ITFG1 inhibitory nucleic acids

Abstract

Provided herein are nucleic acids for reducing ITFG1 expression and methods of use thereof, including methods of medical treatment and prophylaxis.

Description

[0001] This application claims priority to GB2209462.7 filed on June 28, 2022 and GB2302067.0 filed on February 14, 2023, the contents and elements of which are hereby incorporated by reference into this document for all purposes. Technical Field

[0002] The present disclosure relates to the field of molecular biology, more specifically the field of nucleic acid technology. The present disclosure also relates to methods of medical treatment and prevention. Background Art

[0003] The increasing prevalence 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 problem. The main potential causes of end-stage liver disease are hepatitis virus infection (especially hepatitis B and C), drug- and alcohol-induced liver damage, and non-alcoholic fatty liver disease (NAFLD; which is associated with obesity and progresses to non-alcoholic steatohepatitis (NASH)). The burden of hepatitis virus infection is particularly high in Asia (WHO), and the incidence of NAFLD is also increasing. 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 expected to increase, mainly due to the obesity epidemic and aging societies.

[0004] Currently, the only cure for end-stage liver disease is liver transplantation. However, donor organs are limited, and patients with end-stage liver disease may also experience complications that prevent them from undergoing major surgery. Therefore, alternative strategies to delay or reverse end-stage liver disease are being sought. These strategies include cell transplantation, artificial liver devices, and enhancing the endogenous regenerative capacity of the organ.

[0005] The liver is the only internal organ with an extraordinary regenerative capacity. It is known that only 25% of the original liver mass can regenerate back to its full size. Adult hepatocytes are long-lived and usually do not undergo cell division (G0). However, when the liver is damaged, they are able to enter the cell cycle and proliferate. Once cell proliferation is complete, the newly divided cells undergo reconstruction, as well as other regeneration-related processes such as angiogenesis and reorganization of the extracellular matrix, to complete the regeneration process.

[0006] Despite this amazing capacity, the liver's regenerative capacity appears to be limited, especially under conditions of chronic injury. The liver's regenerative capacity is central to liver homeostasis. As the liver is the primary site of drug detoxification, it is exposed to many chemicals in the body that can cause cell death and damage. In addition, through the enterohepatic circulation, it is exposed to microbiota-associated metabolites. The liver can rapidly regenerate damaged tissue, thereby preventing functional failure. Liver regeneration is also crucial in patients who have had a partial liver removal for tumor resection or living donor transplantation.

[0007] WO 2022 / 025827 A1 identifies ITFG1 as an inhibition target to promote proliferation / expansion of cells including hepatocytes, pneumocytes and myoblasts, wound healing and regeneration of liver tissue, and treatment / prevention of diseases characterized by fibrosis, such as liver fibrosis. Summary of the invention

[0008] In a first aspect, the present disclosure provides inhibitory nucleic acids that reduce gene and / or protein expression of ITFG1.

[0009] The present disclosure also provides an inhibitory nucleic acid that reduces gene and / or protein expression of ITFG1, wherein the inhibitory nucleic acid comprises or encodes an antisense nucleic acid targeting a nucleotide sequence comprising or consisting of one of SEQ ID NO: 26, 33, 13 to 25, 27 to 32, 33 to 44, 128, 135, 115 to 127, 129 to 134, or 136 to 146.

[0010] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid targeting a nucleotide sequence comprising or consisting of SEQ ID NO: 26, 33, 128, or 135.

[0011] 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 one of SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178.

[0012] 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 SEQ ID NO:58, 65, 160, or 167.

[0013] In some embodiments, the inhibitory nucleic acid comprises: (i) a nucleic acid comprising a nucleotide sequence of: one of SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178, or a nucleotide sequence having at least 75% sequence identity to: one of SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178; and (ii) a nucleic acid comprising a nucleotide sequence having the reverse complement of the nucleotide sequence of (i), or a nucleotide sequence having at least 75% sequence identity to the reverse complement of the nucleotide sequence of (i).

[0014] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table I, and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table I, wherein the sequences in columns A and B are selected from the same row of Table I.

[0015] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table II, and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table II, wherein the sequences in columns A and B are selected from the same row of Table II.

[0016] In some embodiments, the inhibitory nucleic acid comprises one or more modified nucleotides selected from the group consisting of 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'-phosphate, 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'-phosphate and 2'-fluorouridine-3'-phosphorothioate.

[0017] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising a nucleotide sequence shown in column A of Table III (including modifications thereof), and (ii) a nucleic acid comprising a nucleotide sequence shown in column B of Table III (including modifications thereof), wherein the sequences in columns A and B are selected from the same row of Table III.

[0018] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 235 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 254 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 255 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 256 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 215 (including modifications thereof) and (ii) SEQ ID NO: 257 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 216 (including modifications thereof) and (ii) SEQ ID NO: 235 (including modifications thereof). In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) SEQ ID NO: 226 (including modifications thereof) and (ii) SEQ ID NO: 240 (including modifications thereof).

[0019] In some embodiments, the inhibitory nucleic acid comprises a moiety that promotes uptake of the inhibitory nucleic acid by hepatocytes, optionally wherein the moiety that promotes uptake of the inhibitory nucleic acid by hepatocytes is or comprises an N-acetylgalactosamine (GalNAc) moiety.

[0020] In some embodiments, the inhibitory nucleic acid is siRNA.

[0021] The present disclosure also provides a nucleic acid, optionally isolated, encoding an inhibitory nucleic acid of the present disclosure.

[0022] The present disclosure also provides an expression vector, which comprises the nucleic acid described in the present disclosure.

[0023] The present disclosure also provides a composition comprising the inhibitory nucleic acid, nucleic acid or expression vector described in the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0024] The present invention also provides a cell, which comprises the inhibitory nucleic acid, nucleic acid or expression vector described in the present disclosure.

[0025] The present disclosure also provides a method for reducing the gene and / or protein expression of ITFG1 in cells in vitro or in vivo, comprising introducing the inhibitory nucleic acid, nucleic acid or expression vector described in the present disclosure into the cells.

[0026] The present disclosure also provides the use of the inhibitory nucleic acid, nucleic acid, expression vector or composition of the present disclosure for reducing the gene and / or protein expression of ITFG1 in vitro or in vivo.

[0027] The present disclosure also provides the inhibitory nucleic acid, nucleic acid, expression vector or composition of the present disclosure for use in a method of medical treatment or prevention.

[0028] The present disclosure also provides an inhibitory nucleic acid, nucleic acid, expression vector or composition of the present disclosure for use in a method of treating or preventing a disease / disorder selected from the group consisting of a disease / disorder characterized by fibrosis, a disease / disorder characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), or SH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, hepatic ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small-for-size liver syndrome syndrome), split liver grafts, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome syndrome), myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy,ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture), skin keloid, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental insults, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, Peyronie's disease disease), renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

[0029] The present disclosure also provides the use of the inhibitory nucleic acid, nucleic acid, expression vector or composition of the present disclosure in the preparation of a medicament for treating or preventing a disease / disorder selected from the following: a disease / disorder characterized by fibrosis, a disease / disorder characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure liver failure, acute liver disease, viral hepatitis, hepatic ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia , pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, and systemic lupus-related kidney disease inflammation, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, (e.g., posterior capsule after cataract surgery,Postoperative fibrosis after trabeculectomy for glaucoma (filtering blebs), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental damage, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis chemosis, Peyronie's disease, renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

[0030] The present disclosure also provides a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of an inhibitory nucleic acid, nucleic acid, expression vector or composition of the present disclosure, wherein the disease or condition is selected from the group consisting of a disease / condition characterized by fibrosis, a disease / condition characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), hepatocellular carcinoma (HCC), hepatitis, ... CC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, hepatic ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease COPD, emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic renal Glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis,Postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental insults, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, fibrosis, myelofibrosis, Peyronie's disease, renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

[0031] In some embodiments of various aspects of the present disclosure, the disease / disorder is a disease / disorder characterized by fibrosis, or a disease / disorder characterized by damage and / or death of liver cells.

[0032] In some embodiments, the disease / disorder characterized by fibrosis is selected from the group consisting of chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILD), etc. LI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker's muscular dystrophy dystrophic disease (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS),Chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental insults, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, and Peyronie's disease.

[0033] In some embodiments, the disease / disorder characterized by damage and / or death of hepatocytes is selected from the group consisting of chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, and cholestatic liver disease.

[0034] describe

[0035] ITFG1

[0036] The present disclosure is particularly directed to inhibiting the expression of genes and / or proteins containing integrin alpha FG-GAPrepeat containing 1 (ITFG1; also known as TIP, Linkin / LNKN-1). Human ITFG1 isoform 1 is a 612 amino acid protein comprising a FG-GAP repeat sequence (SEQ ID NO:2), an N-terminal signal peptide (SEQ ID NO:3), and a transmembrane region near the C-terminus (SEQ ID NO:4). The amino acid sequence of mature human ITFG1 isoform 1 - after processing to remove the signal peptide - is shown in SEQ ID NO:5. The amino acid sequence of human ITFG1 isoform 2 is shown in SEQ ID NO:6.

[0037] ITGF1 is a highly conserved protein that is believed to have immunomodulatory functions. It is reported that ITFG1 can promote the secretion of IFNγ, TNFα and IL-10 by human and mouse primary T cells in vitro and improve in vivo pathology in a mouse acute graft-versus-host disease model (GVHD; Fiscella et al., Nat Biotechnol. (2003) 21 (3): 302-7). It is reported that the interaction between ITFG1 and the ATPase RUVBL1 is essential for breast cancer cell invasion and progression (Fan et al., Biochim. Biophys. Acta. Gen. Subj. (2017) 1861 (7): 1788-1800). ITFG1 is present in recurrent amplification of hepatocellular carcinoma (Rudalska et al., Nat. Med. (2014) 20 (10): 1138-1146). As mentioned above, WO2022 / 025827 A1 identifies ITFG1 as an inhibitory target to promote cell proliferation / amplification, wound healing and regeneration of liver tissue, and to treat / prevent diseases characterized by fibrosis, such as liver fibrosis. Functional genetic in vivo RNAi screening in FAH- / - mice determined that shRNA targeting Itfg1 promoted hepatocyte proliferation / amplification, thereby promoting liver tissue regeneration (Example 1 of WO 2022 / 025827 A1). It was found that shRNA targeting Itfg1 accelerated wound healing and liver regeneration in vitro and in vivo (Example 2 of WO 2022 / 025827A1). In addition, RNAi-mediated knockdown of Itfg1 was also shown to attenuate the development of fibrosis in a mouse model of non-alcoholic fatty liver disease (NAFLD) (Example 2 of WO 2022 / 025827 A1). It was also found that RNAi-mediated knockdown of Itfg1 enhanced the proliferation of pneumocytes and myoblasts (Example 2 of WO2022 / 025827 A1).

[0038] In the present specification, reference to "ITFG1" encompasses: human ITFG1 subtype 1, subtypes of human ITFG1 subtype 1 (e.g., human ITFG1 subtype 2), homologs of human ITFG1 subtype 1 (i.e., encoded by the genome of a non-human animal), and variants thereof. In some embodiments, the ITFG1 of the present disclosure comprises or consists of an amino acid sequence having at least 70% or higher amino acid sequence identity (preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity) with the following: SEQ ID NO: 1, 5 or 6. In some embodiments, ITFG1 comprises or consists of an amino acid sequence having 70% or more amino acid sequence identity (preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity) to the following: SEQ ID NO:1.

[0039] Homologs of human ITFG1 subtype 1 may be from any animal. In some embodiments, homologs of human ITFG1 subtype 1 may be from a mammal. In some embodiments, the mammal may be a non-human mammal, such as a primate (e.g., a non-human primate, such as a macaque (e.g., a cynomolgus monkey (Macaca fascicularis), a macaque (Macaca mulatta)), such as a non-human hominid (e.g., a chimpanzee (Pan troglodytes)). In some embodiments, the mammal may be a rabbit, a guinea pig, a rat, a mouse or a rodent, a cat, a dog, a pig, a sheep, a goat, a bovine (e.g., a cow), an equine (e.g., a horse) or a donkey.

[0040] Homologs of human ITFG1 isoform 1 may optionally be characterized as having 70% or more amino acid sequence identity, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. Variants of human ITFG1 isoform 1 may optionally be characterized as having 70% or more amino acid sequence identity, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0041] The amino acid sequence of cynomolgus monkey has 99.3% sequence identity (608 / 612 amino acids) with SEQ ID NO: 1, as shown in SEQ ID NO: 7. The amino acid sequence of Mus musculus ITFG1 has 89.2% sequence identity (546 / 612 amino acids) with SEQ ID NO: 1, as shown in SEQ ID NO: 8.

[0042] Inhibitory nucleic acids and related products

[0043] Aspects and embodiments of the present disclosure relate to inhibitory nucleic acids.As used herein, "inhibitory nucleic acid" refers to a nucleic acid that is capable of reducing or preventing gene and / or protein expression of one or more given target genes / proteins.

[0044] The inhibitory nucleic acids disclosed herein are suitable for reducing the gene and / or protein expression of ITFG1. It should be understood that when the inhibitory nucleic acid is described as reducing the gene expression of ITFG1, it means inhibiting the expression of the gene encoding ITFG1. That is, the inhibition of the gene expression of ITFG1 mentioned herein covers the inhibition of the expression of ITFG1.

[0045] In some embodiments, the inhibitory nucleic acids of the present disclosure may:

[0046] Reducing / preventing the expression of the gene encoding ITFG1;

[0047] Reducing / preventing transcription of a nucleic acid encoding ITFG1 (e.g. from DNA encoding ITFG2 to RNA encoding ITFG3);

[0048] Reduce the level of RNA encoding ITFG1;

[0049] Increased degradation of RNA encoding ITFG1;

[0050] Reduced levels of ITFG1 protein;

[0051] Reduce / prevent normal splicing of the pre-mRNA encoding ITFG1;

[0052] Reduce / prevent translation of the mRNA encoding ITFG1;

[0053] Reduced functional levels of ITFG1; and / or

[0054] Increased cell proliferation / population expansion (e.g., cells expressing ITFG1, such as hepatocytes).

[0055] It should be understood that a given nucleic acid can exhibit more than one characteristic described in the preceding paragraphs. Suitable assays can be used to evaluate the characteristics described in the preceding paragraphs of a given nucleic acid. The assay can be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay. The assay can be, for example, an in vivo assay, i.e., an assay performed in a non-human animal.

[0056] When the assays are cell-based assays, they may include treating the cells with nucleic acids to determine whether the nucleic acids display one or more of the properties. The assays may use species labeled with a detectable entity to facilitate their detection. The assays may include evaluating the properties after treating the cells with a series of quantities / concentrations (e.g., a dilution series) of a given nucleic acid, respectively. It should be understood that the cells are preferably cells expressing ITFG1, such as cells of the liver (e.g., HepG2 cells or HuH7 cells).

[0057] Analysis of the results of such assays can include determining the concentration at which 50% of the maximal level of the relevant activity is achieved. The concentration of the nucleic acid at which 50% of the maximal level of the relevant activity is achieved can be referred to as the "half maximal effective concentration" of the nucleic acid for the relevant activity, which can also be referred to as the "EC 50 For example, the EC of a given inhibitory nucleic acid that increases the degradation of RNA encoding ITFG1 50 It may be a concentration at which 50% of the maximum degradation level of the RNA encoding ITFG1 is achieved.

[0058] Based on the characteristics, EC 50 It may also be called the “half maximal inhibitory concentration” or “IC 50 ”, which is the concentration of nucleic acid at which 50% of the maximum level of inhibition of a given property is observed. For example, the IC of a given inhibitory nucleic acid that reduces the expression of the gene encoding ITFG1 is 50 It may be a concentration that achieves 50% of the maximum inhibition level of gene expression.

[0059] Analyzing the results of such assays can include determining the concentration that produces a specific effect in 50% of the treated population, which concentration can also be referred to as the "ED50". Analyzing the results of such assays can include determining the concentration that produces a specific effect in 80% of the treated population, which concentration can also be referred to as the "ED80". For example, the ED50 or ED80 of a given inhibitory nucleic acid that increases degradation of RNA encoding ITFG1 can be a concentration that achieves 50% or 80% (respectively) of the maximum level of degradation of RNA encoding ITFG1.

[0060] Nucleic acids capable of reducing / preventing gene expression of ITFG1 and / or reducing / preventing transcription of nucleic acids encoding ITFG1 and / or reducing RNA levels encoding ITFG1 and / or increasing degradation of RNA encoding ITFG1 can be identified using assays comprising detecting and / or quantifying RNA levels encoding ITFG1. Such assays may comprise quantifying RNA encoding ITFG1 by RT-qPCR (a technique well known to those skilled in the art). The method may use primers and / or probes to detect and / or quantify RNA encoding ITFG1. Such an assay may comprise introducing (i) a putative inhibitory nucleic acid or (ii) a control nucleic acid (e.g., a nucleic acid known not to affect the level of RNA encoding ITFG1) into cells expressing ITFG1 in in vitro culture (e.g., by transfection), subsequently measuring the level of RNA encoding ITFG1 in the cells according to (i) and (ii) (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe a decrease in the level of ITFG1 gene expression / transcription of the nucleic acid encoding ITFG1 / a decrease in the level of RNA encoding ITFG1 or an increase in the level of RNA encoding ITFG1 degradation) and (iii) comparing the detected levels of RNA encoding ITFG1 to determine whether the putative inhibitory nucleic acid decreases / prevents ITFG1 gene expression / transcription of the nucleic acid encoding ITFG1, and / or decreases the level of RNA encoding ITFG1, and / or increases the degradation of RNA encoding ITFG1.

[0061] In specific embodiments, the ability of a nucleic acid to reduce / prevent gene expression of ITFG1 can be assessed as described in Example 2 herein.

[0062] Nucleic acids capable of reducing / preventing normal splicing of a pre-mRNA encoding ITFG1 can be identified using an assay comprising detecting and / or quantifying the level of RNA (e.g., mature mRNA) encoding one or more ITFG1 subtypes. Such assays can include quantifying RNA (e.g., mature mRNA) encoding one or more ITFG1 subtypes by RT-qPCR. The method can use primers and / or probes to detect and / or quantify mature mRNA produced by canonical splicing of a pre-mRNA transcribed from a gene encoding ITFG1, and / or use primers and / or probes to detect and / or quantify mature mRNA produced by alternative splicing of a pre-mRNA transcribed from a gene encoding ITFG1. The mature mRNA produced by canonical splicing of a pre-mRNA transcribed from a gene encoding ITFG1 can be a mature mRNA encoding a major subtype produced by expression of a gene encoding ITFG1. The major subtype can be the most commonly produced / detected subtype. For example, the mature mRNA produced by canonical splicing of the precursor mRNA transcribed from human ITFG1 may be a mature mRNA encoding human ITFG1 subtype 1 (i.e., having an amino acid sequence as shown in SEQ ID NO: 1). The mature mRNA produced by alternative splicing of the precursor mRNA transcribed from the gene encoding ITFG1 may be a mature mRNA encoding a subtype other than the main subtype produced by expression of the gene encoding ITFG1. For example, the mature mRNA produced by alternative splicing of the precursor mRNA transcribed from human ITFG1 may be a mature mRNA encoding a subtype other than human ITFG1 subtype 1 (i.e., having an amino acid sequence different from SEQ ID NO: 1); for example, a mature mRNA encoding human ITFG1 subtype 2 (i.e., having an amino acid sequence as shown in SEQ ID NO: 6). Such an assay may comprise introducing (e.g., by transfection) (i) a putative inhibitory nucleic acid or (ii) a control nucleic acid (e.g., a nucleic acid known not to affect splicing of pre-mRNA encoding ITFG1) into cells expressing ITFG1 in in vitro culture, then measuring (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe an effect on splicing of pre-mRNA encoding ITFG1) the level of mature mRNA encoding one or more isoforms of ITFG1 in the cells according to (i) and (ii), and (iii) comparing the levels of mature mRNA encoding the isoforms to determine whether the putative inhibitory nucleic acid reduces / prevents normal splicing of pre-mRNA encoding ITFG1.

[0063] Nucleic acids capable of reducing ITFG1 protein levels and / or reducing / preventing translation of mRNA encoding ITFG1 can be identified by using an assay that includes detecting ITFG1 protein levels, for example using techniques well known to those skilled in the art, such as antibody / reporter gene-based methods (Western blot, ELISA, immunohistochemistry / cytochemistry, etc.). The method may use antibodies specific for ITFG1. Such an assay may include introducing (e.g., by transfection) (i) a putative inhibitory nucleic acid or (ii) a control nucleic acid (e.g., a nucleic acid known not to affect ITFG1 protein levels) into cells expressing ITFG1 in in vitro culture, followed by measuring (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe a reduction in ITFG1 protein levels) the level of ITFG1 protein in the cells according to (i) and (ii), and (iii) comparing the levels of ITFG1 protein detected to determine whether the putative inhibitory nucleic acid reduces the level of ITFG1 protein and / or reduces / prevents translation of mRNA encoding ITFG1.

[0064] In specific embodiments, the ability of a nucleic acid to reduce / prevent ITFG1 protein levels can be assessed as described in Example 6 herein.

