts encoding proteins described herein. Splice-switching nucleic acids can be designed to target specific regions of a target transcript, for example, to achieve the skipping of a desired exon(s), e.g., an exon encoding a desired domain / region. SSOs often involve modifications to the oligonucleotide sugar-phosphate backbone to reduce / hinder RNase H degradation, such as phosphorothioate bonds, phosphorodiamidate bonds, such as phosphorodiamidate morpholino (PMO), and may also include, for example, peptide nucleic acids (PNA), locked nucleic acids (LNA), methoxyethyl nucleotide modifications, such as 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications and / or 5'-methylcytosine modifications.

[0262] In some embodiments, antisense nucleic acids inhibit / reduce the translation of nucleic acids containing their target nucleotide sequence. In some embodiments, antisense nucleic acids reduce / prevent the association of translation-required factors (e.g., ribosomes) with nucleic acids containing their target nucleotide sequence.

[0263] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene (for example, a gene associated with organ regeneration), including mRNA, which is the product of RNA processing of the primary transcript.

[0264] It can be acknowledged that the target nucleotide sequence to which an antisense nucleic acid binds is a nucleotide sequence that encodes a protein whose expression is to be inhibited. Therefore, in aspects and embodiments of this disclosure, the target nucleotide sequence of the antisense nucleic acid is a nucleotide sequence of a gene encoding one or more of the following: MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0265] In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding one of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB.

[0266] In some embodiments, the target nucleotide sequence is the nucleotide sequence provided in Table 14.

[0267] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001198695.2 (GI:1677501926, version 2), which is the NCBI reference sequence (SEQ ID NO: 7179) or a portion thereof of human MFAP4 transcript variant 1 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_002404.3 (GI:1677501522, version 3), which is the NCBI reference sequence (SEQ ID NO: 7180) or a portion thereof of human MFAP4 transcript variant 2 mRNA.

[0268] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_012203.2 (GI:1519473711, version 2), which is the NCBI reference sequence (SEQ ID NO: 7181) or a portion thereof of human GRHPR transcript variant 1 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_030790.5 (GI:1653961895, version 5), which is the NCBI reference sequence (SEQ ID NO: 7182) or a portion thereof of human ITFG1 transcript variant 1 mRNA.

[0269] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_005845.5 (GI:1813751621, version 5), which is either the NCBI reference sequence (SEQ ID NO: 7183) or a portion thereof of human ABCC4 transcript variant 1 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001105515.3 (GI:1677498821, version 3), which is either the NCBI reference sequence (SEQ ID NO: 7184) or a portion thereof of human ABCC4 transcript variant 2 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001301829.2 (GI:1677530022, version 2), which is either the NCBI reference sequence (SEQ ID NO: 7185) or a portion thereof of human ABCC4 transcript variant 3 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001301830.2 (GI:1677498275, version 2), which is the NCBI reference sequence (SEQ ID NO: 7186) or a portion thereof of human ABCC4 transcript variant 4 mRNA.

[0270] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001128166.3 (GI:1889680926, version 3), which is the NCBI reference sequence (SEQ ID NO: 7187) or a portion thereof of human PAK3 transcript variant 1 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_002578.5 (GI:1519316149, version 5), which is the NCBI reference sequence (SEQ ID NO: 7188) or a portion thereof of human PAK3 transcript variant 2 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001128167.3 (GI:1890283404, version 3), which is the NCBI reference sequence (SEQ ID NO: 7189) or a portion thereof of human PAK3 transcript variant 3 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001128168.3 (GI:1676441496, version 3), which is the NCBI reference sequence (SEQ ID NO: 7190) or a portion thereof of human PAK3 transcript variant 4 mRNA.

[0271] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_001013642.3 (GI:1519242294, version 3), which is the NCBI reference sequence (SEQ ID NO: 7191) or a portion thereof of human TRNP1 mRNA.

[0272] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_017413.5 (GI:1519315208, version 5), which is the NCBI reference sequence (SEQ ID NO: 7192) or a portion thereof of human APLN mRNA.

[0273] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_005733.3 (GI:1519313609, version 3), which is the NCBI reference sequence (SEQ ID NO: 7193) or a portion thereof of human KIF20A transcript variant 1 mRNA.

[0274] In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_002341.2 (GI:1720810086, version 2), which is either the NCBI reference sequence (SEQ ID NO: 7194) or a portion thereof of human LTB transcript variant 1 mRNA. In some embodiments, the target nucleotide sequence is the nucleotide sequence of NM_009588.1 (GI:6996015, version 1), which is either the NCBI reference sequence (SEQ ID NO: 7195) or a portion thereof of human LTB transcript variant 2 mRNA.

[0275] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) calculated, for example, over the length of the antisense nucleic acid or over the length of a portion of the reference sequence, to the reverse complement or portion of any one of sequence numbers 7179-7195.