[0065] Nucleic acids capable of reducing the level of ITFG1 function (e.g., the ITFG1 function described herein) can be identified using an assay that includes detecting the level of the relevant function. Such an assay can include introducing (e.g., by transfection) (i) a putative inhibitory nucleic acid or (ii) a control nucleic acid (e.g., a nucleic acid known not to affect ITFG1 function) into cells expressing ITFG1 in in vitro culture, then measuring the level of ITFG1 function according to (i) and (ii) in the cells (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe a reduction in the level of ITFG1 function), and (iii) comparing the levels of ITFG1 function detected to determine whether the putative inhibitory nucleic acid reduces the level of ITFG1 function.

[0066] Cell proliferation and / or population expansion of a given cell type (eg, cells expressing ITFG1, such as hepatocytes) or population thereof can be assessed in vitro by measuring or monitoring the number or proportion of such cells over a period of time.

[0067] Cell proliferation / amplification can be studied by analyzing cell division or cell number over a period of time. For example, cell division can be analyzed by in vitro analysis of 3H-thymidine incorporation or by CFSE dilution assay, such as Fulcher and Wong, Immunol Cell Biol (1999) 77 (6): 559-564, the entire contents of which are incorporated herein by reference. Proliferated cells can also be identified by appropriate assays for the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), such as Buck et al., Biotechniques, June 2008; 44 (7): 927-9, and Sali and Mitchison, PNAS USA, February 19, 2008; 105 (7): 2415-2420, the entire contents of which are incorporated herein by reference. Other suitable assays include assays for quantification of viable cells, such as the CellTiter-Glo Luminescent Cell Viability Assay (Promega) or the CCK8 assay (Dojindo).

[0068] Nucleic acids capable of increasing cell proliferation and / or population expansion of a given cell type (e.g., cells expressing ITFG1, such as hepatocytes) can be identified using an assay comprising introducing (e.g., by transfection) (i) a putative inhibitory nucleic acid, or (ii) a control nucleic acid (e.g., a nucleic acid known not to increase cell proliferation / population expansion of a given cell type) into cells cultured in vitro, subsequently measuring (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe an increase in cell proliferation / population expansion) the number / ratio of cells according to (i) and (ii) or some other relevant cell proliferation / population expansion, and (iii) comparing the detected numbers / ratios / proliferation / population expansion to determine whether the putative inhibitory nucleic acid increases cell proliferation and / or population expansion.

[0069] In certain embodiments, the ability of a nucleic acid to increase cell proliferation / population expansion (eg, cells expressing ITFG1, such as hepatocytes) can be assessed as described in Examples 3 or 7 herein.

[0070] In some embodiments, the inhibitory nucleic acids of the present disclosure can reduce the expression of a gene encoding ITFG1 in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the expression level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to inhibit the expression of the gene of interest. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the expression of a gene encoding ITFG1 in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the expression level observed in the absence of the inhibitory nucleic acid or in the presence of an equal amount of a control nucleic acid known not to inhibit the expression of the gene of interest.

[0071] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of RNA encoding ITFG1 in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce the level of RNA encoding ITFG1. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of RNA encoding ITFG1 in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce the level of RNA encoding ITFG1.

[0072] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of transcription of a nucleic acid encoding ITFG1 in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce transcription of the nucleic acid encoding ITFG1. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of transcription of a nucleic acid encoding ITFG1 in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1%, of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce transcription of the nucleic acid encoding ITFG1.

[0073] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of ITFG1 protein in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce the level of ITFG1 protein. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of ITFG1 protein in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid or in the presence of an equal amount of a control nucleic acid known not to reduce the level of ITFG1 protein.

[0074] In some embodiments, an inhibitory nucleic acid of the present disclosure is capable of reducing the level of ITFG1 function in a given assay to less than 1 fold, e.g., ≤0.99 fold, ≤0.95 fold, ≤0.9 fold, ≤0.85 fold, ≤0.8 fold, ≤0.75 fold, ≤0.7 fold, ≤0.65 fold, ≤0.6 fold, ≤0.55 fold, ≤0.5 fold, ≤0.45 fold, ≤0.4 fold, ≤0.35 fold, ≤0.3 fold, ≤0.25 fold, ≤0.2 fold, ≤0.15 fold, ≤0.1 fold, ≤0.05 fold, or ≤0.01 fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of an equal amount of a control nucleic acid known not to reduce the level of ITFG1 function. In some embodiments, an inhibitory nucleic acid of the present disclosure is capable of reducing the level of ITFG1 function in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid or in the presence of an equal amount of a control nucleic acid known not to reduce the level of ITFG1 function.

[0075] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing normal splicing of pre-mRNA encoding ITFG1 in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce normal splicing of pre-mRNA encoding ITFG1. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing the level of normal splicing of pre-mRNA encoding ITFG1 in a given assay to less than 100%, for example, ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5% or ≤1% of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce normal splicing of pre-mRNA encoding ITFG1.

[0076] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing translation of mRNA encoding ITFG1 in a given assay by less than 1 fold, e.g., ≤0.99 fold, ≤0.95 fold, ≤0.9 fold, ≤0.85 fold, ≤0.8 fold, ≤0.75 fold, ≤0.7 fold, ≤0.65 fold, ≤0.6 fold, ≤0.55 fold, ≤0.5 fold, ≤0.45 fold, ≤0.4 fold, ≤0.35 fold, ≤0.3 fold, ≤0.25 fold, ≤0.2 fold, ≤0.15 fold, ≤0.1 fold, ≤0.05 fold, or ≤0.01 fold, compared to the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce translation of mRNA encoding ITFG1. In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of reducing translation of mRNA encoding ITFG1 in a given assay to less than 100%, e.g., ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to reduce translation of mRNA encoding ITFG1.

[0077] The preferred reduced level according to the preceding seven paragraphs is reduced to less than 0.5 times / ≤50%, such as less than 0.4 times / ≤40%, less than 0.3 times / ≤30%, less than 0.2 times / ≤20%, less than 0.15 times / ≤15%, or less than 0.1 times / ≤10%.

[0078] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of increasing the degradation of RNA encoding ITFG1 in a given assay to greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to increase the degradation of RNA encoding ITFG1.

[0079] In some embodiments, the inhibitory nucleic acids of the present disclosure are capable of increasing cell proliferation / population expansion (e.g., cells expressing ITFG1, such as hepatocytes) in a given assay by greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same amount of a control nucleic acid known not to increase cell proliferation / population expansion of the cell type of interest.

[0080] In some embodiments, the inhibitory nucleic acids of the present disclosure prevent or silence the expression of a gene encoding ITFG1. In some embodiments, the inhibitory nucleic acids of the present disclosure prevent or silence the expression of ITFG1 at the protein level. As used herein, the expression of a given gene / protein can be considered to be "prevented" or "silenced" when the expression level is reduced to less than 0.1 times / ≤10% of the level observed in the absence of the inhibitory nucleic acid or in the presence of the same number of control nucleic acids known not to be inhibitors of the expression of the relevant gene / protein.

[0081] In preferred embodiments, the inhibitory nucleic acids (e.g., siRNA) disclosed herein inhibit gene and / or protein expression of ITFG1 in a given assay by greater than 50%, e.g., ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥100%, ≥101%, ≥102%, ≥103%, ≥104%, ≥105%, ≥106%, ≥107%, ≥108%, ≥109%, ≥109%, ≥101%, ≥102%, ≥103%, ≥104%, ≥105%, ≥106%, ≥107%, ≥108%, ≥109%, ≥10 %, ≥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%.

[0082] In preferred embodiments, the inhibitory nucleic acids (e.g., siRNAs) disclosed herein inhibit gene expression of ITFG1 in a given assay (e.g., the assay described in Example 2 herein) by greater than 50%, e.g., ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥10 ... %, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%.

[0083] In preferred embodiments, the inhibitory nucleic acids (e.g., siRNA) disclosed herein inhibit protein expression of ITFG1 in a given assay (e.g., the assay described in Example 2 herein) by greater than 50%, e.g., ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥666%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥767%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥868%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥969%, ≥97%, ≥98%, ≥99%, ≥99%, ≥10 ... %, ≥6%, ≥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%.

[0084] In some embodiments, the inhibitory nucleic acids (e.g., siRNAs) of the present disclosure can be expressed at an IC 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, ≤3 nM, ≤2 nM, ≤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 Inhibit gene and / or protein expression of ITFG1.

[0085] In some embodiments, inhibitory nucleic acids (e.g., siRNAs) of the present disclosure can be expressed at an IC 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 Inhibiting gene expression of ITFG1 (eg, as measured by qRT-PCR).

[0086] In some embodiments, inhibitory nucleic acids (e.g., siRNAs) of the present disclosure can be expressed at an IC 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 Inhibits protein expression of ITFG1 (eg, as measured by ELISA).

[0087] WO 2022 / 025827 A1 (the entire contents of which are incorporated herein by reference) describes inhibitory nucleic acids against ITFG1. In some embodiments, the inhibitory nucleic acid described in WO 2022 / 025827 A1 can be selected from: siRNA having a guide strand / antisense sequence of one of SEQ ID NOs: 457 to 1482 of WO 2022 / 025827 A1 (see Table 4 thereof); siRNA having a guide strand / antisense sequence of SEQ ID NOs: 7 or 8 of WO 2022 / 025827 A1 (see Table 1 thereof); siRNA formed by SEQ ID NOs: 7095 and 7144 of WO 2022 / 025827 A1 (see Table 11 thereof); siRNA formed by SEQ ID NOs: 7096 and 7145 of WO 2022 / 025827 A1 (see Table 11 thereof); siRNA formed by SEQ ID NOs: 7149 and 7154 of WO 2022 / 025827 A1 (see Table 11 thereof); siRNA formed by SEQ ID NO: 7150 and 7155 of WO 2022 / 025827 A1 (see Table 11 thereof); shRNA encoded by a nucleotide sequence of one of SEQ ID NO: 7109 to 7114 of WO 2022 / 025827 A1 (see Table 12 thereof); and shRNA encoded by a nucleotide sequence of one of SEQ ID NO: 7131 to 7140 of WO 2022 / 025827A1 (see Table 13 thereof).

[0088] The inhibitory nucleic acids disclosed herein may not be identical to the inhibitory nucleic acids disclosed in WO 2022 / 025827 A1. In preferred embodiments, the inhibitory nucleic acids disclosed herein have novel and / or improved properties compared to the inhibitory nucleic acids disclosed in WO 2022 / 025827 A1.

[0089] In some embodiments, the inhibitory nucleic acids of the present disclosure:

[0090] The expression of the gene encoding ITFG1 is reduced to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0091] Reducing transcription of a nucleic acid encoding ITFG1 (e.g., from a DNA encoding ITFG1 to an RNA encoding ITFG1) to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0092] The level of RNA encoding ITFG1 is reduced to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0093] Increases degradation of RNA encoding ITFG1 to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0094] The level of ITFG1 protein is reduced to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0095] Compared to the inhibitory nucleic acid described in WO 2022 / 025827 A1, the normal splicing of the pre-mRNA encoding ITFG1 is reduced to a greater extent;

[0096] reduces translation of mRNA encoding ITFG1 to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0097] The functional level of ITFG1 is reduced to a greater extent than the inhibitory nucleic acid described in WO 2022 / 025827 A1;

[0098] Increases cell proliferation / population expansion (e.g., cells expressing ITFG1, such as hepatocytes) to a greater extent than the inhibitory nucleic acids described in WO 2022 / 025827 A1; and / or

[0099] Compared to the inhibitory nucleic acids described in WO 2022 / 025827 A1, reduced toxicity (e.g., in vitro cytotoxicity, e.g., in vivo toxicity to a subject to whom the inhibitory nucleic acid is administered) is exhibited.

[0100] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce the expression of a gene encoding ITFG1 at a comparable concentration in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, at a level lower than the level at which the inhibitory nucleic acids described in WO 2022 / 025827 A1 reduce the expression of the relevant gene.

[0101] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce transcription of a nucleic acid encoding ITFG1 at a comparable concentration in a given assay to less than 1-fold the level at which the inhibitory nucleic acids described in WO 2022 / 025827 A1 reduce nucleic acid transcription, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[0102] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce the level of RNA encoding ITFG1 at a comparable concentration in a given assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[0103] In some embodiments, the inhibitory nucleic acids of the present disclosure increase the degradation of RNA encoding ITFG1 in a given assay at a comparable concentration to greater than 1 times the level at which the inhibitory nucleic acids described in WO 2022 / 025827 A1 increase RNA degradation, for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times, or ≥10 times.

[0104] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce the level of ITFG1 protein in a given assay at a comparable concentration to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[0105] In some embodiments, the inhibitory nucleic acid of the present disclosure reduces the normal splicing of pre-mRNA encoding ITFG1 to less than 1 times the normal splicing level of pre-mRNA encoding ITFG1 reduced by the inhibitory nucleic acid described in WO 2022 / 025827 A1 at a comparable concentration in a given assay, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times or ≤0.01 times.

[0106] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce the translation of mRNA encoding ITFG1 in a given assay at a comparable concentration to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[0107] In some embodiments, the inhibitory nucleic acids of the present disclosure reduce the functional level of ITFG1 at a comparable concentration in a given assay to less than 1 times the relevant functional level reduced by the inhibitory nucleic acids described in WO 2022 / 025827 A1, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times or ≤0.01 times.

[0108] In some embodiments, the inhibitory nucleic acids of the present disclosure increase cell proliferation / population expansion (e.g., cells expressing ITFG1, such as hepatocytes) in a given assay at comparable concentrations to greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, at levels at which the inhibitory nucleic acids described in WO 2022 / 025827A1 increase cell proliferation / population expansion of such cells.

[0109] The toxicity of a given nucleic acid molecule can be studied by analyzing the toxicity related substances after the nucleic acid molecule treatment cell or the subject. Cytotoxicity can be evaluated by contacting the cell with the nucleic acid molecule (e.g., in vitro), then detecting and / or quantifying the number ratio of living cells and / or dead / dead cells after an appropriate time period. This type of analysis can include analyzing the expression or activity level of one or more cysteine ​​proteases, and / or can include detecting and / or quantifying living cells, dead cells, and / or apoptotic cells. Detecting / quantifying apoptosis can include detecting and / or quantifying one or more apoptosis markers (e.g., phosphatidylserine (PS) exposure, Bcl-2 family proteins (e.g., Bax, Bak, Bid) activation, ROS generation, cysteine ​​protease activation, mitochondrial membrane permeabilization, or DNA fragmentation).

[0110] Cytotoxicity can be studied using any of the methods reviewed in, for example, Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, the entire contents of which are incorporated herein by reference. Examples of in vitro assays for cytotoxicity / cell killing include release assays, e.g. 51 Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cytotoxicity based on detecting factors released from lysed cells. Other suitable assays include assays for quantifying viable cells, such as CellTiter-Glo luminescent cell viability assay (Promega) or CCK8 assay (Dojindo).

[0111] In certain embodiments, the cytotoxicity of the nucleic acid (eg, cells expressing ITFG1, such as hepatocytes) can be assessed as described in Example 8 herein.

[0112] The toxicity of a subject treated with a given nucleic acid molecule can be evaluated by analyzing the level of one or more related substances of cell / tissue damage (e.g., in a sample obtained from a subject) after the nucleic acid molecule is applied to the subject for an appropriate period of time. The related substance of cell / tissue damage can be a related substance of an inflammatory immune response, or a biomarker of cell type / tissue / organ damage or impaired function. In some embodiments, the organ can be the liver, and the related substance of liver cell and / or tissue damage can be the level of aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) in the serum of the subject.

[0113] In certain embodiments, the toxicity of a nucleic acid to a subject can be assessed as described in Examples 11, 17, or 19 herein.

[0114] In some embodiments, the level of cytotoxicity exhibited by the inhibitory nucleic acids of the present disclosure at comparable concentrations in a given assay (e.g., in vitro, for example, against cells expressing ITFG1, such as hepatocytes) is less than 1 times, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times, as compared to the level of cytotoxicity exhibited by the inhibitory nucleic acids described in WO 2022 / 025827A1.

[0115] In some embodiments, the inhibitory nucleic acids of the present disclosure exhibit a level of toxicity at a comparable concentration in a given assay (e.g., in vivo, e.g., in a subject to which the inhibitory nucleic acid is administered) that is less than 1 times the level of toxicity exhibited by the inhibitory nucleic acids described in WO 2022 / 025827A1, e.g., ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times.

[0116] In some embodiments, in a given assay, the level of AST and / or ALT in the serum of a subject administered the inhibitory nucleic acid is less than 1 times the level of liver damage-related correlates in the serum of a subject administered a comparable concentration of the inhibitory nucleic acid described in WO 2022 / 025827 A1, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times.

[0117] The inhibitory nucleic acid of the present disclosure may comprise or consist of DNA and / or RNA. The inhibitory nucleic acid may be single-stranded (e.g., in the case of antisense oligonucleotides (e.g., gapmers)). The inhibitory nucleic acid may be double-stranded or may comprise a double-stranded region (e.g., in the case of siRNA, shRNA, etc.). The inhibitory nucleic acid may comprise double-stranded and single-stranded regions (e.g., in the case of shRNA and pre-miRNA molecules, which are double-stranded in the stem region of the hairpin structure and single-stranded in the loop region of the hairpin structure).

[0118] In some embodiments, the inhibitory nucleic acid of the present disclosure can be an antisense nucleic acid as described herein. In some embodiments, the inhibitory nucleic acid can include an antisense nucleic acid as described herein. In some embodiments, the inhibitory nucleic acid can encode an antisense nucleic acid as described herein.

[0119] 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., RNA encoding ITFG1). The antisense nucleic acid of the present disclosure is preferably a single-stranded nucleic acid and binds to the target nucleotide sequence by complementary Watson-Crick base pairing. Complementary base pairing may involve hydrogen bonds between complementary base pairs. Antisense nucleic acids may be provided as single-stranded molecules, such as in the case of antisense oligonucleotides, or may be contained in double-stranded molecule species, such as in the case of siRNA, shRNA, and pre-miRNA molecules.

[0120] The complementary base pairing between antisense nucleic acid and its target nucleotide sequence can be complete. In such embodiments, the antisense nucleic acid comprises or is composed of the reverse complementary sequence of its target nucleotide sequence, and complementary base pairing occurs between the nucleotides of each target nucleotide sequence and the complementary nucleotides in the antisense nucleic acid. Alternatively, the complementary base pairing between antisense nucleic acid and its target nucleotide sequence can be incomplete / partial. In such embodiments, complementary base pairing occurs between the nucleotides of some (but not all) target nucleotide sequences and the complementary nucleotides in the antisense nucleic acid.

[0121] This binding between nucleic acids through complementary base pairing may be referred to as “hybridization.” By binding to its target nucleotide sequence, the 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.

[0122] The nucleotide sequence of antisense nucleic acid is fully complementary to its target nucleotide sequence so that it is combined with or hybridizes to the target nucleotide sequence.It should be understood that antisense nucleic acid preferably has a high degree of sequence identity with the reverse complementary sequence of its target nucleotide sequence.In some embodiments, the antisense nucleic acid comprises or consists of a nucleotide sequence with 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 one of higher sequence identity) with the reverse complementary sequence of its target nucleotide sequence.

[0123] In some embodiments, the antisense nucleic acid of the present disclosure comprises: a nucleotide sequence that is reverse complementary to its target nucleotide sequence, or a nucleotide sequence that comprises 1 to 10 (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions relative to the nucleotide sequence that is reverse complementary to its target nucleotide sequence.

[0124] In some embodiments, the target nucleotide sequence of the antisense nucleic acid of the present disclosure comprises or consists of 5 to 100 (e.g., one of 10 to 80, 12 to 50, or 15 to 30 (e.g., 20 to 27, e.g., ~21) nucleotides) nucleotides. In some embodiments, the target nucleotide sequence of the antisense nucleic acid of the present disclosure comprises or consists of DNA and / or RNA. In some embodiments, the target nucleotide sequence of the antisense nucleic acid of the present disclosure comprises or consists of RNA.

[0125] In some embodiments, the antisense nucleic acid reduces / prevents transcription of a nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces / prevents the association of factors (e.g., enhancers, RNA polymerases) required for normal transcription with a nucleic acid comprising its target nucleotide sequence.

[0126] In some embodiments, the antisense nucleic acid increases / enhances the degradation of the nucleic acid comprising its target nucleotide sequence, for example, by RNA interference. In some embodiments, the antisense nucleic acid reduces / prevents the translation of the nucleic acid comprising its target nucleotide sequence, for example, by RNA interference or by the antisense degradation of Rnase H.

[0127] RNA interference is described in, for example, Agrawal et al., Microbiol.Mol.Bio.Rev.(2003)67(4):657-685 and Hu et al., Sig.Transduc.Tar.Ther.(2020)5(101), the entire contents of which are incorporated herein by reference. In short, double-stranded RNA molecules are recognized by the argonaute components of RNA-induced silencing complexes (RISC). RISC loading complexes (RISC-Loading Complex, RLC) separate double-stranded RNA into single strands and integrate them into active RISC. RISC integration chains are combined with their target RNA by complementary base pairing and according to the recognition of RISC integration RNA and the degree of complementarity with target RNA, and then RISC cuts the target RNA to cause its degradation, or otherwise blocks the entry of ribosomes, thereby preventing its translation. RNAi-based therapies have been approved for a variety of indications (Kim, Chonnam Med J.(2020)56(2):87-93).

[0128] In some embodiments, the antisense nucleic acid reduces / prevents normal post-transcriptional processing (e.g., splicing and / or translation) of a nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces or changes the splicing of a precursor mRNA comprising its target nucleotide sequence into a mature mRNA. In some embodiments, the antisense nucleic acid reduces the translation of an mRNA comprising its target nucleotide sequence into a protein.

[0129] In some embodiments, the antisense nucleic acid reduces / prevents the association of factors required for normal post-transcriptional processing (e.g., components of the spliceosome) with a nucleic acid comprising its target nucleotide sequence. In this case, the antisense nucleic acid may be referred to as a "splice-switching" nucleic acid.

[0130] Splice switching nucleic acids are reviewed in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549-6563, the entire contents of which are incorporated herein by reference. 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 components of the splicing machinery and pre-mRNA. Splice switching nucleic acids can be used to alter the number / ratio of mature mRNA transcripts encoding ITFG1. Splice switching nucleic acids can be designed to target specific regions of the target transcript, for example, to achieve skipping of exons of interest (e.g., exons encoding domains / regions of interest). SSOs typically comprise alterations to the oligonucleotide sugar phosphate backbone to reduce / prevent RNAse H degradation, such as phosphorothioate bonds, phosphodiamidate bonds such as phosphodiamidate morpholine (PMO), and may comprise, 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.

[0131] In some embodiments, the antisense nucleic acid inhibits / reduces the translation of a nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces / prevents the association of factors (e.g., ribosomes) required for translation with a nucleic acid comprising its target nucleotide sequence.

[0132] It should be understood that the target nucleotide sequence to which the antisense nucleic acid binds is a nucleotide sequence encoding a protein whose expression is to be inhibited. Therefore, in aspects and embodiments of the present disclosure, the target nucleotide sequence of the antisense nucleic acid is a nucleotide sequence of a gene encoding ITFG1.

[0133] In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoded by a gene encoding ITFG1. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoding ITFG1. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of exons of RNA encoding ITFG1. In some embodiments, the target nucleotide sequence is a nucleotide sequence of exons of RNA encoding ITFG1.

[0134] In some embodiments, the target nucleotide sequence is a nucleotide sequence of SEQ ID NO: 9, which is the corresponding RNA sequence of NCBI reference sequence: NM_030790.5, which is a cDNA sequence encoding human ITFG1 subtype. In some embodiments, the target nucleotide sequence is a nucleotide sequence of SEQ ID NO: 10, which is the corresponding RNA sequence of NCBI reference sequence: NM_001305002.2, which is a cDNA sequence encoding human ITFG1 subtype 2.

[0135] In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 1st and 226th (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 227th and 299th (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 300th and 445th (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 446th and 503rd (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 504th and 578th (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 579th and 673rd (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between the 674th and 738th (inclusive) of SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 739 and 820 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 821 and 915 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 916 and 1088 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1089 and 1239 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1240 and 1348 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1349 and 1392 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1393 and 1471 (inclusive) from SEQ ID NO:9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1472 and 1596 (inclusive) from SEQ ID NO: 9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1597 and 1679 (inclusive) from SEQ ID NO: 9. In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1680 and 1797 (inclusive) from SEQ ID NO: 9.In some embodiments, the target nucleotide sequence is a nucleotide sequence between positions 1798 and 3170 (inclusive) from SEQ ID NO:9.

[0136] In some embodiments, the target nucleotide sequence is or comprises a nucleotide sequence of one of SEQ ID NOs: 13 to 44. In some embodiments, the target nucleotide sequence is or comprises a nucleotide sequence of one of SEQ ID NOs: 115 to 146. In some embodiments, the target nucleotide sequence is or comprises a nucleotide sequence of SEQ ID NOs: 26 or 33. In some embodiments, the target nucleotide sequence is or comprises a nucleotide sequence of SEQ ID NOs: 128 or 135.

[0137] In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to the reverse complement of one of SEQ ID NOs: 13 to 44. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to the reverse complement of one of SEQ ID NOs: 115 to 146. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to the reverse complement of SEQ ID NO: 26 or 33. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having 75% sequence identity (e.g., at least one of 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 more sequence identity) to the reverse complement of SEQ ID NO: 128 or 135.

[0138] In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to one of SEQ ID NOs:45 to 76. In some embodiments, the antisense nucleic acid comprises or consists 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 more sequence identity) to one of SEQ ID NOs: 147 to 178. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to SEQ ID NO:58 or 65. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to SEQ ID NO: 160 or 167.

[0139] In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to one of SEQ ID NOs: 211 to 230. In some embodiments, the antisense nucleic acid comprises or consists 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 more sequence identity) to one of SEQ ID NOs: 211 to 220. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to one of SEQ ID NOs: 221 to 230.

[0140] In some embodiments, the inhibitory nucleic acid is selected from: siRNA, dsiRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA, snoRNA, or antisense oligonucleotides (e.g., gapmers) or nucleic acids encoding them. In some embodiments, the inhibitory nucleic acid is selected from: siRNA, dsiRNA, miRNA, shRNA. In some embodiments, the inhibitory nucleic acid is siRNA.

[0141] In some embodiments, the inhibitory nucleic acid can include an antisense nucleic acid described herein, for example, as part of a larger nucleic acid species. For example, in some embodiments, the inhibitory nucleic acid can be an siRNA, dsiRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA, or snoRNA comprising an antisense nucleic acid described herein.

[0142] In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA). As used herein, "siRNA" refers to a double-stranded RNA molecule with a length between 17 and 30 (e.g., 20 to 27, e.g., ~21) base pairs, which can participate in the RNA interference (RNAi) pathway to target the degradation of target RNA. Double-stranded siRNA molecules can form nucleic acid complexes with highly complementary RNA chains. In some embodiments, the siRNA molecule comprises a symmetrical 3' overhang, for example, comprising one or two nucleotides (e.g., 'UU' 3' overhang). The chain with complementarity to the target nucleotide sequence (i.e., antisense nucleic acid) in the double-stranded siRNA molecule can be referred to as a "guide" chain, and the other chain can be referred to as a "passenger" chain. The structure and function of siRNA are described in Kim and Rossi's Biotechniques. April 2008; 44 (5): 613-616.

[0143] In some embodiments, the guide strand of a siRNA of the present disclosure may comprise or consist of an antisense nucleic acid according to the embodiments of the antisense nucleic acid described herein.

[0144] 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 of ~27 base pairs in length, which is processed into siRNA by Dicer for RNAi-mediated target RNA degradation. DsiRNA is described in, for example, Raja et al., Asian J Pharm Sci. (2019) 14 (5): 497-510, the entire contents of which are incorporated herein by reference. DsiRNA is optimized for Dicer processing, and compared with 21-mer siRNA, it can have higher efficacy (see, for example, Kim et al., Nat Biotechnol. (2005) 23 (2): 222-226), which may be related to the connection between Dicer-mediated nuclease activity and RISC load.

[0145] In some embodiments, the inhibitory nucleic acid is a microRNA (miRNA), or its precursor (e.g., pri-miRNA or pre-miRNA). The miRNA molecule has a structure similar to that of the siRNA molecule, but is endogenously encoded and derived from the processing of short hairpin RNA molecules. They are initially expressed as long primary transcripts (pri-miRNA), which are processed into hairpins (pre-miRNA) of 60 to 70 nucleotides in the nucleus, which are further processed into smaller species that interact with RISC and target mRNA in the cytoplasm. The miRNA contains a "seed sequence" that is essential for binding to the target mRNA. The seed sequence generally contains six nucleotides, located at the 2nd to 7th position of the 5' end of the miRNA.

[0146] In some embodiments, the inhibitory nucleic acid is a short hairpin RNA (shRNA). The shRNA molecule comprises a nucleotide sequence with a high degree of complementarity, which is associated with each other through complementary base pairing to form a stem region of a hairpin. The nucleotide sequence with a high degree of complementarity can be connected by one or more nucleotides forming a hairpin loop region. The shRNA molecule can be processed (e.g., by DICER catalytic cleavage) to form siRNA or miRNA molecules. The length of the shRNA molecule can be between 35 and 100 (e.g., 40 to 70) nucleotides. The length of the stem region of the hairpin can be between 17 and 30 (e.g., 20 to 27, e.g., ~21) base pairs. The stem region can include GU pairing to stabilize the hairpin structure.

[0147] Can be identified / designed according to principle and / or use the tool well known to those skilled in the art for targeting inhibition of ITFG1 gene and / or protein expression siRNA, dsiRNA, miRNA and shRNA. Parameters and tools for designing siRNA and shRNA molecules are described in, for example, Fakhr et al., Cancer Gene Therapy (2016) 23: 73-82 (the entire contents of which are incorporated herein by reference). Software that can be used by those skilled in the art for designing such molecules is summarized in Fakhr et al., Cancer Gene Therapy (2016) 23: Table 1 of 73-82, and includes, for example, siRNA Wizard (InvivoGen). Detailed information on the preparation of such molecules can be found on the websites of commercial suppliers such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine.

[0148] In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide (ASO). ASO is a single-stranded nucleic acid molecule comprising or consisting of an antisense nucleic acid directed against a target nucleotide sequence. The antisense oligonucleotide of the present disclosure may comprise or consist of an antisense nucleic acid as described herein.

[0149] ASO can change the expression of RNA molecules containing its target nucleotide sequence by changing splicing or by recruiting RNase H to degrade RNA containing the target nucleotide sequence. When ASO binds to RNA containing its target nucleotide sequence, RNase H recognizes the formed nucleic acid complex molecule. The ASO of the present disclosure may contain or consist of antisense nucleic acids of the present disclosure. ASO may contain nucleotides of 17 to 30 (e.g., 20 to 27, e.g., ~21) in length. Many ASOs are designed as chimeras, which contain a mixture of bases with different chemical properties, or as gapmers, which contain a central DNA portion surrounded by a "wing" of modified nucleotides. ASOs are described in, for example, Scoles et al., Neurol Genet. April 2019; 5(2): e323. ASOs sometimes contain alterations to the sugar phosphate backbone to increase their stability and / or reduce / prevent RNAse H degradation, such as phosphorothioate bonds, phosphorodiamidate bonds, such as phosphorodiamidate morpholine (PMO), and may contain, 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.

[0150] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of one of SEQ ID NOs: 45 to 76, or a nucleic acid 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 more sequence identity) to one of SEQ ID NOs: 45 to 76; and (ii) a nucleic acid comprising a reverse transcriptase of the nucleotide sequence of (i). A nucleic acid having a nucleotide sequence that is the complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0151] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of one of SEQ ID NOs: 147 to 178, or a nucleic acid 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 more sequence identity) to one of SEQ ID NOs: 147 to 178; and (ii) an inhibitory nucleic acid comprising a nucleotide sequence of (i). or a nucleic acid having a nucleotide sequence that is the reverse complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0152] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:58 or 65, or a nucleic acid having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to SEQ ID NO:58 or 65; and (ii) a nucleic acid comprising a reverse reciprocal of the nucleotide sequence of (i). or a nucleic acid having a nucleotide sequence that is the complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0153] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 160 or 167, or a nucleic acid having at least 75% sequence identity (e.g., at least one of 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 more sequence identity) to SEQ ID NO: 160 or 167; and (ii) a nucleic acid comprising the reverse transcriptase of the nucleotide sequence of (i). A nucleic acid having a nucleotide sequence that is the complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0154] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of one of SEQ ID NOs: 211 to 230, or a nucleic acid 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 more sequence identity) to one of SEQ ID NOs: 211 to 230; and (ii) an inhibitory nucleic acid comprising a nucleotide sequence of (i). or a nucleic acid having a nucleotide sequence that is the reverse complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0155] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of one of SEQ ID NOs: 211 to 220, or a nucleic acid 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 more sequence identity) to one of SEQ ID NOs: 211 to 220; and (ii) an inhibitory nucleic acid comprising a nucleotide sequence of (i). or a nucleic acid having a nucleotide sequence that is the reverse complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0156] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of one of SEQ ID NOs: 221 to 230, or a nucleic acid 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 more sequence identity) to one of SEQ ID NOs: 221 to 230; and (ii) an inhibitory nucleic acid comprising a nucleotide sequence of (i). or a nucleic acid having a nucleotide sequence that is the reverse complement of the nucleotide sequence of (i), or a nucleic acid having a nucleotide sequence that has 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 more sequence identity) to the reverse complement of the nucleotide sequence of (i).

[0157] In some embodiments according to the preceding seven paragraphs, the nucleotide sequence of (i) and the nucleotide sequence of (ii) may be provided on different nucleic acids (i.e., separate oligonucleotides). Thus, the nucleic acids of (i) and (ii) may be different nucleic acids. In such embodiments, the inhibitory nucleic acid may comprise or consist of a nucleic acid duplex formed by complementary base pairing between different nucleic acids comprising the nucleotide sequences of (i) and (ii).

[0158] Alternatively, in some embodiments, the nucleotide sequence of (i) and the nucleotide sequence of (ii) can be provided on the same nucleic acid (i.e., a single oligonucleotide). That is, the nucleic acids of (i) and (ii) can be the same nucleic acid. In such embodiments, the nucleotide sequence of (i) and the nucleotide sequence of (ii) can be connected by one or more linker nucleotides. The inhibitory nucleic acid can include a nucleic acid duplex region formed by complementary base pairing between the nucleotide sequences of (i) and (ii), and the linker region can form a single-stranded loop region.

[0159] The inhibitory nucleic acid of the present disclosure can include chemically modified nucleotides, for example, wherein phosphate and / or ribose and / or base are chemically modified. Such modifications can affect the activity, specificity and / or stability of the nucleic acid. One or more (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 all) nucleotides of the inhibitory nucleic acid can include such chemical modifications.

[0160] As used herein, the term "including modifications thereof" may refer to any nucleotide modification (eg, methylation), backbone modification (eg, phosphorothioate), and / or targeted modification (eg, the monomers described herein).

[0161] Modifications contemplated for the inhibitory nucleic acids of the present disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101) (incorporated herein by reference), in particular Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101) Figure 2 Further modifications contemplated for the inhibitory nucleic acids of the present disclosure include those described in Selvam et al., Chem Biol Drug Des. (2017) 90(5):665-678 (the entire contents of which are incorporated herein by reference).

[0162] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises one or more nucleotides comprising phosphate modification. In some embodiments, the phosphate modification can be selected from: phosphorothioate (e.g., Rp isomer, Sp isomer), phosphorodithioate, methyl phosphate, methoxypropyl phosphate, 5'-(E)-vinyl phosphate, 5'-methyl phosphate, (S)-5'-C-methyl phosphate, 5'-phosphorothioate and peptide nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more nucleotides comprising phosphate thioate modification.

[0163] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises one or more nucleotides comprising ribose modification. In some embodiments, the ribose modification can be selected from: 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluorine, 2'-deoxy-2'-fluorine, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabinose-fluorine, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclic-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid and ethylene glycol nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more nucleotides comprising 2'-O-methyl modification. In some embodiments, the inhibitory nucleic acid comprises one or more nucleotides comprising 2'-fluorine modification.

[0164] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises one or more nucleotides comprising base modifications. In some embodiments, the base modifications may be selected from the group consisting of 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'-difluorotolyl ribonucleoside, and 5'-nitroindole.

[0165] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises: 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.

[0166] In some embodiments, the inhibitory nucleic acids of the present disclosure comprise one or more modified nucleotides selected from the group consisting of 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-methyl Guanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 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.

[0167] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence comprising 3 to 10 (e.g., one of 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides comprising 2'-fluoro modifications. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence comprising 4 to 15 (e.g., one of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides comprising 2'-fluoro modifications. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence comprising 2 to 6 (e.g., one of 2, 3, 4, 5, or 6) nucleotides comprising thiophosphate modifications. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises 5 to 20 (e.g., one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides comprising 2'-O-methyl modifications. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence comprising 2 to 6 (e.g., one of 2, 3, 4, 5, or 6) nucleotides comprising 2'-O-methyl and phosphorothioate modifications. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence comprising 1 to 4 (e.g., one of 1, 2, 3, or 4) nucleotides comprising 2'-fluoro and phosphorothioate modifications.

[0168] In various aspects and embodiments, the present disclosure provides inhibitory nucleic acids comprising a pattern of modified nucleotides.

[0169] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises (one or more) nucleotide sequences having nucleotides comprising a modification pattern according to one of rows 1 to 29 of Table A below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises (one or more) nucleotide sequences having nucleotides comprising a modification pattern according to one of rows 1 to 24 of Table A below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises an antisense nucleic acid (e.g., a guide strand) having (one or more) nucleotide sequences having nucleotides comprising a modification pattern according to one of rows 1 to 24 of Table A below.

[0170] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises (one or more) nucleotide sequences having nucleotides comprising a modification pattern according to one of rows 25 to 29 of Table A below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a positive nucleic acid (e.g., a positive strand) having (one or more) nucleotide sequences having nucleotides comprising a modification pattern according to one of rows 25 to 29 of Table A below.

[0171] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to one of rows 1 to 24 of Table A below; and (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to one of rows 25 to 29 of Table A below.

[0172] Table A: Modification patterns.

[0173]

[0174]

[0175] In the above table, mN = nucleotides containing 2'-O-methyl modification, fN = nucleotides containing 2'-deoxy-2'-fluoro modification, ps = phosphorothioate bond. If not specified, the two nucleotides are phosphate bonds. [Ligand] = represents any ligand conjugation, for example, it includes at least one GalNAc part, such as 'GalNAc monomer 3'ps'GalNAc monomer 3'ps'GalNAc monomer 3'.

[0176] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to one of rows 1 to 24 of Table A; and (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to one of rows 25 to 29 of Table A, following the pattern pairs in Table B below.

[0177] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) comprising a nucleic acid (e.g., antisense nucleic acid / guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern shown in column 1 of Table B, and (ii) comprising a nucleic acid (e.g., sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern shown in column 2 of Table B, wherein the sequences in columns 1 and 2 are selected from the same row of Table B. The modification pattern is as described in Table A above.

[0178] Table B: Combinations of modification patterns from Table A.

[0179]

[0180]

[0181] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 1 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0182] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 2 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0183] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 3 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0184] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 4 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0185] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 5 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0186] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 6 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0187] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 7 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0188] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 8 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0189] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 9 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0190] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 10 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0191] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 11 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0192] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 12 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0193] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 13 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0194] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 14 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0195] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 15 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0196] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 16 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0197] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 17 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0198] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 18 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0199] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 19 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0200] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 20 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0201] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 21 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0202] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 22 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0203] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 23 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0204] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 24 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 26 of Table A.

[0205] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to any one of rows 1-24 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 25 of Table A.

[0206] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to any one of rows 1-24 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 27 of Table A.

[0207] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to any one of rows 1-14 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 28 of Table A.

[0208] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to any one of rows 1-24 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 29 of Table A.

[0209] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises (i) an antisense nucleic acid (e.g., a guide strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 11 of Table A; and / or (ii) a sense nucleic acid (e.g., a sense strand) having a nucleotide sequence having nucleotides comprising a modification pattern according to row 29 of Table A.

[0210] The modified nucleic acids of the present disclosure can have advantageous properties compared to unmodified nucleic acids, including one or more of increased potency, increased bioavailability, reduced toxicity, reduced off-target effects, increased stability (e.g., resistance to nuclease degradation), improved affinity, expanded chemical functionality, and / or improved targeting to an organ or tissue of interest.

[0211] In embodiments where the inhibitory nucleic acid comprises chemically modified nucleotides as described herein, the nucleotide sequence is evaluated for purposes of sequence comparison as if an equivalent unmodified nucleotide were present in the position in place, in accordance with the present disclosure.

[0212] Nucleic acids comprising nucleotides comprising a modified phosphate group are evaluated for the purposes of nucleotide sequence comparison as if the nucleotide comprising the modified phosphate group instead comprised the equivalent unmodified phosphate group. Nucleic acids comprising nucleotides comprising a modified ribose group are evaluated for the purposes of nucleotide sequence comparison as if the nucleotide comprising the modified ribose group instead comprised the equivalent unmodified ribose group. Nucleic acids comprising nucleotides comprising a modified base are evaluated for the purposes of nucleotide sequence comparison as if the nucleotide comprising the modified base instead comprised the equivalent unmodified base.

[0213] For example, nucleic acids comprising nucleotides comprising pseudouridine, 2-thiouridine and / or 5'-fluoro-2'-deoxyuridine are evaluated for the purpose of nucleotide sequence comparison as if nucleotides comprising uridine were present in their respective positions instead. For example, nucleic acids comprising nucleotides comprising 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 nucleotides comprising adenine were present in their respective positions instead. For example, nucleic acids comprising nucleotides comprising 5'-methylcytidine and / or 6'-phenylpyrrolocytosine are evaluated for the purpose of nucleotide sequence comparison as if nucleotides comprising cytosine were present in their respective positions instead.

[0214] In some embodiments, an inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence set forth in one of SEQ ID NOs: 211 to 241 or 254 to 257 (including modifications thereof).

[0215] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 211 to 220 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 221 to 230 (including modifications thereof).