[0276] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) calculated, for example, over the length of the antisense nucleic acid or over the length of a portion of the reference sequence, for any one or a portion of any of sequence numbers 7179 to 7195.

[0277] In some embodiments, the portion of the antisense nucleic acid and / or reference sequence is 5–50, 5–40, 8–30, 8–25, 10–25, 15–25, or 19–22 nucleotides in length. The antisense nucleic acids described herein may contain thymine or uracil residues. If the antisense nucleic acids described herein are defined by reference to sequence identity with a reference sequence, the nucleic acid may contain a uracil residue in place of any thymine residue in the reference sequence, and vice versa.

[0278] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to a sequence in any one or more of the sequences in Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13, or with respect to the reverse complement of the sequence, calculated over the length of the antisense nucleic acid derived from the table or over the length of the reference sequence.

[0279] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 1 to 7155, calculated over the length of the antisense nucleic acid or the length of the reference sequence.

[0280] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence keys 14-7114 or 7141-7155, calculated over the length of the antisense nucleic acid or the length of the reference sequence.

[0281] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to any one of sequence numbers 1-13, or the reverse complement of any one of sequence numbers 1-13, calculated over the length of the antisense nucleic acid or over the length of the reference sequence.

[0282] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to any one of sequence numbers 7115-7140, or the reverse complement of any one of sequence numbers 7115-7140, calculated over the length of the antisense nucleic acid or over the length of the reference sequence.

[0283] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of sequence IDs 14-347, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of MFAP4, for example, human MFAP4.

[0284] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater) with respect to sequence numbers 1, 2, 15, 19, or 25, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of MFAP4, for example, human MFAP4.

[0285] In some embodiments, the antisense nucleic acid is calculated, for example, over the length of the antisense nucleic acid or over the length of the reference sequence, for sequence numbers 7092, 7093, 7141, 7142, 7146, 7147, 7151, 7152 and / or 7097-7102, and / or sequence numbers 7092, 7093, 7141, 7142, 7146, 7147, 7151, 7152 and / or Alternatively, the antisense nucleic acid may contain or consist of sequences having at least 75% sequence identity with respect to the reverse complement of 7097-7102 (e.g., one of at least 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 greater sequence identity). The antisense nucleic acid may be capable of reducing the gene and / or protein expression of MFAP4, for example, human MFAP4.

[0286] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to sequence number 7097 or 7100, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of MFAP4, for example, human MFAP4.

[0287] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences 7115-7120 and / or their reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of MFAP4, for example, mouse MFAP4.

[0288] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences of sequence numbers 348-456, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of GRHPR, for example, human GRHPR.

[0289] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., at least 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 greater sequence identity) with respect to one or more of SEQ ID NOs: 3, 4, 5, 349, 350, and / or 351 reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of GRHPR, for example, human GRHPR.

[0290] In some embodiments, the antisense nucleic acid comprises or comprises sequences having at least 75% sequence identity (e.g., at least 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 greater sequence identity) with respect to one or more of the sequence numbers 7094, 7143, 7148, 7153, and / or the reverse complements of one or more of the sequence numbers 7094, 7143, 7148, 7153, and / or 7103–7108, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. Antisense nucleic acids may be capable of reducing the gene and / or protein expression of GRHPR, for example, human GRHPR.

[0291] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) to one or more of the sequence sequences of sequence numbers 7121-7129, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of GRHPR, for example, mouse GRHPR.

[0292] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of sequence IDs 457-1482, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of ITFG1, for example, human ITFG1.

[0293] In some embodiments, the antisense nucleic acid comprises or comprises sequences having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 6, 7, 457, 465, 468, 470, and / or 473 reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. Antisense nucleic acids may be capable of reducing the gene and / or protein expression of ITFG1, for example, human ITFG1.

[0294] In some embodiments, the antisense nucleic acid is calculated, for example, over the length of the antisense nucleic acid or over the length of the reference sequence, for one or more of the sequence numbers 7095, 7096, 7144, 7145, 7149, 7150, 7154, 7155 and / or 7109-7114, and / or sequence numbers 7095, 7096, 7144, 7145, 7149, 7150, 7154, 7155 and / or The antisense nucleic acid may contain or consist of a sequence having at least 75% sequence identity (e.g., at least 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 greater sequence identity) with respect to one or more reverse complements of 7109-7114. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of ITFG1, e.g., human ITFG1.

[0295] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 7130-7140 and / or their reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of ITFG1, for example, mouse ITFG1.

[0296] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences of sequence numbers 1483-2208, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of ABCC4, for example, human ABCC4.

[0297] In some embodiments, the antisense nucleic acid comprises or comprises sequences having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 1483, 1485, 1486, 1488, 1489, and / or 1490 reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. Antisense nucleic acids may be capable of reducing the gene and / or protein expression of ABCC4, for example, human ABCC4.