[0216] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 231 to 241 or 254 to 257 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 231 to 235, 241, or 254 to 257 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 236 to 240 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 235, 241, 255, 256, or 257 (including modifications thereof).

[0217] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence shown in one of SEQ ID NOs: 211 to 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence shown in one of SEQ ID NOs: 231 to 241 or 254 to 257 (including modifications thereof).

[0218] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 211 to 220 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 231 to 235, 241, or 254 to 257 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 221 to 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence as shown in one of SEQ ID NOs: 236 to 240 (including modifications thereof).

[0219] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table I (as follows), and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table I, wherein the sequences in columns A and B are selected from the same row of Table I.

[0220] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table II (as follows), and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table II, wherein the sequences in columns A and B are selected from the same row of Table II.

[0221] In some embodiments, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table III (including modifications thereof) (as follows), and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table III (including modifications thereof), wherein the sequences in columns A and B are selected from the same row of Table III.

[0222] For example, when the nucleotide sequences of (i) and (ii) are selected from row 1 of Table I, the inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:45; and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:77. For further example, the inhibitory nucleic acid designated as "CG_200135" described in Example 1 herein is formed by: (i) an oligonucleotide having a nucleotide sequence of SEQ ID NO:147, and (ii) an oligonucleotide having a nucleotide sequence of SEQ ID NO:179; this embodiment corresponds to row 1 of Table II. For further example, the inhibitory nucleic acid labeled 'CG_200197' described in Example 1 herein is formed by: (i) an oligonucleotide having a nucleotide sequence of SEQ ID NO:211 (including modifications thereof), and (ii) an oligonucleotide having a nucleotide sequence of SEQ ID NO:231 (including modifications thereof); this embodiment corresponds to row 1 of Table III.

[0223] Table I:

[0224]

[0225]

[0226] Table II:

[0227] Column A Column B 1 SEQ ID NO:147 SEQ ID NO:179 2 SEQ ID NO:148 SEQ ID NO:180 3 SEQ ID NO:149 SEQ ID NO:181 4 SEQ ID NO:150 SEQ ID NO:182 5 SEQ ID NO:151 SEQ ID NO:183 6 SEQ ID NO:152 SEQ ID NO:184 7 SEQ ID NO:153 SEQ ID NO:185 8 SEQ ID NO:154 SEQ ID NO:186 9 SEQ ID NO:155 SEQ ID NO:187 10 SEQ ID NO:156 SEQ ID NO:188 11 SEQ ID NO:157 SEQ ID NO:189 12 SEQ ID NO:158 SEQ ID NO:190 13 SEQ ID NO:159 SEQ ID NO:191 14 SEQ ID NO:160 SEQ ID NO:192 15 SEQ ID NO:161 SEQ ID NO:193 16 SEQ ID NO:162 SEQ ID NO:194 17 SEQ ID NO:163 SEQ ID NO:195 18 SEQ ID NO:164 SEQ ID NO:196 19 SEQ ID NO:165 SEQ ID NO:197 20 SEQ ID NO:166 SEQ ID NO:198 21 SEQ ID NO:167 SEQ ID NO:199 22 SEQ ID NO:168 SEQ ID NO:200 23 SEQ ID NO:169 SEQ ID NO:201 24 SEQ ID NO:170 SEQ ID NO:202 25 SEQ ID NO:171 SEQ ID NO:203 26 SEQ ID NO:172 SEQ ID NO:204 27 SEQ ID NO:173 SEQ ID NO:205 28 SEQ ID NO:174 SEQ ID NO:206 29 SEQ ID NO:175 SEQ ID NO:207 30 SEQ ID NO:176 SEQ ID NO:208 31 SEQ ID NO:177 SEQ ID NO:209 32 SEQ ID NO:178 SEQ ID NO:210

[0228] Table III:

[0229]

[0230]

[0231]

[0232]

[0233] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 212 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 213 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 214 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 217 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 218 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 219 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 220 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 231 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 232 (including modifications thereof).In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 233 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 234 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 213 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 232 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 213 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 233 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 213 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 234 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 232 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 233 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 234 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 218 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 232 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 218 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 233 (including modifications thereof).In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 218 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 234 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 211 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 212 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 213 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 214 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 217 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 218 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 219 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof).In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 220 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 241 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 228 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 236 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 226 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 236 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 236 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 239 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 228 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 239 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 237 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 228 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 238 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 226 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 237 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 226 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 238 (including modifications thereof).In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 230 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 238 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 226 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 239 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 235 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 216 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 235 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 226 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 240 (including modifications thereof). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254 (including modifications thereof).

[0234] The inhibitory nucleic acids described herein may include a GalNAc targeting moiety, such as any GalNAc moiety or monomer described herein. In some embodiments, the inhibitory nucleic acids disclosed herein include: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 235 or 234 (including modifications thereof), wherein the nucleic acid further includes a GalNAc moiety (preferably a triantennary GalNAc moiety, or at least one GalNAc monomer, such as described using the formula provided below, e.g., "GalNAc monomer 1", "GalNAc monomer 2", "GalNAc monomer 3", "GalNAc monomer 4" or "GalNAc monomer 5"). In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 254, 241 or 231 (including modifications thereof), wherein the nucleic acid further comprises a GalNAc moiety (preferably a triantennary GalNAc moiety, or at least one GalNAc monomer, as described using the formula provided below, e.g., "GalNAc monomer 1", "GalNAc monomer 2", "GalNAc monomer 3", "GalNAc monomer 4" or "GalNAc monomer 5"). In some embodiments, the inhibitory nucleic acid comprises three GalNAc moieties, e.g., in series. For example, three "GalNAc monomer 1", three "GalNAc monomer 2", three "GalNAc monomer 3", three "GalNAc monomer 4", or three "GalNAc monomer 5". In some embodiments, the inhibitory nucleic acid comprises a combination of GalNAc moieties selected from two or more of "GalNAc monomer 1," "GalNAc monomer 2," "GalNAc monomer 3," "GalNAc monomer 4," and "GalNAc monomer 5."

[0235] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 235 or 234 (including modifications thereof), wherein the nucleic acid further comprises a triantennary GalNAc moiety. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 235 (including modifications thereof), wherein the nucleic acid further comprises at least one (preferably 3) "GalNAc monomer 3" as described below.

[0236] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 241 or 231 (including modifications thereof), wherein the nucleic acid further comprises at least one "GalNAc monomer 3" as described below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 241 or 231 (including modifications thereof), wherein the nucleic acid further comprises three (or at least three) "GalNAc monomers 3" as described below.

[0237] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 255 or 231 (including modifications thereof), wherein the nucleic acid further comprises at least one "GalNAc monomer 5" as described below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 255 or 231 (including modifications thereof), wherein the nucleic acid further comprises three (or at least three) "GalNAc monomers 5" as described below.

[0238] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 256 or 231 (including modifications thereof), wherein the nucleic acid further comprises at least one "GalNAc monomer 4" as described below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 256 or 231 (including modifications thereof), wherein the nucleic acid further comprises three (or at least three) "GalNAc monomers 4" as described below.

[0239] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 257 or 231 (including modifications thereof), wherein the nucleic acid further comprises at least one "GalNAc monomer 1" as described below. In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 254, 257 or 231 (including modifications thereof), wherein the nucleic acid further comprises three (or at least three) "GalNAc monomers 1" as described below.

[0240] In some embodiments, the inhibitory nucleic acid of the present disclosure comprises: (i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 215 (including modifications thereof); and / or (ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 235, 241, 255, 256 or 257 (including modifications thereof and targeting portions).

[0241] Two or more GalNAc monomers can be connected by one or more phosphodiester bonds and / or phosphorothioate bonds. At least two monomers can be connected by phosphodiester bonds. At least two monomers can be connected by phosphorothioate bonds. In some cases, all GalNAc monomers are connected by phosphorothioate bonds.

[0242] The inhibitory nucleic acids disclosed herein can be produced according to techniques well known to those skilled in the art.

[0243] For example, the inhibitory nucleic acid can be recombinantly produced by transcription of a nucleic acid sequence encoding the inhibitory nucleic acid. The nucleic acid encoding the inhibitory nucleic acid of the present disclosure can, for example, be contained in an expression vector for expressing the inhibitory nucleic acid.

[0244] Transcription can be carried out in a cell-free transcription reaction, using a recombinase (e.g., RNA polymerase) to transcribe the inhibitory nucleic acid. Alternatively, the production of the inhibitory nucleic acid of the present invention can be carried out in a cell comprising a nucleic acid encoding the inhibitory nucleic acid, and cellular enzymes (e.g., RNA polymerase) can be used for transcription. Producing the inhibitory nucleic acid of the present invention by intracellular expression can include transcribing from a vector. In order to produce the inhibitory nucleic acid of the present invention, the introduction of nucleic acid / vector can be carried out in any manner known in the art (e.g., transfection, transduction, electroporation, etc.). The expression of the inhibitory nucleic acid can be regulated using a cell-specific promoter (e.g., a hepatocyte-specific promoter).

[0245] For example, the shRNA molecules of the present disclosure can be produced in cells by transcription from a vector encoding the shRNA. The shRNA can be produced in cells by transfecting cells with a vector encoding the shRNA sequence under the control of an RNA polymerase promoter.

[0246] The siRNA molecules of the present disclosure can be produced intracellularly by transcription from a vector encoding a shRNA that encodes / comprising the siRNA, followed by processing of the shRNA molecule by the cellular DICER to form the siRNA molecule.

[0247] Inhibitory nucleic acids can also be synthesized using standard solid phase or liquid phase synthesis techniques well known in the art. Solid phase synthesis can use phosphoramidite chemistry. In short, the solid supported nucleotide can be detritylated and then coupled with appropriately activated nucleoside phosphoramidites to form phosphite triester bonds. End-capping can then be used, followed by oxidation of the phosphite triester with an oxidant (usually iodine). The cycle can then be repeated to produce polynucleotides.

[0248] The present disclosure provides nucleic acids comprising or encoding the inhibitory nucleic acids of the present disclosure. In some embodiments, the nucleic acids comprising or encoding the inhibitory nucleic acids comprise or consist of DNA and / or RNA.

[0249] The present disclosure also provides nucleic acids comprising a vector comprising or encoding the inhibitory nucleic acid of the present disclosure.

[0250] The nucleic acids and vectors of the present disclosure can be provided in purified or isolated form, ie, from other nucleic acids, or from naturally occurring biological material.

[0251] The nucleotide sequence of the nucleic acid containing or encoding the inhibitory nucleic acid of the present disclosure can be contained in a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a carrier for transferring exogenous nucleic acid into a cell. A vector can be a vector for expressing nucleic acid in a cell. Such vectors can include a promoter sequence that is operably connected to a nucleotide sequence encoding a sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express nucleic acids from the vector of the present disclosure.

[0252] The term "operably linked" may include situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in a manner that places the expression of the nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of affecting the transcription of the nucleic acid sequence.

[0253] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0254] In some embodiments, the vector can be a eukaryotic vector, such as a vector comprising elements necessary for expressing nucleic acids from the vector in eukaryotic cells. In some embodiments, the vector can be a mammalian vector, such as comprising a cytomegalovirus (CMV) or SV40 promoter to drive expression. In some embodiments, the vector comprises a cell or tissue specific promoter. In some embodiments, the vector comprises a hepatocyte specific promoter.

[0255] The present disclosure also provides a plurality of inhibitory nucleic acids of the present disclosure. The present disclosure also provides nucleic acids and vectors comprising or encoding a plurality of inhibitory nucleic acids of the present disclosure.

[0256] Individual inhibitory nucleic acids in a plurality of inhibitory nucleic acids of the present disclosure may be identical or non-identical. Similarly, in embodiments where a nucleic acid / vector comprising or encoding an inhibitory nucleic acid of the present disclosure comprises / encodes more than one inhibitory nucleic acid of the present disclosure, the inhibitory nucleic acids comprised / encoded by the nucleic acid / vector may be identical or non-identical.

[0257] In some embodiments, the nucleic acid / vector may encode one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 inhibitory nucleic acids of the disclosure. In some embodiments, the nucleic acid / vector may encode multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) copies of a given inhibitory nucleic acid of the disclosure.

[0258] In some embodiments, a plurality of inhibitory nucleic acids of the present disclosure may be a plurality of non-identical inhibitory nucleic acids. In some embodiments, a plurality of inhibitory nucleic acids may comprise one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-identical inhibitory nucleic acids. In some embodiments, a nucleic acid / vector may comprise / encode a plurality of non-identical inhibitory nucleic acids of the present disclosure.

[0259] According to embodiments of multiple inhibitory nucleic acids of the present disclosure, and according to embodiments of nucleic acids / vectors comprising / encoding multiple non-identical inhibitory nucleic acids of the present disclosure, the following two paragraphs further define multiple non-identical inhibitory nucleic acids.

[0260] In some embodiments, the non-identical inhibitory nucleic acids comprise or encode non-identical antisense nucleic acids. In such embodiments, the non-identical antisense nucleic acids can each independently conform to any embodiment of the antisense nucleic acids described above.

[0261] In some embodiments, different inhibitory nucleic acids may comprise or encode antisense nucleic acids targeting different target nucleotide sequences. In such embodiments, different target nucleotide sequences may each independently conform to any embodiment of the target nucleotide sequence of the antisense nucleic acid described above.

[0262] The present disclosure also provides a cell comprising or expressing (i) an inhibitory nucleic acid of the present disclosure, (ii) a nucleic acid comprising or encoding an inhibitory nucleic acid of the present disclosure, and / or (iii) a vector comprising a nucleic acid comprising or encoding an inhibitory nucleic acid of the present disclosure.

[0263] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including rodents), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows or any animal in the order Bovis), horse (including any animal in the order Equine), donkey, and non-human primate). In a preferred embodiment, the cell can be a human cell. In some embodiments, the cell can be a hepatocyte.

[0264] The present disclosure also provides a method for producing a cell comprising a nucleic acid or vector of the present disclosure, comprising introducing the nucleic acid or vector of the present disclosure into a cell. In some embodiments, introducing the nucleic acid or vector of the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g., retroviral transduction).

[0265] The present disclosure also provides a method for producing an inhibitory nucleic acid of the present disclosure or a nucleic acid comprising or encoding an inhibitory nucleic acid of the present disclosure, comprising culturing a cell comprising a nucleic acid under conditions suitable for the cell to express the nucleic acid or vector, wherein the nucleic acid comprises or encodes the inhibitory nucleic acid of the present disclosure or the vector of the present disclosure. In some embodiments, the method is performed in vitro.

[0266] The present disclosure also provides compositions comprising the nucleic acids (including inhibitory nucleic acids, nucleic acids comprising / encoding inhibitory nucleic acids, expression vectors comprising / encoding such nucleic acids) or cells of the present disclosure.

[0267] In therapeutic and preventive applications, the compositions of the present disclosure are preferably formulated as medicaments or pharmaceutical compositions (suitable for clinical use). Such compositions may include nucleic acids or cells and one or more other pharmaceutically acceptable ingredients well known to those skilled in the art.

[0268] The compositions of the present disclosure may comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silicon dioxide, stearic acid, vegetable stearates), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0269] The term "pharmaceutically acceptable" as used herein is applicable to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable for contacting tissues of relevant subjects (e.g., human subjects) within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. Various carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, adhesives, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweeteners of the compositions disclosed herein must also be "acceptable", i.e. compatible with other ingredients of the preparation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, adhesives, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweeteners can be found in standard pharmaceutical texts, such as Remington's "Pharmacy Science and Practice (The Science and Practice of Pharmacy)" (A. Adejare, ed.), 23rd edition (2020), Academic Press.

[0270] The compositions of the present disclosure can be prepared by any method well-known in the pharmaceutical field. These methods include the step of combining the active compound with a carrier constituting one or more auxiliary components. Typically, the active compound is uniformly and closely combined with a carrier (e.g., a liquid carrier, a finely ground solid carrier, etc.), and then the product is molded (if necessary) to prepare the preparation.

[0271] The composition can be prepared for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal administration, which may include injection or infusion. Suitable preparations may be included in the selected reagents in sterile or isotonic media. Preparations and modes of administration may be selected according to the medicament to be administered and the disease to be treated / prevented.

[0272] Compositions of the present disclosure can be formulated into liquid forms, including gel forms. Liquid preparations can be formulated for administration to selected organs or regions of human or animal bodies by injection or infusion (e.g., by catheter). Another aspect of the present disclosure relates to a method for preparing or producing a medicine or pharmaceutical composition of the present disclosure, the method comprising preparing a pharmaceutical composition or medicine by mixing an agent with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0273] The nucleic acids (including inhibitory nucleic acids, expression vectors), cells and compositions of the present disclosure may be modified and / or formulated to facilitate delivery to and / or uptake by target cells / tissues (e.g., cells of the liver (hepatocytes) or liver tissue).

[0274] Targeted delivery strategies for such species are reviewed in, for example, Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25 (9): 1602-1608, the entire contents of which are incorporated herein by reference. In particular, the nucleic acids disclosed herein can use the delivery platforms described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5 (101) (incorporated above by reference), or Tatiparti et al., "siRNA Delivery Strategies: A Comprehensive Review of Recent Developments", edited by Thomas Nann, Nanomaterials 7.4 (2017): 77 and Lehto T et al., Adv Drug Deliv Rev. 2016, 106 (Pt A): 172-182, the entire contents of which are incorporated herein by reference.

[0275] In some embodiments, the articles of the present disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of the present disclosure may be associated (covalently or non-covalently) with cell penetrating peptides (e.g., protein transduction domains, trojan peptides, arginine-rich peptides, vectocell peptides), cationic polymers, cationic lipids, or viral vectors.

[0276] Nanoparticles can be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendrimers, and polymer therapeutic agents. Nanoparticles can be inorganic, for example, such as nanotubes or metal particles, optionally with the addition of organic molecules. In some embodiments, the nanoparticles are nanoparticles as described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3: 16023, the entire contents of which are incorporated herein by reference. In some embodiments, the nanoparticles are PLGA, polypeptides, poly (β-amino esters), DOPE, polycations containing β-cyclodextrin, linear PEI, PAMAM dendrimers, branched PEI, chitosan or polyphosphate nanoparticles.

[0277] In some embodiments, nucleic acids of the present disclosure (e.g., inhibitory nucleic acids, nucleic acids comprising / encoding inhibitory nucleic acids, or expression vectors) comprise modifications to incorporate one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest. In some embodiments, nucleic acids of the present disclosure are linked (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest.

[0278] Modification and formulation of nucleic acids to facilitate targeted delivery to cell types and / or tissues of interest are described, for example, in Lorenzer et al., Journal of Controlled Release (2015) 203: 1-15, the entire contents of which are incorporated herein by reference. The moiety that facilitates delivery to and / or uptake by a cell type or tissue of interest can selectively bind to the target cell type / tissue of interest. The moiety can facilitate penetration of the cell membrane of cells of the target cell type and / or target tissue. The moiety can bind to a molecule expressed on the cell surface of the target cell type / tissue of interest. The moiety can facilitate internalization of the nucleic acid by the target cell type / tissue of interest (e.g., by endocytosis).

[0279] Moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest are described in, for example, Benizri et al., Bioconjug Chem. (2019) 30(2): 366-383, the entire contents of which are incorporated herein by reference. Such moieties include, for example, N-acetylgalactosamine (GalNAc), α-tocopherol, cell penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments / derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g., palmitic acid), and core lipid moieties.

[0280] In some embodiments, the moiety can be, for example, a peptide / polypeptide (e.g., an antibody, fragment or derivative thereof, a peptide aptamer or a cell penetrating peptide) or a nucleic acid (e.g., a nucleic acid aptamer) that binds to a target cell type / tissue of interest, for example, by interacting with a molecule expressed on the cell surface of the target cell type / tissue of interest.

[0281] In some embodiments, the nucleic acid of the present disclosure comprises a portion that facilitates delivery to liver cells (e.g., hepatocytes) and / or liver tissue and / or absorption by liver cells (e.g., hepatocytes) and / or liver tissue. In such embodiments, the portion can facilitate penetration of the cell membrane of the hepatocyte. The portion can bind to a molecule expressed on the cell surface of the hepatocyte. In some embodiments, the molecule expressed on the cell surface of the hepatocyte is an asialoglycoprotein receptor, such as ASGR1 or ASGR2. The portion can facilitate internalization of the nucleic acid by the hepatocyte (e.g., by endocytosis).

[0282] In some embodiments, the moiety can be, for example, a peptide / polypeptide (e.g., an antibody, fragment or derivative thereof, a peptide aptamer or a cell penetrating peptide) or a nucleic acid (e.g., a nucleic acid aptamer) that binds to hepatocytes and / or liver tissue, for example by interacting with a molecule expressed on the cell surface of hepatocytes (e.g., an asialoglycoprotein receptor, such as ASGR1 or ASGR2).

[0283] In some embodiments, the moiety is or comprises GalNAc. In some embodiments, the nucleic acid is conjugated to GalNAc. GalNAc interacts with asialoglycoprotein receptors expressed by hepatocytes. After GalNAc binds to ASGPR, nucleic acids conjugated to GalNAc are effectively internalized by hepatocytes through receptor-mediated endocytosis (see, e.g., Nair et al., J. Am. Chem. Soc. (2014) 136 (49): 16958-16961). In some embodiments, the nucleic acid is conjugated to one or more (e.g., 1, 2, 3, 4 or more) GalNAc moieties. In some embodiments, one or more GalNAc moieties may be covalently associated with the 5' or 3' end of one or more nucleic acid chains. In some embodiments, the nucleic acid is conjugated to a triantennary GalNAc carbohydrate moiety (such moieties are described, e.g., in Nair et al., supra)

[0284] In some embodiments, the moiety is or comprises α-tocopherol (i.e., vitamin E). In some embodiments, the nucleic acid is conjugated to α-tocopherol. Nucleic acid-α-tocopherol conjugates have been used to target nucleic acid delivery to the liver (see, e.g., Nishina et al., Mol Ther. (2008) 16(4): 734-740). In some embodiments, the nucleic acid is conjugated to one or more (e.g., 1, 2, 3, 4 or more) α-tocopherol moieties. In some embodiments, one or more α-tocopherol moieties can be covalently associated with the 5' or 3' end of one or more nucleic acid chains.

[0285] The nucleic acid of the present disclosure (e.g., the inhibitory nucleic acid of the present disclosure) may comprise a GalNAc monomer as described below and in PCT / EP2022 / 072271, the entire contents of which are incorporated herein by reference. It should be understood that, depending on the nature of R1, the following description relates to both monomers suitable for oligonucleotide synthesis and GalNAc-oligonucleotide conjugates prepared using the GalNAc monomer, e.g., nucleic acids with GalNAc, such as siRNAs for reducing gene and / or protein expression of ITFG1.

[0286] The GalNAc monomer may be a compound represented by Formula 1, Formula 2 or Formula 3

[0287]

[0288]

[0289] or a pharmaceutically acceptable salt thereof;

[0290] in

[0291] R1 is O-PN(C 1-4 alkyl)2OCH2CH2CN, OH, phosphoramidite bonds to oligonucleotides, or polystyrene beads or long-chain alkylamine controlled-pore glass beads (LCAA-CPG) linked via succinic acid, diglycolic acid, or hydroquinone-O,O'diacetic acid bonds;

[0292] R2 is H or a protecting group;

[0293] Each R3 is independently C 1-4 Alkyl, OC 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Haloalkyl or H;

[0294] R4 is H, OH, OC 1-4Alkyl or halogen;

[0295] L is -(WY) k -WX-;

[0296] k is 0 to 5;

[0297] Each W is independently L1 or L2;

[0298] Each L1 is (CH2) n , wherein n is independently 1 to 25;

[0299] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is independently 1 to 24, and Het is independently a heteroatom;

[0300] X is a bond, Het, -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2C≡C-, or *-CH2C≡C-, wherein *, if present, indicates the site of attachment to W; and

[0301] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, wherein Z is H, C 1-4 alkyl or a protecting group; and wherein

[0302] GalNAc can be protected or deprotected.

[0303] In some embodiments, X is a bond, Het, -CH2-, -CO-, *-(Het)CH2C≡C-, or *-CH2C≡C-, wherein *, if present, indicates the site of attachment to W; and

[0304] Each Y is independently CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0305] In some embodiments, when the compound is a compound represented by Formula 1, Formula 2 or Formula 3,

[0306] L is a linker selected from the group consisting of -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X- or -L2-Y-L2-X-;

[0307] L1 is (CH2) n , wherein n is 2 to 25;

[0308] L2 is CH2CH2 (HetCH2CH2) m, wherein m is 1 to 12, and Het is a heteroatom;

[0309] X is a bond, Het, -CH2-, -CO-, *-(Het)CH2C≡C-, or *-CH2C≡C-; and

[0310] Y is CONZ, NZCO, SO2NZ or NZSO2, where Z is H, C 1-4 Alkyl or protecting group.

[0311] In some embodiments, when the compound is a compound represented by Formula 1, Formula 2 or Formula 3,

[0312] L is a linker selected from the group consisting of -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X- or -L2-Y-L2-X-;

[0313] L1 is (CH2) n , wherein n is 2 to 25;

[0314] L2 is CH2CH2 (HetCH2CH2) m , wherein m is 1 to 12, and Het is a heteroatom;

[0315] X is a bond, Het, -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2C≡C-, or *-CH2C≡C-; and

[0316] Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0317] In some embodiments, the compound is a compound represented by Formula 1

[0318]

[0319] or a pharmaceutically acceptable salt thereof;

[0320] in

[0321] R1 is O-PN(C 1-4 alkyl)2OCH2CH2CN, OH, phosphoramidite bonds to oligonucleotides, or polystyrene beads or long-chain alkylamine controlled pore glass beads (LCAA-CPG) linked via succinic acid, diglycolic acid, or hydroquinone-O,O'diacetic acid bonds;

[0322] R2 is H or a protecting group;

[0323] Each R3 is independently C1-4 Alkyl, OC 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Haloalkyl or H;

[0324] R4 is H, OH, OC 1-4 Alkyl or halogen;

[0325] L is -(WY) k -WX-;

[0326] k is 0 to 5;

[0327] Each W is independently L1 or L2;

[0328] Each L1 is (CH2) n , wherein n is independently 1 to 25;

[0329] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is independently 1 to 24, and Het is independently a heteroatom;

[0330] X is a bond, Het, -CH2-, -CO- or *O-CH2-CO, for example a bond, Het, -CH2- or -CO-; and

[0331] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group;

[0332] And the GalNAc can be protected or deprotected.

[0333] In some embodiments, the compound is a compound represented by Formula 2

[0334]

[0335] or a pharmaceutically acceptable salt thereof;

[0336] in

[0337] R1 is O-PN(C 1-4 alkyl)2OCH2CH2CN, OH, phosphoramidite bonds to oligonucleotides, or polystyrene beads or long-chain alkylamine controlled pore glass beads (LCAA-CPG) linked via succinic acid, diglycolic acid, or hydroquinone-O,O'diacetic acid bonds;

[0338] R2 is H or a protecting group;

[0339] Each R3 is independently C 1-4 Alkyl, OC 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Haloalkyl or H;

[0340] R4 is H, OH, OC 1-4 Alkyl or halogen;

[0341] L is -(WY) k -WX-;

[0342] k is 0 to 5;

[0343] Each W is independently L1 or L2;

[0344] Each L1 is (CH2)n ， wherein n is independently 1 to 25;

[0345] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is independently 1 to 24, and Het is independently a heteroatom;

[0346] X is a bond, Het, -CH2-, -CO- or *O-CH2-CO, for example a bond, Het, -CH2- or -CO-; and

[0347] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group;

[0348] And the GalNAc can be protected or deprotected.

[0349] In some embodiments, the compound is a compound represented by formula 3a

[0350]

[0351] or a pharmaceutically acceptable salt thereof;

[0352] in

[0353] R1 is O-PN(C 1-4 alkyl)2OCH2CH2CN, OH, phosphoramidite bonds to oligonucleotides, or polystyrene beads or long-chain alkylamine controlled pore glass beads (LCAA-CPG) linked via succinic acid, diglycolic acid, or hydroquinone-O,O'diacetic acid bonds;

[0354] R2 is H or a protecting group;

[0355] Each R3 is independently C 1-4 Alkyl, OC 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Haloalkyl or H;

[0356] R4 is H, OH, OC 1-4 Alkyl or halogen;

[0357] L is -(WY) k -WX-;

[0358] k is 0 to 5;

[0359] Each W is independently L1 or L2;

[0360] Each L1 is (CH2) n , wherein n is independently 1 to 25;

[0361] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is independently 1 to 24, and Het is independently a heteroatom;

[0362] X is a bond, Het, -CH2-, -CO- or *O-CH2-CO, for example a bond, Het, -CH2- or -CO-; and

[0363] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group;

[0364] And the GalNAc can be protected or deprotected.

[0365] In some embodiments, when the compound is a compound represented by Formula 1, Formula 2 or Formula 3a,

[0366] L is a linker selected from the group consisting of -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X- or -L2-Y-L2-X-;

[0367] L1 is (CH2) n , wherein n is 2 to 25;

[0368] L2 is CH2CH2 (HetCH2CH2) m , wherein m is 1 to 12, and Het is a heteroatom;

[0369] X is a bond, Het, -CH2-, -CO- or *O-CH2-CO, for example a bond, Het, -CH2- or -CO-; and

[0370] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0371] It should be understood that reference to a compound includes, where appropriate, pharmaceutically acceptable salts, hydrates and solvates thereof.

[0372] GalNAc

[0373] GalNAc (IUPAC name N-acetylgalactosamine) is an amino sugar derivative of galactose. It is used as a targeting ligand in antisense and saRNA and siRNA liver therapies.

[0374]

[0375] In the monomers and conjugates described herein, the GalNAc is attached via C1. It will be appreciated by those skilled in the art that, as is conventional in carbohydrate chemistry, α- and β-stereochemistry are possible. Both stereochemistries are conceivable. In some embodiments, α-stereochemistry is used. In some embodiments, and as exemplified herein, β-stereochemistry is used.

[0376] In the synthesis of oligonucleotides and oligonucleotide conjugates, GalNAc can be protected, for example, with an acetyl group. Therefore, it should be understood that the term GalNAc used herein refers to both the structure shown with free hydroxyl groups and the structure in which these hydroxyl groups are protected by suitable protecting groups.

[0377] That is, unless otherwise indicated, the GalNAc written in the formula described herein can be a moiety as shown below, wherein P is hydrogen or a protecting group (eg, acetyl).

[0378]

[0379] Usually, when R1 is O-PN (C 1-4 In the structure of (alkyl) 2OCH2CH2CN, OH, polystyrene beads or LCAA-CPG, GalNAc is protected, thereby protecting GalNAc to react to form an oligonucleotide or oligonucleotide conjugate. For example, GalNAc can be completely protected by acetyl. That is, each P can be a protecting group, such as acetyl.

[0380] In structures where R1 is a phosphoramidite bond to an oligonucleotide, the GalNAc may be protected as described above (eg, immediately after synthesis) or may be unprotected as is normal for such a final product. That is, each P may be hydrogen.

[0381] R1 Group

[0382] R1 represents a chemical moiety used to attach the monomer to an oligonucleotide chain, a precursor group, or an attachment site for an oligonucleotide chain. As described herein, phosphoramidite chemistry is preferred. Thus, in the monomer unit, R1 is suitably O-PN (C 1-4 Alkyl) 2OCH2CH2CN, wherein the alkyl group may be linear or branched, preferably isopropyl.

[0383] For example, R1 can be

[0384] In some embodiments, R1 is OH, and the free hydroxyl group is suitable for reaction with, for example, a chlorophosphoramidite (eg, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite).

[0385] In some embodiments, R1 is a phosphoramidite bond to the oligonucleotide.

[0386] In some embodiments, R1 is a long chain alkylamine controlled pore glass bead (LCAA-CPG) linked through succinic acid, diglycolic acid, or hydroquinone-O,O'diacetic acid linkages.

[0387] R2 Group

[0388] The monomers disclosed herein may include protected hydroxyl groups during the synthesis steps. It should be understood that the present invention extends to these free hydroxyl groups and protected forms thereof. Thus, R2 may be a protecting group or H.

[0389] In some cases, R2 is selected from Tr, MMTr, DMTr or TMTr protecting groups. Tr is trityl. MMTr is 4'-methoxytrityl. DMTr is dimethoxytrityl (IUPAC name bis-(4-methoxyphenyl)-phenylmethyl). TMTr is 4',4',4'-trimethoxytrityl. They are widely used protecting groups for protecting the 5'-hydroxyl in nucleosides, particularly in oligonucleotide synthesis. Those skilled in the art will appreciate that other suitable protecting groups may be used.

[0390] R3 Group

[0391] When present, each R3 may be H or a suitable substituent. Suitably, R3 is C 1-4 Alkyl, OC 1-4Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Haloalkyl or H.

[0392] In some embodiments, R3 is methyl, methoxy, or H. In some embodiments, R3 is H.

[0393] R4 Group

[0394] The monomers disclosed herein may include substituents commonly used in oligonucleotide monomers. This is represented by R4. Therefore, R4 can be a substituent or H.

[0395] Suitable R4 substituents include hydroxyl, OC 1-4 Alkyl (preferably Ome) and halogen (preferably F).

[0396] Linker

[0397] The general formula includes a linker. Collectively referred to herein as L. The linkers may be the same or different. For example, different general formulas described herein may preferably have different linkers, and when multiple linkers are present in the general formula, these linkers may be the same or different.

[0398] Suitably, when the compound is a compound represented by Formula 1, Formula 2 or Formula 3,

[0399] L is -(WY) k -WX-;

[0400] in

[0401] k is 0 to 5;

[0402] Each W is independently L1 or L2;

[0403] Each L1 is (CH2) n , where n is 1 to 25;

[0404] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is 1 to 24, and Het is a heteroatom;

[0405] X is a bond, a heteroatom (Het), -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2C≡C-, or *-CH2C≡C-, where *, if present, indicates the site of attachment to W; and

[0406] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0407] In some embodiments,

[0408] k is 0 to 5;

[0409] Each W is independently L1 or L2;

[0410] Each L1 is (CH2) n , where n is 1 to 25;

[0411] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is 1 to 24, and Het is a heteroatom;

[0412] X is a bond, a heteroatom (Het), -CH2-, -CO-, *-(Het)CH2C≡C-, or *-CH2C≡C-, where *, if present, indicates the site of attachment to W; and

[0413] Each Y is independently CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0414] In some embodiments, k is 0 to 2. In some embodiments, k is 0 or 1.

[0415] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, -L2-Y-L2-X-, or -L1-Y-L2-Y-L1-X-.

[0416] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

[0417] in

[0418] Each L1 is (CH2) n , wherein n is 2 to 25;

[0419] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is 1 to 12, and Het is a heteroatom;

[0420] X is a bond, a heteroatom (Het), -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2C≡C-, or *-CH2C≡C-; for example, a bond, a heteroatom (Het), -CH2-, -CO-, *-(Het)CH2C≡C-, or *-CH2C≡C-; and

[0421] Each Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group; for example, Y is CONZ, NZCO, SO2NZ or NZSO2.

[0422] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

[0423] Each L1 is (CH2) n , wherein n is 2 to 10;

[0424] Each L2 is CH2CH2(OCH2CH2) m , where m is 1 to 5;

[0425] X is a bond, -CH2-, -CO-, *O-CH2-CO, -O-, *-OCH2C≡C-, or *-CH2C≡C-, e.g., a bond, -CH2-, -CO-, -O-, *-OCH2C≡C-, or *-CH2C≡C-; and

[0426] Y is CONZ or O-CH2-CONZ, such as O-CH2-CONZ, wherein Z is H, C 1-4 Alkyl or protecting group.

[0427] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

[0428] Each L1 is (CH2) n , wherein n is 2 to 10;

[0429] Each L2 is CH2CH2(OCH2CH2) m , where m is 1 to 5;

[0430] X is a bond, -CO-, *O-CH2-CO, -O-, *-OCH2C≡C-, or *-CH2C≡C-, for example, a bond, -CO-, -O-, *-OCH2C≡C-, or *-CH2C≡C-; and

[0431] Y is CONZ or O-CH2-CONZ, such as O-CH2-CONZ, wherein Z is H, C 1-4 Alkyl or protecting group.

[0432] Suitably, when the compound is a compound represented by Formula 1, Formula 2 or Formula 3a,

[0433] L is -(WY) k -WX-;

[0434] in

[0435] k is 0 to 5;

[0436] Each W is independently L1 or L2;

[0437] Each L1 is (CH2) n , where n is 1 to 25;

[0438] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is 1 to 24, and Het is a heteroatom;

[0439] X is a bond, a heteroatom (Het), -CH2-, *O-CH2-CO or -CO-, for example a bond, a heteroatom (Het), -CH2- or -CO-; and

[0440] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0441] In some embodiments, k is 0 to 2. In some embodiments, k is 0 or 1.

[0442] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, -L2-Y-L2-X-, or -L1-Y-L2-Y-L1-X-.

[0443] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

[0444] in

[0445] Each L1 is (CH2) n , wherein n is 2 to 25;

[0446] Each L2 is CH2CH2(HetCH2CH2) m , wherein m is 1 to 12, and Het is a heteroatom;

[0447] X is a bond, Het, -CH2-, *O-CH2-CO or -CO-, for example a bond, Het, -CH2- or -CO-; and

[0448] Each Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example CONZ, NZCO, SO2NZ or NZSO2, wherein Z is H, C 1-4 Alkyl or protecting group.

[0449] In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

[0450] Each L1 is (CH2) n , wherein n is 2 to 10;

[0451] Each L2 is CH2CH2(OCH2CH2) m , where m is 1 to 5;

[0452] X is a bond, -CO-, *O-CH2-CO or -O-, for example a bond, -CO- or -O-; and

[0453] Y is CONZ or O-CH2-CONZ, such as O-CH2-CONZ, wherein Z is H, C 1-4 Alkyl or protecting group.

[0454] It should be understood that when the linker is connected to GalNAc, the arrangement is GalNAc-L-. It should be understood that when the linker is connected to R1, the arrangement is R1-L-. That is, the directionality of the linker should be read as GalNAc-L1-X-, GalNAc-L1-Y-L1-X-, GalNAc-L1-Y-L2-X-, GalNAc-L2-X-, GalNAc-L2-Y-L1-X-, GalNAc-L2-Y-L2-X-, R1-L1-X-, R1-L1-Y-L1-X-, R1-L1-Y-L2-X-, R1-L2-X-, R1-L2-Y-L1-X-, R1-L2-Y-L2-X-, etc.

[0455] In some embodiments, the linker L is selected from -L1-X-, -L1-Y-L1-X-, and -L1-Y-L2-X-.

[0456] L1

[0457] L1 is (CH2) nThat is, when present, L1 is an alkylene chain. Alternatively, the alkylene chain may be optionally substituted with one or more substituents selected from the group consisting of halogen (e.g., F or Cl), C 1-4 Alkyl, C 1-4 Halogenated alkyl, OC 1-4 Alkyl and OC 1-4 Halogenated alkyl.

[0458] n is from 1 to 25. That is, the alkylene chain contains 1 to 25 carbon atoms. In some embodiments, n is from 2 to 25. That is, the alkylene chain contains 2 to 25 carbon atoms. In some embodiments, n is from 2 to 10. That is, the alkylene chain contains 2 to 10 carbon atoms. In some embodiments, n is 2; that is, the alkylene chain is ethylene. In some embodiments, n is from 3 to 10. In some embodiments, n is from 8 to 10. In some embodiments, n is from 3 to 6.

[0459] In some embodiments, n is 4 to 9. In some embodiments, n is 4; that is, the alkylene chain is butylene. In some embodiments, n is 5; that is, the alkylene chain is pentylene. In some embodiments, n is 6; that is, the alkylene chain is hexylene. In some embodiments, n is 7; that is, the alkylene chain is heptylene. In some embodiments, n is 8; that is, the alkylene chain is octylene. In some embodiments, n is 9; that is, the alkylene chain is nonylene.

[0460] L2

[0461] L2 is CH2CH2 (HetCH2CH2) m ; That is, if L2 is present, it is a (HetCH2CH2) chain, m is 1 to 24, and Het is a heteroatom, so that in addition to the leading ethylene group, the linker also contains 1 to 24 repeating (HetCH2CH2) units. The other valences on the heteroatoms, if present, can be replaced by H or C 1-4 The alkyl group (preferably H) occupies 1. Exemplary heteroatoms include O, S, and N (eg, NH).

[0462] In some embodiments, m is 1 to 12. In some embodiments, Het is an oxygen atom.

[0463] When m is 1 and Het is an oxygen atom, L2 is -CH2CH2OCH2CH2-; when m is 2 and Het is an oxygen atom, L2 is -CH2CH2OCH2CH2OCH2CH2-, and so on. In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1, 2, or 3. In some embodiments, m is 1 or 2. In some embodiments, m is 2.

[0464] X Group

[0465] In some embodiments, X is -CO-. In other words, the linker is connected through an acyl group. In some embodiments, the linker is -L1-CO-, such as pentanoyl (-CO(CH2)4-) or decanoyl (-CO(CH2)9-).

[0466] In some embodiments, X is a bond. In other words, the linker can be connected to the leading CH2- of L1 or L2 as desired. For example, the linker can be -L1-, -L1-Y-L1-, -L1-Y-L2-, -L2-, -L2-Y-L1-, or L2-Y-L2-. In some embodiments, the linker is -L1-; for example, ethylene.

[0467] In some embodiments, X is a heteroatom (Het). The other valences on the heteroatom, if present, can be replaced by H or C 1-4 Alkyl (preferably H) occupies. Exemplary heteroatoms include O, S and N (eg, NH). In some preferred embodiments, X is -O-.

[0468] In some embodiments, X is *-(Het)CH2C≡C-, where * is the site of attachment to W. The remaining valences on the heteroatoms, if present, may be replaced by H or C 1-4 Alkyl (preferably H) occupies. Exemplary heteroatoms include O, S and N (eg, NH). In some preferred embodiments, X is *-OCH2C≡C-.

[0469] In some embodiments, X is *-CH2C≡C-, wherein * is the site of attachment to W. In other words, the linker is attached via a propargyl group. In some embodiments, the linker is -L1-CH2C≡C-.