[0298] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences 2209-5060, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of PAK3, for example, human PAK3.

[0299] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 2209, 2225, and / or the reverse complements of one or more of the sequence numbers 2209, 2225, and / or 2234, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of PAK3, for example, human PAK3.

[0300] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., at least 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 greater sequence identity) with respect to one or more of the sequence numbers 5061-5389, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of TRNP1, for example, human TRNP1.

[0301] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of SEQ ID NOs: 5061 and / or 5062 reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of TRNP1, for example, human TRNP1.

[0302] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 5390-5966, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of APLN, for example, human APLN.

[0303] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., at least 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 greater sequence identity) with respect to one or more of the sequence numbers 5390, 5391, 5392, and / or the reverse complement of one or more of the sequence numbers 5390, 5391, 5392, and / or 5393, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of APLN, for example, human APLN.

[0304] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences of sequence numbers 5967-6974, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of KIF20A, for example, human KIF20A.

[0305] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 5967, 5970, and / or the reverse complements of one or more of the sequence numbers 5967, 5970, and / or 5971, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing the gene and / or protein expression of KIF20A, for example, human KIF20A.

[0306] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence sequences of sequence numbers 6975-7091, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing gene and / or protein expression in LTBs, e.g., human LTBs.

[0307] In some embodiments, the antisense nucleic acid comprises or comprises a sequence having at least 75% sequence identity (e.g., one of at least 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 greater sequence identity) with respect to one or more of the sequence numbers 6977, 6978, and / or 6993 reverse complements, calculated over the length of the antisense nucleic acid or over the length of the reference sequence. The antisense nucleic acid may be capable of reducing gene and / or protein expression in LTBs, e.g., human LTBs.

[0308] Antisense nucleic acids may contain, or consist of, sequences that hybridize to any of the sequences listed in Tables 1 to 14, or sequences that hybridize to the complements of any of the sequences listed in Tables 1 to 14.

[0309] In some embodiments, the nucleic acid inhibitor is an antisense oligonucleotide (ASO). An ASO is a single-stranded nucleic acid molecule containing or comprising an antisense nucleic acid against a target nucleotide sequence. The antisense oligonucleotides according to this disclosure may contain or consist of antisense nucleic acids as described herein.

[0310] An ASO can modify the expression of an RNA molecule containing the target nucleotide sequence by altering its splicing or by recruiting an RNase H to degrade the RNA containing the target nucleotide sequence. The RNase H recognizes the nucleic acid complex molecule formed when the ASO binds to the RNA containing the target nucleotide sequence. The ASOs according to this disclosure may comprise or consist of the antisense nucleic acids according to this disclosure. The ASOs may have nucleotide lengths of 10 to 40 (e.g., 17 to 30, 20 to 27, 21 to 23). Numerous ASOs have been designed as chimeras containing mixtures of bases with different chemistry, or as gapmers containing a central DNA portion surrounded by "wings" of modified nucleotides. ASOs are described, for example, by Scoles et al., Neurol Genet. 2019 Apr; 5(2):e323. ASOs may also include modifications to the sugar-phosphate backbone to increase their stability and / or reduce / hinder RNase H degradation, such as phosphorothioate bonds, phosphorodiamidate bonds, such as phosphorodiamidate morpholino (PMO), and may also include, for example, peptide nucleic acids (PNA), locked nucleic acids (LNA), methoxyethyl nucleotide modifications, such as 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications and / or 5'-methylcytosine modifications.

[0311] In some embodiments, the nucleic acid inhibitor is selected from siRNA, dsiRNA, miRNA, shRNA, pre-miRNA, pre-miRNA, saRNA, snoRNA, or antisense oligonucleotides (e.g., gapmers) or nucleic acids encoding them. In some embodiments, the nucleic acid inhibitor is selected from siRNA, dsiRNA, miRNA, and shRNA. In some embodiments, the nucleic acid inhibitor is siRNA. In some embodiments, the nucleic acid inhibitor is shRNA.

[0312] Nucleic acid inhibitors may be RNAi agents (e.g., siRNA, shRNA, or miRNA-based shRNA or CRISR / CAS9 knockout gRNA) or nucleic acids encoding RNAi agents that reduce the expression of one or more genes / mRNAs, such as MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, and / or LTB.

[0313] In some embodiments, the inhibitory nucleic acid may include, for example, the antisense nucleic acid described herein as part of a larger nucleic acid species. For example, in some embodiments, the inhibitory nucleic acid may be siRNA, dsiRNA, miRNA, shRNA, pre-miRNA, pre-miRNA, saRNA, or snoRNA, including the antisense nucleic acid described herein.