[0470] In some embodiments, X is -CH2-.

[0471] In some embodiments, X is *O-CH2-CO. It will be appreciated that in the unit of formula L2-X, X being *O-CH2-CO may be suitable because the synthetic route may include oxidation of the terminal alcohol of the PEG chain.

[0472] Y Group

[0473] Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, wherein Z is H, C 1-4 In some embodiments, Y is CONZ or SO2NZ, wherein Z is H, C 1-4 An alkyl group (eg, methyl) or a protecting group.

[0474] Similarly, it will be appreciated that in the unit of formula L2-Y, Y being O-CH2-CONZ or O-CH2SO2NZ may be suitable, as the synthetic route may include oxidation of the terminal alcohol of the PEG chain.

[0475] In some embodiments where more than one Y is present, each Y is the same. In some embodiments, Y is CONZ; in other words, Y provides an amide bond that can be optionally protected. Suitable nitrogen protecting groups are known in the art and include benzyl (Bn). In some embodiments, Z is H, Bn, or C 1-4 alkyl.

[0476] In some embodiments, Y is CONH. That is, the linker is selected from -L1-CONH-L1-X-, -L1-CONH-L2-X-, -L2-CONH-L1-X-, and -L2-CONH-L2-X-; preferably -L1-CONH-L1-X- and -L1-CONH-L2-X-; more preferably -L1-CONH-L2-X-.

[0477] In a preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2OCH2CH2-; that is, the linker is -L1-Y-L2-X-, L1 is present and n is 4, Y is CONH, L2 is present and m is 1, and X is a bond.

[0478] In a preferred embodiment, the linker is -(CH2)5-CONH-(CH2)4-CO-; that is, the linker is -L1-Y-L1-X-, wherein X is -CO-, Y is CONH, two L1 chains are present, and one n is 4 and the other n is 5.

[0479] In a preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2-(OCH2CH2)2-CONH-(CH2)5-; that is, the linker is -L1-Y-L2-Y-L1-X-, wherein X is a bond, each Y is CONH, n of the first L1 is 4, n of the second L1 is 5, and m is 2.

[0480] In a preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-(CH2)C≡C-; that is, the linker is -L1-Y-L2-X-, L1 is present and n is 4, Y is CONH, L2 is present and m is 1, and X is *-O(CH2)C≡C-.

[0481] Some preferred linkers

[0482] In some embodiments, the linker L is -L1-Y-L1-X- (so that k is 1 and both W are L1), wherein X is -CO-, Y is CONH, one n is 4, and the other n is 5. That is, the linker is -(CH2)4-CONH-(CH2)5-CO-. [Linker 1]

[0483] In some embodiments, the linker L is -L1-Y-L2-X- (so k is 1, one W is L1, and the other W is L2), wherein n is 4, Y is CONH, m is 4, Het is O, and X is -CO-. That is, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)4-CO-. [Linker 2]

[0484] In some embodiments, the linker L is -L1-X- (so k is 0 and W is L1), wherein n is 9 and X is -CO-. That is, the linker is -(CH2)9-CO-. [Linker 3]

[0485] In some embodiments, the linker L is -L1-Y-L2-X- (so k is 1, one W is L1, and the other W is L2), wherein n is 4, Y is CONH, m is 1, Het is O, and X is -O-CH2-CO. That is, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-CH2-CO-. [Linker 4]

[0486] In some embodiments, the linker L is -L2-X- (so k is 0, W is L2), wherein m is 3, Het is O, and X is -O-CH2-CO-. That is, the linker

[0487] -CH2CH2(OCH2CH2)3-O-CH2-CO-. [Linker 5]

[0488] In some embodiments, the linker L is -L2-Y-L1-X- (so k is 1, and one W is L2 and the other W is L1), wherein m is 1, Het is O, Y is O-CH2-CONH (ie, Z is H), n is 5, and X is -CO-. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)5-CO-.

[0489] [Linker 6]

[0490] In some embodiments, the linker L is -(L1-Y)2-L1-X- (so k is 2, and all three Ws are L1), one n is 4, another n is 3, the last n is 1, one Y is CONH (ie, Z is H), another Y is NHCO (ie, Z is H), and X is -CH2C≡C-. That is, the linker is -(CH2)4-CONH-(CH2)3-NHCO-CH2-CH2C≡C-. [Linker 7]

[0491] In some embodiments, the linker L is -(L1-Y)2-L1-X- (so k is 2 and all three Ws are L1), one n is 4, another n is 3, the last n is 4, one Y is CONH (ie, Z is H), another Y is NHCO (ie, Z is H), and X is a bond. That is, the linker is -(CH2)4-CONH-(CH2)3-NHCO-(CH2)4-. [Linker 7 - Hydrogenation].

[0492] In some embodiments, the linker L is -L2-Y-L2-X- (so that k is 1 and both W are L2), one m is 1 and the other m is 1, both Het are O, Y is O-CH2-CONH (i.e., Z is H), and X is -OCH2C≡C- (i.e., Het is O). That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)-OCH2C≡C-. [Linker 8].

[0493] In some embodiments, the linker L is -L2-Y-L2-X- (so k is 1, and both W are L2), one m is 1, the other m is 2, both Het are O, Y is O-CH2-CONH (ie, Z is H), and X is -CH2-. That is, the linker is

[0494] -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)2-CH2-. [Linker 8 – Hydrogenation]

[0495] In some embodiments, the linker L is -L2-Y-L1-Y-L1-X- (so k is 2, and one W is L2, and the other two Ws are L1), wherein m is 1, one Y is O-CH2-CONH (ie, Z is H), and one Y is NHCO (ie, Z is H), one n is 3, the other n is 1, and X is -CH2C≡C-. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-CH2-CH2C≡C-.

[0496] [Linker 9]

[0497] In some embodiments, the linker L is -L2-Y-L1-Y-L1-X- (so k is 2, and one W is L2 and the other two W are L1), wherein m is 1, one Y is O-CH2-CONH (ie, Z is H), and one Y is NHCO (ie, Z is H), one n is 3, the other n is 4, and X is a bond. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-(CH2)4-. [Linker 9 - Hydrogenation]

[0498] In some embodiments, the linker L is -L1-Y-L2-X- (so k is 1, and one W is L1 and the other W is L2), wherein n is 4, Y is CONH (ie, Z is H), m is 1, and X is -OCH2C≡C- (ie, Het is O). That is, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-OCH2C≡C-. [Linker 10]

[0499] In some embodiments, the linker L is -L1-Y-L2-X- (so k is 1, and one W is L1 and the other W is L2), wherein n is 4, Y is CONH (ie, Z is H), m is 2, and X is -CH2-. That is, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)2-CH2-. [Linker 10 - Hydrogenation]

[0500] Formula 1

[0501] In some embodiments, the compound is a compound of Formula 1. Suitably, the two linkers are different and can be distinguished as L a and L b , as shown in Formula 1a. In some embodiments, the stereochemistry is as shown in Formula 1b.

[0502]

[0503] Preferably, L a is a linker represented by the formula -L1-X-, optionally, wherein n is 2 and X is an oxygen atom. a It is -(ethylene)O-, and the substituent is R1-(ethylene)O-.

[0504] That is, the compound may be a compound represented by the following formula:

[0505]

[0506] In some alternative embodiments, L a is L2, optionally wherein m is 1 or 2, preferably 1.

[0507] Preferably, L b is a linker represented by the formula -L1-X-, wherein n is 4 and X is -CO-. b It is -(butylene)CO-.

[0508] That is, the compound may be a compound represented by the following formula:

[0509]

[0510] In some embodiments, the present invention provides a monomer represented by the following formula (GalNAc monomer 1):

[0511]

[0512] That is, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; the first linker (L a ) is -L1-X-, wherein n=2 and X is an oxygen atom; the second linker (L b ) is -L1-X-, wherein X is -CO- and n is 4; GalNAc is protected by acetyl (ie, P is Ac).

[0513] Formula 2

[0514] In some embodiments, the compound is a compound represented by Formula 2.

[0515]

[0516] Preferably, each R3 is independently methyl, methoxy or H. More preferably, R3 is H.

[0517] In some embodiments, the compound is a compound of Formula 2a or 2b:

[0518]

[0519] Preferably, the linker is -L1-X-, wherein X is -CO-. That is, the linker is connected to the amine of the core motif via an amide bond. In some embodiments, n is 8 to 10. In some embodiments, n is 9; that is, the linker is decanoyl.

[0520] In some embodiments, the linker is -L1-Y-L1-X-, wherein X is -CO- and Y is CONZ. That is, the linker is connected to the amine of the core motif via an amide bond. In some embodiments, the L1 group can have different n values. In some embodiments, each n is independently 4 or 5. In some embodiments, the linker is -(CH2)5-Y-(CH2)4-X-, for example, -(CH2)5-CONH-(CH2)4-CO-.

[0521] In some embodiments, the present invention provides a monomer (GalNAc monomer 2) represented by the following formula:

[0522]

[0523] That is, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; both R3 are H; the linker is -L1-X-, wherein X is -CO-, n is 9, and GalNAc is protected by acetyl (ie, P is Ac).

[0524] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH, DMTr); both R3 are H; the linker L is -L1-Y-L1-X- (so k is 1 and both W are L1), wherein X is -CO-, Y is CONH, one n is 4 and the other n is 5 (i.e., the linker is -(CH2)4-CONH-(CH2)5-CO-) [Linker 1], and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0525] In some embodiments, the present invention provides a monomer represented by the following formula (GalNAc monomer 4):

[0526]

[0527] That is, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; both R3 are H; the linker is -L1-Y-L1-X-, wherein X is -CO-, Y is CONH, one n is 4 and the other n is 5 (that is, the linker is -(CH2)5-CONH-(CH2)4-CO-), and GalNAc is protected by acetyl (that is, P is Ac).

[0528] In some embodiments, the present invention provides a compound shown in Formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L1-Y-L2-X- (so that k is 1, one W is L1, and the other W is L2), wherein n is 4, Y is CONH, m is 4, Het is O, and X is -CO- (i.e., the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)4-CO-[Linker 2]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0529] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L1-X- (so k is 0 and W is L1), wherein n is 9, X is -CO- (ie, the linker is -(CH2)9-CO-[Linker 3]), and GalNAc is protected by an acetyl group (ie, P is Ac).

[0530] In some embodiments, the present invention provides a compound shown in Formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L1-Y-L2-X- (so that k is 1, one W is L1, and the other W is L2), wherein n is 4, Y is CONH, m is 1, Het is O, and X is -O-CH2-CO (i.e., the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-CH2-CO-[Linker 4]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0531] In some embodiments, the present invention provides a compound shown in Formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L2-X- (so k is 0 and W is L2), wherein m is 3, Het is O, X is O-CH2-CO (i.e., the linker is -CH2CH2(OCH2CH2)3-O-CH2-CO-[Linker 5]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0532] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L2-Y-L1-X- (so k is 1, and one W is L2 and the other W is L1), wherein m is 1, Het is O, Y is O-CH2-CONH (i.e., Z is H), n is 5, X is -CO- (i.e., the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)5-CO-[Linker 6]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0533] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -(L1-Y)2-L1-X- (so k is 2 and all three Ws are L1), one n is 4, another n is 3, the last n is 1, one Y is CONH (ie, Z is H), another Y is NHCO (ie, Z is H), X is -CH2C≡C- (ie, the linker is -(CH2)4-CONH-(CH2)3-NHCO-CH2-CH2C≡C- [Linker 7]), and GalNAc is protected by an acetyl group (ie, P is Ac).

[0534] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -(L1-Y)2-L1-X- (so k is 2 and all three Ws are L1), one n is 4, another n is 3, the last n is 4, one Y is CONH (ie, Z is H), another Y is NHCO (ie, Z is H), and X is a bond (ie, the linker is

[0535] -(CH2)4-CONH-(CH2)3-NHCO-(CH2)4-[Linker 7-hydrogenated]), and GalNAc is protected by an acetyl group (ie, P is Ac).

[0536] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L2-Y-L2-X- (so k is 1, and both W are L2), one m is 1, the other m is 1, both Het are O, Y is O-CH2-CONH (ie, Z is H), and X is -OCH2C≡C- (ie, Het is O) (ie, the linker is

[0537] -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)-OCH2C≡C-[Linker 8]), and GalNAc is protected by acetyl group (ie, P is Ac).

[0538] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L2-Y-L2-X- (so k is 1, and both W are L2), one m is 1, the other m is 2, both Het are O, Y is O-CH2-CONH (i.e., Z is H), X is -CH2- (i.e., the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)2-CH2-[linker 8-hydrogenated]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0539] In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected via a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L2-Y-L1-Y-L1-X- (so that k is 2, one W is L2, and the other two W are L1), wherein m is 1, one Y is O-CH2-CONH (ie, Z is H), one Y is NHCO (ie, Z is H), one n is 3, another n is 1, X is -CH2C≡C- (ie, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-CH2-CH2C≡C-[Linker 9]), and GalNAc is protected by an acetyl group (ie, P is Ac). In some embodiments, the present invention provides a compound of formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond;

[0540] R2 is OH or DMTr; both R3 are H; the linker L is -L2-Y-L1-Y-L1-X- (so k is 2, and one W is L2 and the other two W are L1), wherein m is 1, one Y is O-CH2-CONH (i.e., Z is H), one Y is NHCO (i.e., Z is H), one n is 3 and the other n is 4, X is a bond (i.e., the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-(CH2)4-[Linker 9-hydrogenated]), and GalNAc is protected by acetyl (i.e., P is Ac).

[0541] In some embodiments, the present invention provides a compound shown in Formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L1-Y-L2-X- (so k is 1, one W is L1 and the other W is L2), wherein n is 4, Y is CONH (i.e., Z is H), m is 1, X is -OCH2C≡C- (i.e., Het is O) (i.e., the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-OCH2C≡C- [Linker 10]), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0542] In some embodiments, the present invention provides a compound shown in Formula 2, wherein R1 is a phosphoramidite bond connected to an oligonucleotide, or a polystyrene bead or a long-chain alkylamine controlled pore glass bead (LCAA-CPG) connected by a succinic acid, diglycolic acid or hydroquinone-O,O'diacetic acid bond; R2 is OH or DMTr; both R3 are H; the linker L is -L1-Y-L2-X- (so k is 1, one W is L1, and the other W is L2), wherein n is 4, Y is CONH (i.e., Z is H), m is 2, X is -CH2- (i.e., the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)2-CH2-), and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0543] Formula 3

[0544] In some embodiments, the compound is a compound represented by Formula 3.

[0545]

[0546] Preferably, R4 is H, OH, OC 1-4 More preferably, R4 is H.

[0547] Preferably, X is *-OCH2C≡C-, wherein * represents the connection site with W. In some embodiments, the linker is -L1-Y-L2-X-, for example -L1-Y-L2-OCH2C≡C- (i.e., X is *-OCH2C≡C-). In some embodiments, Y is CONH. In some embodiments, n is 4, Y is CONH, and m is 1.

[0548] In some embodiments, the compound is a compound of formula 3a

[0549]

[0550] Preferably, X is an oxygen atom. In some embodiments, the linker is -L1-Y-L2-X-, for example -L1-Y-L2-O- (i.e., X is an oxygen atom). In some embodiments, Y is CONH. In some embodiments, n is 4, Y is CONH, and m is 1.

[0551] In some embodiments, the linker is -(CH2)4-CONH-(CH2)5-CO-[Linker 1]

[0552] In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)4-CO-[Linker 2]

[0553] In some embodiments, the linker is -(CH2)9-CO-[Linker 3]

[0554] In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-CH2-CO-[Linker 4]

[0555] In some embodiments, the linker is -CH2CH2(OCH2CH2)3-O-CH2-CO-[Linker 5]

[0556] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)5-CO-[Linker 6]

[0557] In some embodiments, the linker is -(CH2)4-CONH-(CH2)3-NHCO-CH2-CH2C≡C-[Linker 7]

[0558] In some embodiments, the linker is -(CH2)4-CONH-(CH2)3-NHCO-(CH2)4-[Linker 7-Hydrogenated]

[0559] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)-OCH2C≡C-[Linker 8]

[0560] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)2-CH2-[Linker 8-Hydrogenated]

[0561] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-CH2-CH2C≡C-[Linker 9]

[0562] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-(CH2)4-[Linker 9-Hydrogenated]

[0563] In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-OCH2C≡C-[Linker 10]

[0564] In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)2-CH2-[Linker 10-Hydrogenated]

[0565] In some embodiments, the present invention provides a monomer (GalNAc monomer 3) represented by the following formula:

[0566]

[0567] That is, in Formula 3, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; R4 is H; the linker L is -L1-Y-L2-X-, wherein n is 4, Y is CONH, m is 1, X is *-OCH2C≡C-, and GalNAc is protected by an acetyl group (i.e., P is Ac). That is, in Formula 3a, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; R4 is H; the linker L is -L1-Y-L2-X-, wherein n is 4, Y is CONH, m is 1, X is an oxygen atom, and GalNAc is protected by an acetyl group (i.e., P is Ac).

[0568] In some embodiments, the present invention provides a monomer represented by the following formula (GalNAc monomer 5):

[0569]

[0570] That is, in Formula 3, R1 is O-PN(iPr)2OCH2CH2CN; R2 is DMTr; R4 is H; the linker L is -L1-Y-L2-X-, wherein n is 4, Y is CONH, m is 2, X is -CH2-, and GalNAc is protected by an acetyl group (i.e., P is Ac). That is, L2 is -CH2CH2OCH2CH2OCH2CH2-.

[0571] The term "monomer residue" refers to a monomer unit attached at one or more positions in an oligonucleotide chain. In the structures below, wavy lines indicate attachment sites within an oligonucleotide chain or the termini of an oligonucleotide chain, as applicable.

[0572] Thus, the inhibitory nucleic acids of the present disclosure may comprise at least one GalNAc monomer residue as described herein.

[0573] Suitably, the inhibitory nucleic acid (oligonucleotide) of the present disclosure can include adjacent GalNAc monomer residues as described herein. In some embodiments, the inhibitory nucleic acid preferably includes two adjacent monomer residues, more preferably three adjacent monomer residues. It should be understood that the monomer residue can be located at any site in the nucleotide chain. In some preferred embodiments, the monomer residue is located at or near the 3' end of the oligonucleotide. In some embodiments, the monomer residue is located at or near the 5' end of the oligonucleotide.

[0574] In some embodiments, the present disclosure provides oligonucleotides (eg, inhibitory nucleic acids) comprising at least one monomer residue having the formula:

[0575]

[0576] Optionally, three copies of the monomer residue are included; that is, three copies of the monomer shown in formula (A), (B) or (C). Suitably, the copies are continuous. In other words, the oligonucleotide comprises three adjacent monomer residues, and the monomer residues are selected from one of formula (A), (B) or (C). In some embodiments, X is O. In some embodiments, X is S. The oligonucleotides and siRNAs exemplified herein comprise three monomer residues shown in formula (A), wherein X is O, X is S and X is S, respectively. Other oligonucleotides and siRNAs exemplified herein comprise three monomer residues shown in formula (B), wherein X is O, X is S and X is S, respectively. Other oligonucleotides and siRNAs exemplified herein comprise three monomer residues shown in formula (C), wherein X is O, X is S and X is S, respectively. The monomer residue may be located at the 3' end of the oligonucleotide. Alternatively, the monomer residue may be located at the 5' end of the oligonucleotide or at another site in the chain.

[0577] In some embodiments, the inhibitory nucleic acids of the present disclosure are selected from Fig.18A , 18B or those drawn in 18C. The wavy lines represent nucleotide chains. In some embodiments, the inhibitory nucleic acids (e.g., siRNAs) disclosed herein are selected from Fig.19A , 19B or those drawn in 19C. The wavy lines represent RNA strands. That is, the inhibitory nucleic acids of the present disclosure may include Fig.18A , B and C or Fig.19A , B and C show three consecutive monomers.

[0578] In some embodiments, the monomer is selected from:

[0579]

[0580]

[0581] Also, when the monomer contains an acetylenic bond, the bond in the corresponding monomer is completely hydrogenated.

[0582] Conjugates of biomolecules can be produced using "click chemistry", as described in, for example, I and Brechbiel Cancer Biother Radiopharm. (2009) 24 (3): 289-302 and Astakhova et al., Mol Pharm. (2018) 15 (8): 2892-2899, both of which are incorporated herein by reference in their entirety. In some embodiments, conjugation can employ alkyne-azide or thiomaleimide methods. In some embodiments, nucleic acids can be conjugated to portions that facilitate delivery to target cell types or tissues and / or absorption by target cell types or tissues, for example at the 3' and / or 5' ends of one or more chains of nucleic acids.

[0583] The nucleic acid can be conjugated to one or more moieties that facilitate delivery to and / or uptake by a target cell type or tissue via a linker. In some embodiments, the linker can be or comprise a nucleotide sequence. The nucleotide sequence of the linker can comprise one or more modified nucleotides described herein.

[0584] Therapeutic and preventive applications

[0585] The inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein can be used in therapeutic and prophylactic methods.