[0314] In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA). As used herein, “siRNA” refers to a double-stranded RNA molecule having a base pair length between 17 and 30 (e.g., 20–27, e.g., about 21–23) that can participate in the RNA interference (RNAi) pathway for targeted degradation of target RNA. Double-stranded siRNA molecules can be formed as nucleic acid complexes of highly complementary RNA strands. The strand of a double-stranded siRNA molecule that is complementary to the target nucleotide sequence (i.e., the antisense nucleic acid) may be called the “guide” strand, and the other strand may be called the “passenger” strand. The structure and function of siRNA are described, for example, in Kim and Rossi, Biotechniques. 2008 Apr; 44(5):613–616.

[0315] RNAi agents may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands, for example, containing one, two, or three nucleotides (e.g., a "UU" 3' overhang, a "TT" 3' overhang, or a "CCA" 5' overhang). The overhangs may be 1 to 6 nucleotides long, for example, 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhangs may result from one strand being longer than the other, or from two strands of equal length being twisted. The overhangs may form a mismatch with the target mRNA, or they may be complementary to the targeted gene sequence, or they may be a different sequence. The first and second strands may also be joined by additional bases to form a hairpin, or by other non-base linkers.

[0316] In some embodiments, the passenger strand of the siRNA according to this disclosure may include a 5' terminal "CCA" modification, i.e., the addition of the nucleotide "CCA". In some embodiments, the passenger strand of the siRNA according to this disclosure may include a 3' terminal "TT" modification, for example, replacing two nucleotides at the 3' end.

[0317] In some embodiments, the guide strand of siRNA according to this disclosure may include or consist of an antisense nucleic acid conforming to the embodiments of antisense nucleic acids described herein.

[0318] In some embodiments, the siRNAs of this disclosure (e.g., in Tables 1-11) may be contained within longer shRNA sequences (e.g., in Tables 12 and 13) that are processed to form the siRNA.

[0319] The terms “RNAi agent” or “RNAi,” when used synonymously herein, refer to agents containing RNA that mediates targeted cleavage of RNA transcripts by the RNA-induced silencing complex (RISC) pathway, as defined herein. RNAi agents direct sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). RNAi agents modulate, for example, inhibit the expression of genes associated with organ regeneration in cells, for example, in cells within a subject such as a mammalian subject. The term “RNAi agent” includes both shRNA (e.g., in Table 12 or 13) or precursor RNA processed into siRNA by RISC (e.g., in Tables 1-11), as well as the siRNA itself that inhibits the expression of endogenous genes.

[0320] The present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene in vivo. The RNAi agent may comprise a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 12 to 30 nucleotides in length. For example, each strand may be between 14 to 30 nucleotides, 17 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides in length.

[0321] The sense and antisense strands typically form a double-stranded RNA ("dsRNA"). The double-stranded region of an RNAi agent can be 12–30 nucleotide pairs long. For example, the double-stranded region may be between 14–30 nucleotide pairs, 17–30 nucleotide pairs, 27–30 nucleotide pairs, 17–23 nucleotide pairs, 17–21 nucleotide pairs, 17–19 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 19–21 nucleotide pairs, 21–25 nucleotide pairs, or 21–23 nucleotide pairs. In another example, the double-stranded region may be selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0322] In some embodiments, the inhibitory nucleic acid is a Dicer small interfering RNA (dsiRNA). As used herein, “dsiRNA” refers to a double-stranded RNA molecule having a length of approximately 27 base pairs that is processed by Dicer into siRNA for RNAi-mediated degradation of target RNA. dsiRNA is described, for example, in Raja et al., Asian J Pharm Sci. (2019) 14(5): 497–510, the entire text of which is incorporated herein by reference. dsiRNA may be optimized for Dicer processing and may have increased potency compared to 21-mer siRNA (see, for example, Kim et al., Nat Biotechnol. (2005) 23(2): 222–226), which may be related to the interaction between Dicer-mediated nuclease activity and RISC loading.

[0323] In some embodiments, inhibitory nucleic acids are microRNAs (miRNAs) or their precursors (e.g., pre-miRNAs). miRNA molecules have a similar structure to siRNA molecules but are endogenously encoded and originate from the processing of short hairpin RNA molecules. They are initially expressed as long primary transcripts (pre-miRNAs), which are processed in the nucleus into 60-70 nucleotide hairpins (pre-miRNAs), and further processed in the cytoplasm into smaller species that interact with RISC and target mRNA. miRNAs contain a "seed sequence" essential for binding to target mRNA. The seed sequence typically consists of six nucleotides and is located at positions 2-7 of the 5' end of the miRNA.