[0586] The present disclosure provides inhibitory nucleic acids, nucleic acids, expression vectors or compositions described herein for use in medical treatment or prevention methods. Also provided is the use of inhibitory nucleic acids, nucleic acids, expression vectors or compositions described herein in the preparation of a medicament for treating or preventing a disease or condition. Also provided is a method for treating or preventing a disease or condition, comprising administering to a subject a therapeutically or preventively effective amount of an inhibitory nucleic acid, nucleic acid, expression vector or composition described herein.

[0587] The terms "disorder," "disease," and "condition" are used interchangeably to refer to a pathological problem with a body part, organ, or system characterized by an identifiable group of signs or symptoms.

[0588] The therapeutic or preventive intervention of the present disclosure can effectively reduce the development or progress of disease / illness, alleviate the symptoms of disease / illness or reduce the pathology of disease / illness. Intervention can effectively prevent the progress of disease / illness, such as preventing the deterioration of disease / illness or slowing down the development speed of disease / illness. In some embodiments, intervention can lead to the improvement of disease / illness, such as reducing the symptoms of disease / illness or reducing some other related factors of the severity / activity of disease / illness. In some embodiments, intervention can prevent disease / illness from developing to late stage (such as more serious stage or chronic stage).

[0589] Terms such as "develop," "developing," and "development" of a disease, as used herein, refer to both the onset of the disease and the progression, worsening, or worsening of the disease state / events associated therewith.

[0590] It should be understood that the articles of manufacture of the present disclosure (inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein) can be used to treat / prevent any disease / disorder that can benefit from reducing the gene and / or protein expression level of ITFG1, and / or reducing the functional level of ITFG1.

[0591] According to the present disclosure, the disease / disorder to be treated / prevented may be a disease / disorder in which ITFG1 gene and / or protein expression and / or ITFG1 function is associated with the pathology. For example, the disease / disorder may be a disease / disorder in which increased expression of ITFG1 gene and / or protein and / or increased level of ITFG1 function is associated with the pathology of the disease / disorder.

[0592] The disease / disorder may be characterized by an increased expression level of the ITFG1 gene and / or protein, or an increased level of ITFG1 function (e.g., compared to the expression level or associated function in the absence of the disease / disorder). The disease / disorder may be a disease / disorder in which an increased expression level of the ITFG1 gene and / or protein and / or an increased level of ITFG1 function is positively correlated with the onset, development or progression of the disease / disorder. The disease / disorder may be a disease / disorder in which an increased expression level of the ITFG1 gene and / or protein and / or an increased level of ITFG1 function is positively correlated with the severity of one or more symptoms of the disease / disorder. The disease / disorder may be a disease / disorder in which an increased expression level of the ITFG1 gene and / or protein and / or an increased level of ITFG1 function is a risk factor for the onset, development or progression of the disease / disorder.

[0593] According to the preceding paragraphs, the increase in the expression level of the ITFG1 gene and / or protein, and / or the increase in the level of ITFG1 function, may be in cells, tissues and / or organs that exhibit one or more symptoms of the disease / disorder. In some embodiments, the increase in the expression level of the ITFG1 gene and / or protein, and / or the increase in the level of ITFG1 function, may be in liver cells (e.g., hepatocytes), liver tissue and / or liver.

[0594] According to the present disclosure, therapeutic or preventive interventions can achieve reductions in ITFG1 gene and / or protein expression levels, and / or reductions in ITFG1 functional levels (i.e., in the subject being treated). In some embodiments, therapeutic / preventive interventions can achieve reductions in ITFG1 gene and / or protein expression levels, and / or reductions in ITFG1 functional levels in cells, tissues, and / or organs that exhibit one or more symptoms of the disease / disorder. In some embodiments, therapeutic / preventive interventions can achieve reductions in ITFG1 gene and / or protein expression levels, and / or reductions in ITFG1 functional levels in liver cells (e.g., hepatocytes), liver tissues, and / or liver.

[0595] The products of the present disclosure can be used for therapeutic / preventive intervention to treat or prevent fibrosis. WO 2022 / 025827A1 (incorporated herein by reference as above) shows that RNAi-mediated knockdown of ITFG1 expression attenuates the development of fibrosis, particularly liver fibrosis in a NAFLD mouse model (see Example 2 of WO 2022 / 025827A1).

[0596] Thus, in some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is fibrosis, or a disease / disorder characterized by fibrosis. As used herein, a disease / disorder "characterized by fibrosis" is a disease in which fibrosis is a symptom of the disease / disorder.

[0597] Fibrosis can be initiated by pathological conditions, such as conditions, infections or disease states that lead to the production of profibrotic factors (e.g., TGFβ1). Fibrosis can be initiated by physical injury / irritation, chemical injury / irritation or environmental injury / irritation. Physical injury / irritation can occur during surgery, such as iatrogenic causes. Chemical injury / irritation can include drug-induced fibrosis, such as chronic administration of drugs such as bleomycin, cyclophosphamide, amiodarone, procainamide, penicillamine, gold and nitrofurantoin (Daba et al., Saudi Med J. (2004) 25 (6): 700-706). Environmental injury / irritation can include contact with asbestos fibers or silica.

[0598] Fibrosis can be in any tissue / organ of the body. In some embodiments, fibrosis is in the lungs (e.g., bronchioles, alveoli), airways (e.g., nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi), heart, kidneys, liver, skeletal muscle, blood vessels, eyes, skin, pancreas, intestines, small intestine, large intestine, colon, joints, brain, or bone marrow. Fibrosis can also occur in multiple tissues / organs simultaneously.

[0599] In some embodiments, fibrosis may be in an organ or tissue of the respiratory system, such as the lungs (e.g., bronchioles, alveoli) or airways (e.g., nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, fibrosis may be in an organ or tissue of the cardiovascular system, such as the heart or blood vessels. In some embodiments, fibrosis may be in an organ or tissue of the gastrointestinal system, such as the liver, intestines, small intestine, large intestine, colon, or pancreas. In some embodiments, fibrosis may be in the eyes. In some embodiments, fibrosis may be in the skin. In some embodiments, fibrosis may be in an organ or tissue of the nervous system, such as the brain. In some embodiments, fibrosis may be in the bone marrow. In some embodiments, fibrosis may be in the joints. In some embodiments, fibrosis may be in an organ or tissue of the urinary system, such as the kidneys. In some embodiments, fibrosis may be in an organ or tissue of the musculoskeletal system, such as muscle tissue. In some embodiments, fibrosis may be in an organ or tissue of one or more organ systems.

[0600] Diseases and conditions characterized by fibrosis include, but are not limited to:

[0601] Diseases / conditions affecting the liver, such as chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, liver cancer, and hepatocellular carcinoma (HCC);

[0602] Diseases / conditions affecting the respiratory system such as pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, and asthma;

[0603] Diseases / conditions affecting the cardiovascular system such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, and cerebral infarction;

[0604] Diseases / conditions affecting the kidneys, such as tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, and nephritis associated with systemic lupus;

[0605] Diseases / conditions affecting the pancreas, such as pancreatic fibrosis, cystic fibrosis, and chronic pancreatitis;

[0606] Diseases / disorders affecting the nervous system, such as gliosis, Alzheimer’s disease, and multiple sclerosis;

[0607] Diseases / disorders affecting the musculoskeletal system, such as muscular dystrophies, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and fibrotic myopathies;

[0608] Diseases / conditions affecting the gastrointestinal system, such as inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, and primary sclerosing cholangitis (PSC);

[0609] Diseases / conditions that affect the skin, such as scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, and skin keloids;

[0610] Diseases / conditions affecting the eye, such as Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, and subconjunctival fibrosis;

[0611] Diseases / conditions affecting the joints, such as arthrofibrosis, arthritis, and adhesive capsulitis;

[0612] Diseases / disorders affecting multiple tissue / organ systems, including progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD); pre-fibrotic and fibrotic neoplastic diseases, fibrosis induced by chemical or environmental insults (e.g., cancer chemotherapy, pesticides, radiation / cancer radiotherapy);

[0613] Cancers, such as liver, hepatocellular, stomach, esophageal, lung, head and neck, colorectal, pancreatic, cervical, and vulvar cancers;

[0614] Mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, and Peyronie's disease.

[0615] In some embodiments, fibrosis is fibrosis of the liver. In some embodiments, the disease / disorder to be treated is selected from the group consisting of chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, and hepatocellular carcinoma (HCC).

[0616] In aspects and embodiments of the present disclosure, inhibitory nucleic acids of the present disclosure are provided for use in treating or preventing diseases / disorders characterized by fibrosis as described herein. Also provided is the use of inhibitory nucleic acids of the present disclosure in the preparation of a medicament for treating or preventing diseases / disorders characterized by fibrosis as described herein. Also provided is a method for treating or preventing diseases / disorders characterized by fibrosis as described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of an inhibitory nucleic acid of the present disclosure.

[0617] The products of the present disclosure are also used to treat / prevent diseases / disorders that derive therapeutic or preventive benefits from an increase in the number / ratio of cells expressing ITFG1 (e.g., hepatocytes, lung cells, myoblasts) and / or an increase in cell proliferation / population expansion of cells expressing ITFG1 (e.g., hepatocytes, lung cells, myoblasts). WO 2022 / 025827 A1 (incorporated herein by reference as above) shows that RNAi-mediated knockdown of ITFG1 expression promotes proliferation of hepatocytes, lung cells, and myoblasts.

[0618] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder characterized by damage and / or death of cells (e.g., hepatocytes) / liver tissue / liver of the liver. Diseases / disorders characterized by damage and / or death of cells (e.g., hepatocytes) / liver tissue / liver of the liver include chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), blood Trematode liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, hepatic ischemia-reperfusion injury (IRI; for example, "warm" ischemia-reperfusion (WIR)), radiation-induced liver disease (RILD), drug-induced liver injury (DILI; for example, acetaminophen-induced liver injury), autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, and cholestatic liver disease.

[0619] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a non-liver disease / disorder, i.e., it is not related to the liver. Such diseases / disorders may still have the characteristics as described above, for example, characterized by an increase in the expression level of the ITFG1 gene and / or protein, or by an increase in the level of ITFG1 function.

[0620] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder characterized by damage and / or death of respiratory system cells (e.g., lung cells, e.g., lung epithelial cells) / respiratory system tissues (e.g., lung tissues) / respiratory system organs (e.g., lungs). Diseases / disorders characterized by damage and / or death of respiratory system (e.g., lung cells, e.g., lung epithelial cells) / respiratory system tissues (e.g., lung tissues) / respiratory system organs (e.g., lungs) include pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, and asthma.

[0621] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder characterized by damage and / or death of muscle cells / muscle tissue. Diseases / disorders characterized by muscle cells / muscle tissue include muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrotic myopathy, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), myocardial fibrosis, myocarditis, endomyocardial fibrosis, myocardial infarction, and arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0622] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder that obtains therapeutic and / or preventive benefits from the proliferation / survival and / or increase in number / ratio of renal cells (e.g., tubular cells and / or ductal cells). In some embodiments, the disease / disorder is a renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, or acute kidney injury.

[0623] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder that obtains therapeutic and / or preventive benefits from the proliferation / survival and / or increase in number / ratio of glial cells and / or neuronal cells. In some embodiments, the disease / disorder is a neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, multiple sclerosis, frontotemporal dementia, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy or prion disease.

[0624] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder that obtains therapeutic and / or preventive benefit from an increase in the proliferation / survival and / or number / ratio of intestinal epithelial cells and / or enteroendocrine cells of the intestine. In some embodiments, the disease / disorder is inflammatory bowel disease, ulcerative colitis, Crohn's disease, inflammatory bowel syndrome, chronic inflammatory disorders of the gastrointestinal tract, or celiac disease.

[0625] The products disclosed herein can also be used for methods of forming and / or regenerating tissues (e.g., liver tissue, lung tissue, or muscle tissue). The products disclosed herein can be used for methods of healing (i.e., repairing damage) tissues (e.g., liver tissue, lung tissue, or muscle tissue). WO 2022 / 025827 A1 (incorporated herein by reference as above) shows that RNAi-mediated ITFG1 expression knockdown promotes wound healing and liver regeneration in vitro and in vivo by promoting hepatocyte proliferation (Example 2 of WO 2022 / 025827 A1). As described above, WO 2022 / 025827 A1 also shows that RNAi-mediated ITFG1 expression knockdown promotes pneumocyte and myoblast proliferation.

[0626] In certain embodiments, the present disclosure contemplates the use of the preparations of the present disclosure to promote the repair and / or reversal of liver cell / tissue loss. The present disclosure contemplates the use of the preparations of the present disclosure to enhance / improve liver function in subjects with impaired liver function (e.g., due to disease / disorder or other liver damage (e.g., injury, e.g., DILI)).

[0627] The products disclosed herein can be used to promote hepatocyte proliferation to produce functional liver tissue. Therefore, the products disclosed herein provided herein are used to promote hepatocyte proliferation, survival and / or function; promote the growth, maintenance and / or function of liver tissue; promote the regeneration of hepatocytes and / or liver tissue; and / or maintain / improve liver function.

[0628] In addition to other properties described herein, in some embodiments, the articles of manufacture of the present disclosure have one or more of the following properties, for example and optionally after administration to a subject as compared to the subject before administration:

[0629] Reduce / prevent fibrosis;

[0630] Reduce / prevent steatosis;

[0631] Reduce / prevent liver cirrhosis;

[0632] Reduce / prevent inflammation;

[0633] Reduce / prevent NASH;

[0634] Delaying the onset or progression of liver failure;

[0635] Reducing fibrosis scores, such as measured by the Fibrosis-4 (FIB-4) index of liver fibrosis;

[0636] Reduction in steatosis scores, such as measured by the Steatosis-Associated Fibrosis Estimator (SAFE) score or the CAP score;

[0637] Reduction / prevention of hepatic stellate cell activity, e.g., as measured by Acta2 mRNA expression;

[0638] Increase / promote liver function, e.g., as measured by increased albumin expression;

[0639] Reduction / prevention of circulating liver marker levels such as ALT, AST and / or bilirubin; and / or

[0640] Reduction / prevention of hepatotoxicity, for example as measured by reduced ALT, AST and / or bilirubin levels.

[0641] The administration of the preparations disclosed herein is preferably a "therapeutically effective" or "prophylactically effective" amount sufficient to show a therapeutic or prophylactic benefit to the subject. The amount actually administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease / disorder and the specific preparation administered. Treatment prescriptions, such as dosage decisions, etc., are the responsibility of general practitioners and other physicians, and typically take into account the disease / disorder to be treated, the condition of the individual subject, the delivery site, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's "Pharmacy Science and Practice" (Edited by A. Adejare), 23rd edition (2020), Academic Press.

[0642] The administration of the products of the present disclosure can be topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal. The administration can be injection or infusion.

[0643] In some aspects and embodiments of the present disclosure, the inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein can be administered to the liver, for example, to one or more hepatocytes. In some cases, the inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein can be administered subcutaneously or orally to the blood (i.e., intravenously / intra-arterially).

[0644] In some aspects and embodiments of the present disclosure, the products of the present disclosure can be targeted for delivery, that is, relative to other parts of the body, the concentration of the relevant agent in certain parts of the subject's body is increased. In some embodiments, the method includes intravenous, intra-arterial, intramuscular or subcutaneous administration, wherein the relevant products are formulated into a targeted drug delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendritic polymers, antibodies, aptamers, nanotubes or micro-silica rods. Such systems can include magnetic elements to guide the agent to the desired organ or tissue. Suitable nanocarriers and delivery systems are obvious to those skilled in the art.

[0645] In some cases, the related agents are formulated for targeted delivery to specific cells, tissues and / or organs. In some cases, the related agents are formulated for targeted delivery to liver cells (e.g., hepatocytes), liver tissue and / or liver.

[0646] The specific mode and / or site of administration can be selected according to the site where the gene and / or protein expression of ITFG1 needs to be reduced (eg, liver cells (eg, hepatocytes), liver tissue and / or liver).

[0647] In some embodiments, the therapeutic or preventive interventions of the present disclosure may further comprise the administration of another agent to treat / prevent the disease / disorder.

[0648] The administration of the product disclosed herein can be based on the disease to be treated, alone or in combination with other treatments, administered simultaneously or sequentially. Simultaneous administration refers to administration together with another therapeutic agent, for example, as a pharmaceutical composition (combined preparation) comprising two medicaments, or administered immediately after each other, and optionally by the same route of administration (for example, administered to the same tissue, artery, vein or other blood vessel). Sequential administration refers to administration of one medicament, and then another medicament is administered separately after a given time interval. It is not necessary to administer two medicaments by the same route, although this is the case in certain embodiments. The time interval can be any time interval.

[0649] The articles of manufacture of the present disclosure may provide multiple doses. One or more doses or each dose may be administered simultaneously or sequentially with another therapeutic agent.

[0650] Multiple doses may be separated by a predetermined time interval, which may be selected to be 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 31 days, or one of 1, 2, 3, 4, 5, or 6 months. For example, a dose may be administered once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0651] The articles disclosed herein can be formulated into a sustained-release delivery system to release the inhibitory nucleic acid, nucleic acid, expression vector or composition at a predetermined rate. The sustained-release delivery system can maintain a constant drug / therapeutic / prophylactic concentration over a specified period of time. In some embodiments, the inhibitory nucleic acid, nucleic acid, expression vector or composition described herein is formulated into a liposome, a gel, an implant, a device or a drug-polymer conjugate (e.g., a hydrogel).

[0652] According to various aspects of the present invention, methods are provided for or including (e.g., in the context of treatment / prevention of diseases / disorders described herein) reducing gene and / or protein expression of ITFG1 and / or increasing cell proliferation / population expansion of cells (e.g., hepatocytes) expressing ITFG1. Also provided are agents of the present disclosure for use in such methods, and uses of agents of the present disclosure in the preparation of compositions (e.g., drugs) for use in such methods. It should be understood that the methods generally include administering an agent according to the present disclosure to a subject.

[0653] Similarly, following therapeutic or prophylactic intervention according to the present disclosure, a decrease in ITFG1 gene and / or protein expression, and / or an increase in cell proliferation / population expansion of cells expressing ITFG1 (e.g., hepatocytes) may be observed in a subject (e.g., compared to levels prior to intervention).

[0654] In some embodiments, the therapeutic / preventive interventions of the present disclosure may be described as being "associated with" one or more of the effects described in the preceding paragraph. Those skilled in the art can readily assess such properties using techniques routinely practiced in the art.

[0655] Subjects

[0656] The subject of the various aspects of the invention may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use).

[0657] The subject to which the disclosed article is to be administered (e.g., pursuant to a therapeutic or preventive intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient.

[0658] The subject may be suffering from (e.g., may have been diagnosed with) a disease or condition described herein, may be suspected of suffering from such a disease / condition, or may be at risk of developing / infecting such a disease / condition. In embodiments of the present disclosure, a subject may be selected for treatment based on a method based on the characterization of one or more markers of such a disease / condition.

[0659] Reagent test kit

[0660] In some aspects of the present disclosure, a partial kit is provided. In some embodiments, the kit can have at least one container containing a predetermined amount of an inhibitory nucleic acid, nucleic acid, expression vector, cell, or composition described herein.

[0661] In some embodiments, the kits can include materials for producing an inhibitory nucleic acid, nucleic acid, expression vector, cell, or composition described herein.

[0662] The kit may provide an inhibitory nucleic acid, nucleic acid, expression vector, cell or composition and instructions for administration to a patient to treat a particular disease / disorder.

[0663] In some embodiments, the kit may also include at least one container containing a predetermined amount of another therapeutic agent (e.g., as described herein). In such embodiments, the kit may also include a second drug or pharmaceutical composition so that the two drugs or pharmaceutical compositions can be administered simultaneously or separately to provide a combined treatment for a particular disease or condition.

[0664] The kits of the present disclosure may include instructions for use, such as in the form of an instruction booklet or leaflet. The instructions may include protocols for implementing any one or more of the methods described herein.

[0665] Sequence identity

[0666] Pairwise and multiple sequence alignments for determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega ( J. 2005, Bioinformatics 21, 951-960), T-coffee (Noredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780) software. When using such software, for example, for gap penalty and extension penalty, the default parameters are preferably used.

[0667] sequence

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679] In the sequences in the table above, G = guanosine-3'-phosphate, C = cytidine-3'-phosphate, U = uridine-3'-phosphate, T = thymidine-3'-phosphate, A = adenosine-3'-phosphate, mU = 2'-O-methyluridine-3'-phosphate, mA = 2'-O-methyladenosine-3'-phosphate, mG = 2'-O-methylguanosine-3'-phosphate, mC = 2'-O- methylcytidine-3'-phosphate, smU = 2'-O-methyluridine-3'-phosphorothioate, smA = 2'-O-methyladenosine-3'-phosphorothioate, smG = 2'-O-methylguanosine-3'-phosphorothioate, smC = 2'-O-methylcytidine-3'-phosphorothioate, fU = 2'-fluorouridine-3'-phosphate, fA = 2'-fluoroadenosine-3'-phosphate , fG=2'-fluoroguanosine-3'-phosphate, fC=2'-fluorocytidine-3'-phosphate, sfC=2'-fluorocytidine-3'-phosphorothioate, sfG=2'-fluoroguanosine-3'-phosphorothioate, sfA=2'-fluoroadenosine-3'-phosphorothioate, sfU=2'-fluorouridine-3'-phosphorothioate; ps=phosphorothioate bond, triGal=triantennary GalNAc moiety; ptGal=monomer of formula 3 / 3a, for example, "GalNAc monomer 3"; tGal=monomer of formula 3 / 3a, for example, "GalNAc monomer 5"; gGal=monomer of formula 2 / 2a / 2b, for example, "GalNAc monomer 4"; mGal=monomer of formula 1 / 1a / 1b / 1c, for example, "GalNAc monomer 1".