[0324] In some embodiments, the inhibitory nucleic acid is a short hairpin RNA (shRNA), such as those provided in Tables 12 and 13 (showing sense-loop-antisense sequences). The shRNA molecule contains a sequence of highly complementary nucleotides that associate with each other by complementary base pairing to form the stem region of the hairpin. The sequence of highly complementary nucleotides may be linked by one or more nucleotides that form the loop region of the hairpin. The shRNA molecule can be processed (e.g., by catalytic cleavage with a dicer) to form an siRNA or miRNA molecule. The shRNA molecule may have a nucleotide length between 35 and 100 (e.g., 40 to 70). The stem region of the hairpin may have a base pair length between 17 and 30 (e.g., 20 to 27, e.g., about 21 to 23). The stem region may include GU pairing to stabilize the hairpin structure. The shRNA sequences described herein may include sequences that will subsequently be processed into shorter siRNA strands, such as the guide / passenger strands shown in Tables 1-11.

[0325] siRNAs, dsiRNAs, miRNAs, and shRNAs for targeted inhibition of the expression of one or more genes and / or proteins of MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB can be identified / designed in accordance with principles and / or tools well known to those skilled in the art. Parameters and tools for designing siRNA and shRNA molecules are described, for example, in Fakhr et al., Cancer Gene Therapy (2016) 23:73–82 (the entire text of which is incorporated herein by reference). Software that can be used by those skilled in the art for the design of such molecules is summarized in Table 1 of Fakhr et al., Cancer Gene Therapy (2016) 23:73–82, and includes, for example, siRNA Wizard (InvivoGen). Details for constructing such molecules can be found on the websites of commercial suppliers such as Ambion, Dharmacon, GenScript, Invitrogen, and OligoEngine.

[0326] In some embodiments, the inhibitory nucleic acids according to this disclosure are (i) nucleotide sequences having at least 75% sequence identity to one or more of the sequence sequences 1 to 7091 or one of the sequence sequences 1 to 7091 (e.g., at least 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 greater sequence identity). Nucleic acids comprising a nucleotide sequence and a nucleotide sequence having a reverse complement of the nucleotide sequence of (i), or having at least 75% sequence identity to the reverse complement of the nucleotide sequence of (i) (e.g., at least 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 greater sequence identity). Sequence IDs 1 to 7091 are shown in Tables 1 to 10 provided herein. The nucleic acids provided in this disclosure may reduce the expression of one of the following genes and / or proteins: MFAP4, GRHPR, ITFG1, ABCC4, PAK3, TRNP1, APLN, KIF20A, or LTB, according to the headings in the table where the sequence numbers are presented. For example, the sequence numbers presented in Table 2 may reduce the expression of the MFAP4 gene and / or protein.

[0327] In some embodiments, the inhibitory nucleic acids according to this disclosure are (i) having at least 75% sequence identity with any one or more nucleotide sequences of SEQ ID NOs. 7092-7096 or one of SEQ ID NOs. 7092-7096 (e.g., at least 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 greater sequence identity). A nucleic acid comprising a creotide sequence, and a nucleic acid comprising a nucleotide sequence having a reverse complement of the nucleotide sequence of (i), or having at least 75% sequence identity to the reverse complement of the nucleotide sequence of (i) (e.g., at least 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 greater sequence identity).

[0328] In some embodiments, the inhibitory nucleic acids according to the Disclosure are (i) nucleotide sequences of SEQ ID NO: 7092 or nucleotides having at least 75% sequence identity to SEQ ID NO: 7092 (e.g., at least 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 greater sequence identity). (ii) a nucleic acid comprising an ochide sequence, and (ii) a nucleic acid comprising a nucleotide sequence having sequence identity of at least 75% to sequence number 7141 (e.g., one of at least 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 greater sequence identity).

[0329] In some embodiments, the inhibitory nucleic acids according to the Disclosure are (i) nucleotide sequences of SEQ ID NO: 7093 or nucleotides having at least 75% sequence identity to SEQ ID NO: 7093 (e.g., at least 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 greater sequence identity). (ii) a nucleic acid comprising an ochide sequence, and (ii) a nucleic acid comprising a nucleotide sequence having sequence identity of at least 75% to sequence number 7142 (e.g., one of at least 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 greater sequence identity).

[0330] In some embodiments, the inhibitory nucleic acids according to this disclosure are (i) nucleotide sequences of SEQ ID NO: 7094 or nucleotides having at least 75% sequence identity to SEQ ID NO: 7094 (e.g., at least 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 greater sequence identity). (ii) a nucleic acid comprising an ochide sequence, and (ii) a nucleic acid comprising a nucleotide sequence having sequence identity of at least 75% to sequence number 7143 (e.g., one of at least 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 greater sequence identity).

[0331] In some embodiments, the inhibitory nucleic acids according to the Disclosure are (i) nucleotide sequences of SEQ ID NO: 7095 or nucleotides having at least 75% sequence identity to SEQ ID NO: 7095 (e.g., at least 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 greater sequence identity). (ii) a nucleic acid comprising an ochide sequence, and (ii) a nucleic acid comprising a nucleotide sequence having sequence identity of at least 75% to sequence number 7144 (e.g., one of at least 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 greater sequence identity).

[0332] In some embodiments, the inhibitory nucleic acid according to the Disclosure is (i) a nucleotide sequence of SEQ ID NO: 7096 or a nucleotide having at least 75% sequence identity to SEQ ID NO: 7096 (e.g., at least 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 greater sequence identity). (ii) a nucleic acid comprising an ochide sequence, and (ii) a nucleic acid comprising a nucleotide sequence having sequence identity of at least 75% to sequence number 7145 (e.g., one of at least 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 greater sequence identity).

[0333] In some embodiments according to the seven paragraphs above, the nucleotide sequence of (i) and the nucleotide sequence of (ii) may be provided by different nucleic acids (i.e., distinct oligonucleotides). In such embodiments, the nucleic acids of (i) and (ii) may be different nucleic acids. In such embodiments, the inhibitory nucleic acid may include, or consist of, a nucleic acid double helix formed by complementary base pairing between different nucleic acids containing the nucleotide sequences of (i) and (ii).

[0334] Alternatively, in some embodiments, the nucleotide sequences of (i) and (ii) may be provided as the same nucleic acid (i.e., a single oligonucleotide). That is, the nucleic acids of (i) and (ii) may be the same nucleic acid. In such embodiments, the nucleotide sequences of (i) and (ii) may be linked by one or more linker nucleotides. The inhibitory nucleic acid may include a nucleic acid double-strand region formed by complementary base pairing between the nucleotide sequences of (i) and (ii), and the linker region may form a single-strand loop region.

[0335] Disclosed herein are nucleic acid inhibitors comprising, or encoding, an RNAi agent having at least 70%, 80%, 90%, or 95% sequence identity to an RNA sequence listed in any of Tables 1 to 13 (or any combination thereof), or an RNAi agent that hybridizes to a complement of an RNA sequence listed in any of Tables 1 to 13 (or any combination thereof) under stringency conditions.

[0336] Disclosed herein are nucleic acid inhibitors comprising, or encoding, an RNAi agent having at least 70%, 80%, 90%, or 95% sequence identity to any RNA sequence listed in Tables 2 to 12 (or any combination thereof), or an RNAi agent that hybridizes to a complement of any RNA sequence listed in Tables 2 to 12 (or any combination thereof) under stringency conditions.

[0337] Disclosed herein are nucleic acid inhibitors comprising, or encoding, an RNAi agent having at least 70%, 80%, 90%, or 95% sequence identity with the RNA sequences listed in Tables 1 to 13, or an RNAi agent that hybridizes with the complement of the RNA sequences listed in Tables 1 to 13 under stringency conditions.

[0338] The terms “nucleic acid” and “polynucleotide” are used synonymously herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. These terms further include, but are not limited to, mRNA or cDNA containing intronic sequences. The polynucleotide backbone may contain sugars and phosphate groups (as commonly found in RNA or DNA) or modified or substituted sugars or phosphate groups. Alternatively, the polynucleotide backbone may contain synthetic subunits, e.g., polymers of phosphoramidites, and thus may be oligodeoxynucleoside phosphoramidates or mixed phosphoramidate-phosphodiester oligomers. Polynucleotides may include modified nucleotides, e.g., methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups, e.g., fluororibose and thioates, and nucleotide branching. The sequence of nucleotides may be interrupted by non-nucleotide components. After polymerization, polynucleotides may be further modified, such as by conjugation with labeling components. Other types of modifications included in this definition are caps, substitution with one or more analogs of naturally occurring nucleotides, and the introduction of means for attaching polynucleotides to proteins, metal ions, labeling components, other polynucleotides, or solid supports. The term “polynucleotide” also encompasses peptide nucleic acids, PNAs, and LNAs. Polynucleotides may further include genomic DNA, cDNA, or DNA-RNA hybrids.

[0339] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides. The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or DNA transcription, respectively). RNA may also be modified after transcription. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is a single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to mRNA.

[0340] "Stringency conditions" refer to conditions under which a nucleic acid can hybridize to its target polynucleotide sequence, but not to other sequences. Stringency conditions are sequence-dependent (for example, longer sequences hybridize specifically at higher temperatures). Generally, stringency conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of probes complementary to the target sequence hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and polynucleotide concentration). Typically, stringency conditions for short probes (e.g., 10–50 nucleotides) are a salt concentration of at least about 0.01–1.0 M sodium ion concentration (or other salt) at about pH 7.0–8.3, and a temperature of at least about 30°C.

[0341] As used herein, the term “complementary,” when used in reference to nucleic acid sequences, refers to complementary sequences of nucleic acid sequences that are oriented in opposite directions to provide complementarity when folded into a hairpin structure, as determined by base pairing. This term encompasses partial complementarity, where only some of the bases match according to the base pairing rules and the overall complementarity between the two nucleic acid sequences. qualification The nucleic acid inhibitors / inhibitory nucleic acids according to this disclosure may include, for example, chemically modified nucleotide acids in which a phosphonate and / or ribose and / or base is chemically modified. Such modifications may affect the activity, specificity and / or stability of the nucleic acid. One or more nucleotides of the nucleic acid inhibitor (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 or any one of them) may include such chemical modifications.

[0342] Modifications intended in accordance with the nucleic acid inhibitors of this disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101) (incorporated herein by reference above), in particular those shown in Figure 2 of Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101). Further modifications intended in accordance with the nucleic acid inhibitors of this disclosure include those described in Selvam et al., Chem Biol Drug Des. (2017) 90(5):665-678, which are incorporated herein by reference in their entirety.

[0343] In some embodiments, the inhibitory nucleic acid according to this disclosure comprises one or more nucleotides comprising a phosphonate modification. In some embodiments, the phosphonate modification(s) may be selected from phosphorothioates (e.g., Rp isomers, Sp isomers), phosphorodithioates, methylphosphonates, methoxypropylphosphonates, 5'-(E)-vinylphosphonates, 5'-methylphosphonates, phosphate-containing (S)-5'-C-methyl, 5'-phosphorothioates, and peptide nucleic acids. In some embodiments, the nucleic acid inhibitor comprises one or more nucleotides comprising a phosphorothioate modification.

[0344] In some embodiments, the inhibitory nucleic acid according to this disclosure comprises one or more nucleotides comprising a ribose modification. In some embodiments, the ribose modification(s) may be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid, and glycol nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more nucleotides comprising a 2'-O-methyl modification. In some embodiments, the inhibitory nucleic acid comprises one or more nucleotides comprising a 2'-fluoro modification.

[0345] In some embodiments, the inhibitory nucleic acids according to the Disclosure comprise one or more nucleotides comprising a base modification. In some embodiments, the base modification(s) may be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluyl ribonucleoside, and 5'-nitroindole.

[0346] In some embodiments, the inhibitory nucleic acids according to the Disclosure include one or more nucleotides comprising a phosphorothioate modification, one or more nucleotides comprising a 2'-O-methyl modification, and one or more nucleotides comprising a 2'-fluoro modification.

[0347] In some embodiments, the inhibitory nucleic acids according to this disclosure are 2'-O-methyluridine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate It comprises one or more modified nucleotides selected from 2'-fluorouridine-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate.

[0348] In some embodiments, the inhibitory nucleic acid according to the disclosure comprises a nucleotide sequence comprising 3 to 10 nucleotides (e.g., one of 3, 4, 5, 6, 7, 8, 9, or 10) containing 2'-fluoro modifications. In some embodiments, the inhibitory nucleic acid according to the disclosure comprises 4 to 15 nucleotides (e.g., one of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) containing 2'-fluoro modifications. In some embodiments, the inhibitory nucleic acid according to the disclosure comprises a nucleotide sequence comprising 2 to 6 nucleotides (e.g., one of 2, 3, 4, 5, or 6) containing phosphorothioate modifications. In some embodiments, the inhibitory nucleic acid according to the disclosure comprises 5 to 20 nucleotides (e.g., one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) containing 2'-O-methyl modifications. In some embodiments, the inhibitory nucleic acid according to the disclosure comprises a nucleotide sequence comprising 2 to 6 nucleotides (e.g., one of 2, 3, 4, 5, or 6) including 2'-O-methyl and phosphorothioate modifications. In some embodiments, the inhibitory nucleic acid according to the disclosure comprises a nucleotide sequence comprising 1 to 4 nucleotides (e.g., one of 1, 2, 3, or 4) including 2'-fluoro and phosphorothioate modifications.

[0349] In embodiments in which the nucleic acid inhibitor / inhibitory nucleic acid comprises nucleotides having chemical modifications as described herein, the nucleotide sequence is nevertheless evaluated for the purpose of sequence comparison according to this disclosure as if an equivalent unmodified nucleotide were present instead.

[0350] Nucleic acids containing nucleotides(or nucleotides) with modified phosphonate groups are evaluated for the purpose of nucleotide sequence comparison as if they contained equivalent unmodified phosphonate groups instead. Nucleic acids containing nucleotides(or nucleotides) with modified ribose groups are evaluated for the purpose of nucleotide sequence comparison as if they contained equivalent unmodified ribose groups instead. Nucleic acids containing nucleotides(or nucleotides) with modified base groups are evaluated for the purpose of nucleotide sequence comparison as if they contained equivalent unmodified base groups instead.

[0351] As an example, nucleic acids containing nucleotides with pseudouridine, 2-thiouridine, and / or 5'-fluoro-2'-deoxyuridine are evaluated for the purpose of nucleotide sequence comparison as if a uridine-containing nucleotide were instead present at each respective position. As an example, nucleic acids containing nucleotides with N6'-methyladenosine, N-ethylpiperidine 7'-EAA triazole-modified adenine, and / or N-ethylpiperidine 6'-triazole-modified adenine are evaluated for the purpose of nucleotide sequence comparison as if an adenine-containing nucleotide were instead present at each respective position. As an example, nucleic acids containing nucleotides with 5'-methylcytidine and / or 6'-phenylpyrrolo-cytosine are evaluated for the purpose of nucleotide sequence comparison as if a cytosine-containing nucleotide were instead present at each respective position.

[0352] In some embodiments, the inhibitory nucleic acids according to this disclosure include nucleic acids comprising nucleotide sequences (including modifications thereof) shown in Table 11.

[0353] In some embodiments, the inhibitory nucleic acid includes a nucleic acid comprising one or more nucleotide sequences (including modifications thereof) shown in any of the sequence numbers 7146-7155 in Table 11. The following six paragraphs refer to the sequence numbers presented in Table 11.

[0354] In some embodiments, the inhibitory nucleic acids of the present disclosure include (i) a nucleotide sequence (including modifications thereof) having at least 75% sequence identity to any one of the nucleotide sequences of SEQ ID NOs. 7146-7150 (e.g., one of at least 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 greater sequence identity). (ii) a nucleic acid comprising (ii) any one nucleotide sequence (including modifications thereof) of any one of sequence numbers 7151 to 7155 or a nucleotide sequence (including modifications thereof) having at least 75% sequence identity to any one of sequence numbers 7151 to 7155 (e.g., one of at least 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 greater sequence identity).

[0355] In some embodiments, the inhibitory nucleic acid according to the Disclosure is (i) a nucleotide sequence (including modifications thereof) having at least 75% sequence identity to SEQ ID NO: 7146 (e.g., one of at least 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 greater sequence identity) (including modifications thereof) (ii) a nucleic acid comprising (ii) the nucleotide sequence of SEQ ID NO: 7151 (including modifications thereof) or a nucleotide sequence (including modifications thereof) having at least 75% sequence identity to SEQ ID NO: 7151 (e.g., one of at least 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 greater sequence identity).

[0356] In some embodiments, the inhibitory nucleic acid according to the Disclosure is (i) a nucleotide sequence (including modifications thereof) having at least 75% sequence identity to SEQ ID NO: 7147 (e.g., one of at least 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 greater sequence identity) (including modifications thereof) (ii) a nucleic acid...

Claims

1. A pharmaceutical agent for treating or preventing diseases associated with fibrosis, comprising an ITFG1 inhibitor as an active ingredient; The aforementioned fibrosis-related disease is a disease or condition of the liver; The inhibitor of ITFG1 is an inhibitory nucleic acid; and, The inhibitory nucleic acid is (i) an RNAi agent that reduces the expression of ITFG1, or (ii) a nucleic acid that encodes an RNAi agent that reduces the expression of ITFG1. The aforementioned pharmaceutical.

2. The liver disease or condition is selected from acute liver disease, chronic liver disease, metabolic liver disease, fatty liver, hepatic fibrosis, primary sclerosing cholangitis (PSC), cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, primary biliary cirrhosis (PBC), hepatitis, and liver injury. The pharmaceutical product according to claim 1.

3. The pharmaceutical product according to claim 2, wherein the liver fibrosis is virus-induced liver fibrosis.

4. The pharmaceutical product according to claim 2, wherein the hepatitis is alcohol-induced hepatitis.

5. The pharmaceutical product according to claim 2, wherein the liver injury is drug-induced liver injury or virus-induced liver injury.

6. The pharmaceutical product according to any one of claims 1 to 5, wherein the RNAi agent that reduces the expression of ITFG1 is siRNA, shRNA, miRNA-based shRNA, or CRISR / CAS9 knockout gRNA.

7. The pharmaceutical product according to any one of claims 1 to 6, wherein the inhibitory nucleic acid comprises or encodes a nucleotide sequence having at least 90% sequence identity with any one of sequence numbers 457 to 1482.

8. A method for regenerating liver tissue in vitro, The step of inhibiting ITFG1 in tissue cells using an ITFG1 inhibitor; The inhibitor of ITFG1 is an inhibitory nucleic acid; and, The inhibitory nucleic acid is (i) an RNAi agent that reduces the expression of ITFG1, or (ii) a nucleic acid that encodes an RNAi agent that reduces the expression of ITFG1. The aforementioned method.

9. A method for increasing / expanding liver cells in vitro, The step includes inhibiting ITFG1 in hepatocytes using an ITFG1 inhibitor; The inhibitor of ITFG1 is an inhibitory nucleic acid; and, The inhibitory nucleic acid is (i) an RNAi agent that reduces the expression of ITFG1, or (ii) a nucleic acid that encodes an RNAi agent that reduces the expression of ITFG1. The aforementioned method.

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