[0680] The present invention includes any combination of described aspects and preferred features unless such a combination is expressly impermissible or explicitly avoided.

[0681] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0682] Aspects and embodiments of the present disclosure will now be described by way of example with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0683] Throughout this specification (including the claims that follow), unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0684] It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by use of the preposition "about", it will be understood that the particular value forms another embodiment.

[0685] When a nucleic acid sequence is disclosed herein, its reverse complement is also expressly contemplated.

[0686] The methods described herein can be performed in vitro or in vivo. In some embodiments, the methods described herein are performed in vitro. The term "in vitro" is intended to encompass experiments in cultured cells, while the term "in vivo" is intended to encompass experiments in intact multicellular organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0688] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings.

[0689] Figure 1 . A bar graph showing knockdown of ITFG1 gene expression in HuH7 cells by different dsiRNAs. As described in Example 2, HuH7 cells were transfected with the indicated dsiRNAs, and the gene expression of ITFG1 was analyzed by qPCR.

[0690] Figure 2 . Bar graph showing knockdown of ITFG1 gene expression in HuH7 cells by indicated siRNA and dsiRNA molecules. As described in Example 4, HuH7 cells were transfected with indicated siRNAs, and ITFG1 gene expression was analyzed by qPCR.

[0691] Figure 3 . Bar graph showing knockdown of ITFG1 gene expression in HuH7 cells by indicated siRNA and dsiRNA molecules. As described in Example 4, HuH7 cells were transfected with indicated siRNAs, and ITFG1 gene expression was analyzed by qPCR.

[0692] Figure 4 . Bar graph showing knockdown of ITFG1 gene expression in HuH7 cells by indicated siRNA and dsiRNA molecules. As described in Example 4, HuH7 cells were transfected with indicated siRNAs, and ITFG1 gene expression was analyzed by qPCR.

[0693] Figure 5Western blot photographs showing that the expression of ITFG1 at the protein level was knocked down. As described in Example 6, HuH7 cells were transfected with the indicated siRNA at the indicated concentrations and analyzed by Western blot using an anti-ITFG1 antibody to determine the expression level at the protein level.

[0694] Fig. 6A and 6B . Bar graph showing the effect of knocking down ITFG1 expression by the indicated siRNAs on HuH7 cell proliferation. As described in Example 7, HuH7 cells were transfected with the indicated dsiRNAs and analyzed by (6A) CellTiter-Glo luminescent cell viability assay or (6B) CCK8 assay to determine the effect on cell proliferation.

[0695] Figure 7 . A graph showing knockdown of Itfg1 expression in mice. As described in Example 10, mice were administered a single dose of 2 mg / kg of the indicated siRNA, and the expression level of Itfg1 was analyzed in samples obtained after the indicated days.

[0696] Figure 8 . A graph showing knockdown of Itfg1 expression in mice. As described in Example 10, a single dose of 2 mg / kg, 6 mg / kg or 10 mg / kg of the indicated siRNA was administered to mice, followed by analysis of Itfg1 expression levels.

[0697] Fig. 9A and 9B . A diagram showing the results of the evaluation of liver toxicity after administration of the specified siRNA to mice. As described in Example 11, a single dose of 10 mg / kg of the specified siRNA was administered to mice, and the serum levels of (9A) aspartate aminotransferase (AST) and (9B) alanine aminotransferase (ALT) were subsequently analyzed. The threshold of toxicity is indicated.

[0698] Fig.10 . A graph showing the results of the evaluation of Itfg1 knockdown over time in mice following administration of a single 6 mg / kg body weight dose of CG_200277 as described in Example 12.

[0699] Fig.11 . A graph showing knockdown of Itfg1 expression in mice after administration of different doses of CG_200277 and CG_200280 as described in Example 12.

[0700] Fig. 12A and 12B. A graph showing the results of the evaluation of hepatotoxicity after multiple doses of CG_200277 were administered to mice. As described in Example 12, multiple doses of 20 mg / kg of CG_200277 were administered to mice, and serum levels of (12A) aspartate aminotransferase (AST) and (12B) alanine aminotransferase (ALT) were subsequently analyzed. Circles = PBS, squares = CG_200277.

[0701] Fig.13A and 13B Figure (13A) shows the results of Ki67 expressing (Ki67+) cells 42 hours after partial hepatectomy in mice treated with CG_200277 or PBS. The livers were immunostained and analyzed for the proportion of Ki67+ cells as described in Example 15. Figure (13B) shows the liver weight of mice treated with CG_200280 or PBS as absolute weight or ratio to the corresponding mouse body weight.

[0702] Figures 14A to 14I . As described in Example 16.2, a graph showing various in vivo results of WFD-treated mice at baseline and after an additional 12 weeks of CG_200277 or PBS treatment following monthly administration of 6 mg / kg body weight doses of CG_200277 or PBS. (14A) Knockdown of Itfg1 mRNA expression, (14B) Knockdown of ITFG1 protein expression, (14C) quantification of ITFG1 protein, (14D) fibrosis score, (14E) steatosis score, (14F) expression of Ki67, (14G) expression of Acta2 mRNA, (14H) expression of smooth muscle actin, (14I) expression levels of albumin were evaluated as described in Example 16.2.

[0703] Fig.15 Graph showing quantification of Picro Sirius Red stained areas in livers of mice fed a CDHF diet and treated with 6 mg / kg body weight of CG_200280 or PBS for an additional 4 weeks. The livers were processed as described in Example 16.3.

[0704] Figures 16A to 16C.Graphs showing various in vivo results for mice that underwent bile duct ligation (BDL) surgery as described in Example 16.4. (16A) Graph showing body weight of mice that received PBS or a single 6 mg / kg body weight dose of CG_200277 for up to 13 days after bile duct ligation surgery. Circles = PBS, squares = CG_200277. (16B) Graph showing serum correlates of hepatotoxicity for PBS and CG_200277 treated groups (aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin). (16C) Graph showing liver fibrosis scores for mice treated with PBS and CG_200277.

[0705] Figures 17A to 17D Graphs showing in vivo results for mice receiving 6 and 12 repeated monthly injections of 6 mg / kg body weight of CG-200277 or PBS as described in Example 17. (17A) Graph showing the correlation of hepatotoxic AST, ALT, and albumin in the serum of mice treated for 6 months. (17B) Graph showing wet liver weight and liver: body weight ratio of mice treated for 6 months. (17C) Graph showing the correlation of hepatotoxic ALT and AST in the serum of mice treated for 12 months. (17D) Graph showing wet liver weight and liver: body weight ratio of mice treated for 12 months.

[0706] Figures 18A to 18C . Schematic diagram of inhibitory nucleic acids of the present disclosure conjugated to GalNAc monomers described herein. Wavy lines indicate nucleotide chains.

[0707] Figures 19A to 19C Schematic diagram of a double-stranded inhibitory nucleic acid (eg, siRNA) of the present disclosure conjugated to a GalNAc monomer as described herein. The wavy lines indicate nucleotide chains.

[0708] Figures 20A to 20C Graphs showing knockdown of Itfg1 expression in vivo in rats and cynomolgus monkeys following administration of PBS, 2 mg / kg, or 6 mg / kg of CG_200280 as described in Example 18. (20A) Graph showing relative Iftg1 expression in rat liver harvested 7 days after injection. (20B) Graph showing relative Iftg1 expression in rat kidney harvested 7 days after injection. (20C) Graph showing relative Iftg1 expression in cynomolgus monkey liver, quantified from liver biopsies performed 0, 14, 28, 56, and 84 days after injection.

[0709] Fig.21 . A graph showing the results of the evaluation of hepatotoxicity in rats immediately before and 48 hours after administration of a single dose of CG_200280 (30 mg / kg, 100 mg / kg or 400 mg / kg body weight). Example

[0710] In the following examples, the inventors describe the identification and characterization of siRNA molecules for inhibiting gene and protein expression of ITFG1.

[0711] Example 1: Inhibitory Nucleic Acids for RNAi-Mediated Knockdown of ITFG1 Expression

[0712] siRNAs targeting different regions of the mRNA encoding human ITFG1 were designed using a bioinformatics platform with an algorithmic score that predicts the knockdown potency of siRNAs. The platform was also able to filter out mRNA fragments with a minor allele frequency (MAF) < 0.01, a mismatch rate with cynomolgus monkey of 0, and an off-target score < 0.1 for both human and cynomolgus monkey. The off-target score is a composite score that summarizes the following: a score of "1" when the siRNA binds to the off-target gene with 100% identity, a score of "0.1" when the siRNA binds to the off-target gene with 1 mismatch, and a score of "0.01" when the siRNA binds to the off-target gene with 2 mismatches.

[0713] The mRNA sequences of ITFG1 targeted by siRNA are shown in SEQ ID NOs: 13 to 44. The nucleotide sequences of the antisense nucleic acids (i.e., guide strands) of the siRNAs targeting SEQ ID NOs: 13 to 44 are shown in SEQ ID NOs: 45 to 76. The siRNAs are also converted into 27-mer dsiRNA molecules. The mRNA sequences of ITFG1 targeted by dsiRNA are shown in SEQ ID NOs: 115 to 146. The nucleotide sequences of the antisense nucleic acids of the dsiRNAs targeting SEQ ID NOs: 115 to 146 are shown in SEQ ID NOs: 147 to 178.

[0714] The following inhibitory nucleic acids were generated:

[0715] Table 1: dsiRNA targeting ITFG1 mRNA

[0716]

[0717]

[0718] Table 2: siRNAs containing nucleotide modifications targeting ITFG1 mRNA

[0719]

[0720]

[0721]

[0722]

[0723] Example 2: Evaluation of knockdown of ITFG1 expression using dsiRNA

[0724] The ability of the dsiRNAs shown in Table 1 of Example 1 to inhibit ITFG1 gene expression was evaluated as follows.

[0725] Human hepatocellular carcinoma HuH7 cell line (Cellosaurus accession number: CVCL_0336) cells were maintained in vitro in a humidified incubator at 37°C and 5% CO2, and cultured in DMEM high-glucose medium containing 10% FBS, 1% sodium pyruvate, and 1% PenStrep.

[0726] 5,000 HuH7 cells were seeded per well of a 96-well plate and transfected with different ITFG1-targeting dsiRNAs or non-targeting siRNAs (NT1 to NT5) at a final concentration of 20 nM using the transfection agent DharmaFECT3 (Horizon) according to the manufacturer's instructions (quintuplicate for each condition).

[0727] The transfected cells were maintained in culture as described above. Cells were harvested 3 days after transfection for analysis of the mRNA levels encoding ITFG1.

[0728] The cells were washed with PBS and then processed for Cell-to-Ct analysis using the SingleShot Cell Lysis RT-qPCR Kit (Bio-Rad) according to the manufacturer's instructions to quantify the mRNA levels encoding ITFG1. The primers used for qPCR are described in SEQ ID NOs: 242 and 243. The ITFG1 knockdown level was calculated by 2^-ddct, normalized to the housekeeping gene PPIB, and further normalized to the non-targeting siRNA1 (NT1) control. The fold change of ITFG1 mRNA level was calculated as: fold change = 2 -(样品(ITFG1 Ct-PPIB Ct)-NT1(ITFG1Ct-PPIB Ct) .

[0729] The results are as follows Figure 1 shown.

[0730] Example 3: Evaluation of the functional consequences of knocking down ITFG1 expression using dsiRNA

[0731] The effects of knocking down ITFG1 by dsiRNA shown in Table 1 of Example 1 on cell proliferation were investigated as follows.

[0732] 2,500 HuH7 cells were transfected with dsiRNA or non-targeting siRNA (NT1 or NT2) in Table 1 and maintained in culture medium for 3 days as described in Example 2. Cells were then harvested and viable cells were detected using (i) CellTiter-Glo luminescent cell viability assay (Promega), (ii) CCK8 detection kit (Dojindo) or (iii) Ki67 immunostaining according to the manufacturer's instructions.

[0733] For the CellTiter-Glo luminescent cell viability assay, cells were incubated in lysis buffer and CellTiter-Glo for 20 minutes. The resulting luminescence was then read in a plate reader. For the CCK8 assay, cells were incubated with the CCK8 reagent for 1 hour, and the absorbance at 450 nm was measured using a plate reader to determine the concentration of the colored product. For Ki67 immunostaining, cells were fixed in 4% paraformaldehyde, washed with PBS, permeabilized in PBS containing 0.2% TritonX-100, blocked with 2% BSA, incubated overnight with anti-Ki67 antibody, washed with PBS, incubated with anti-rabbit-AF568 secondary antibody and DAPI (nuclear stain), washed with PBS, and 25 images of Ki67 and DAPI staining were obtained per copy by confocal microscopy. The number of Ki67+ cells and DAPI-stained nuclei was analyzed using ImageJ, and the number of Ki67+ cells was divided by the total number of DAPI-stained nuclei to obtain the ratio of Ki67+ cells. Fold changes in luminescence, absorbance, or proportion of Ki67+ cells were determined relative to the values ​​obtained for cells transfected with a non-targeting siRNA control.

[0734] The results are shown in the table below. Several dsiRNAs were found to upregulate the proliferation rate of Huh7 cells by >10% compared to cells transfected with NT1.

[0735]

[0736]

[0737] Example 4: Evaluation of knockdown of ITFG1 expression using siRNA containing nucleotide modifications

[0738] The effect of knocking down ITFG1 by siRNA containing nucleotide modifications on cell proliferation was investigated as follows.

[0739] HuH7 cells were transfected with CG_200198, CG_200199, CG_200200, CG_200201, CG_200202, CG_200203, CG_200204, CG_200205, CG_200206 (siRNAs having the antisense sequence of SEQ ID NO: 58 and comprising modified nucleotides), CG_200148 (dsiRNAs targeting the same region of ITFG1 mRNA) or non-targeting siRNAs (NT1 or NT2) using RNAiMAX (Life Technologies) and maintained in culture for 3 days as described in Example 2. The fold change in the level of mRNA encoding ITFG1 was determined as described in Example 2.

[0740] The results are as follows Figure 2 It was found that certain siRNAs containing nucleotide modifications were more effective in inhibiting ITFG1 expression than dsiRNAs targeting the same region of ITFG1 mRNA.

[0741] In further experiments, HuH7 cells were transfected with CG_200244, CG_200246, CG_200242, CG_200243, CG_200241, CG_200237, CG_200238, CG_200239, CG_200240, CG_200245 (siRNAs having the antisense sequence of SEQ ID NO: 65 and comprising modified nucleotides), CG_200155 (dsiRNAs targeting the same region of ITFG1 mRNA), CG_200199 or non-targeting siRNA (NT) as described above in Example 4 and maintained in culture for 3 days. The fold change in the level of mRNA encoding ITFG1 was determined as described in Example 2.

[0742] The results are as follows Figure 3 It was found that certain siRNAs containing nucleotide modifications were more effective in inhibiting ITFG1 expression than dsiRNAs targeting the same region of ITFG1 mRNA.

[0743] In further experiments, knockdown of ITFG1 by siRNAs containing nucleotide modifications and passenger strand mismatches was investigated. As described in Example 4 above, HuH7 cells were treated with CG_200211, CG_200221, CG_200231, CG_200209, CG_200219, CG_200229, CG_200212, CG_200222, CG_200232, CG_200214, CG_200224, CG_200234, CG_200252, CG_200262, CG_200272, CG_200254, CG_200264, CG_200274, CG_200256, CG_200266, CG_200276 (i.e., siRNAs having SEQ ID NO:58 antisense sequence, comprising modified nucleotides and also comprising passenger strand mismatched siRNA), CG_200201, CG_200199, CG_200202, CG_200204, CG_200242, CG_200244, CG_200246 (corresponding siRNA comprising modified nucleotides and no passenger strand mismatched) or non-targeting siRNA (NT1) and maintained in culture medium for 3 days. The fold change of mRNA level encoding ITFG1 was determined as described in Example 2.

[0744] The results are as follows Figure 4 It was found that certain siRNAs containing mismatched nucleotides in the passenger strand were more effective in inhibiting ITFG1 expression than the corresponding siRNAs without mismatches in the passenger strand.

[0745] Example 5: Evaluation of IC50 value for knockdown of ITFG1 expression

[0746] CG_200231, CG_200232 and CG_200242 (see Table 2 of Example 1) were evaluated to determine their IC50 for knocking down ITFG1 expression in HuH7 cells and in cells of the mouse hepatocyte cell line AML12 (Cellosaurus Accession No.: CVCL_0140).

[0747] In further experiments, knockdown of ITFG1 by siRNAs containing nucleotide modifications and passenger strand mismatches was evaluated to determine IC50 values. As described in Example 4, HuH7 cells were maintained in culture medium for 3 days with CG_200202, CG_200204, CG_200231, CG_200232 (i.e., siRNAs having the antisense sequence of SEQ ID NO: 58, containing modified nucleotides and also containing passenger strand mismatches), CG_200242 (i.e., siRNAs having the antisense sequence of SEQ ID NO: 58, containing modified nucleotides and no passenger strand mismatches) or non-targeting siRNA (NT1) at concentrations ranging from 1.6 pM to 50 nM. As described in Example 2, the fold change of mRNA levels encoding ITFG1 was determined. Experiments in AML12 cells were performed in exactly the same manner as HuH7 cells, except that the primers used for qPCR were shown in SEQ ID NOs: 244 and 245.

[0748] The results are as follows:

[0749] siRNA molecules IC50 (pM) in Huh7 cells IC50 (pM) in AML12 cells CG_200202 13.6 180.4 CG_200204 21.09 Untested CG_200231 32.35 114 CG_200232 27.31 134.9 CG_200242 98.87 184

[0750] It was found that siRNA could effectively inhibit the expression of ITFG1 in human cells and Itfg1 in mouse cells.

[0751] In further experiments, the IC50 of ITFG1 knockdown by CG_200231, CG_200232 and CG_200242 in HuH7 and AML12 cells was investigated again. Briefly, cells were transfected with siRNA at concentrations ranging from 80 fM to 5 nM and maintained in culture medium for 3 days as described above. Fold changes in the levels of mRNA encoding ITFG1 were determined as described in Example 2.

[0752] The results are as follows:

[0753] siRNA IC50 (pM) in Huh7 IC50(pM) in AML12 CG_200231 2.552 18.18 CG_200232 1.485 27.24 CG_200242 17.8 94.65

[0754] Example 6 Evaluation of Inhibition of ITFG1 Protein Expression

[0755] CG_200231, CG_200232, and CG_200242 (see Table 2 of Example 1) were evaluated to confirm their ability to knock down the expression of ITFG1 at the protein level.

[0756] In brief, as described in Example 4 above, HuH7 cells were transfected with 30nM or 1nM siRNA and maintained in culture medium for 3 days. Cells were then harvested in standard RIPA buffer supplemented with a protease inhibitor cocktail and subsequently sonicated. Protein concentration was determined by BCA assay according to the manufacturer's protocol. Reducing buffer was added to each protein sample, which was then boiled to denature the protein. Protein samples were then separated by SDS-PAGE, transferred to a PVDF membrane, blocked, incubated with an anti-ITFG1 antibody, washed, incubated with a secondary anti-detection antibody, washed, and then developed using an ECL substrate. Western blots were imaged on a ChemiDoc imaging system.

[0757] The results are as follows Figure 5 The evaluated siRNAs were found to knock down the expression of ITFG1 at the protein level in a dose-dependent manner.

[0758] Example 7: Evaluation of the functional consequences of knocking down ITFG1 expression using siRNA containing nucleotide modifications

[0759] The effects of knocking down ITFG1 by CG_200231, CG_200232 and CG_200242 (see Table 2 of Example 1) on cell proliferation were investigated as follows.

[0760] As described in Example 3, HuH7 cells were transfected with CG_200231, CG_200232 and CG_200242 or non-targeting siRNA (NT) using RNAiMAX (Life Technologies) and maintained in culture medium for 3 days. Cells were then harvested and viable cells were detected using CellTiter-Glo luminescent cell viability assay (Promega) or (ii) CCK8 assay kit (Dojindo) as described in Example 3.

[0761] The results are as follows Fig. 6A and 6B are shown and summarized in the following table:

[0762]

[0763] It was found that siRNA can effectively promote cell proliferation.

[0764] Example 8: Evaluation of cytotoxicity

[0765] CG_200202, CG_200231, CG_200232, and CG_200242 were studied to determine their cytotoxicity in human Huh7 cells and mouse AML12 cells.

[0766] Briefly, siRNAs were transfected into Huh7 and HepG2 cells at concentrations between 1 and 200 nM and harvested 3 days post-transfection for CCK8 analysis as described in Example 7. The therapeutic index was calculated by dividing the IC50 by the half maximal cytotoxic concentration (CC50).

[0767] The results are as follows:

[0768]

[0769] CG_200202 without passenger chain mismatches was determined to be cytotoxic to mouse AML12 cells, whereas equivalent molecules containing passenger chain mismatches (CG_200231, CG_200232) were not cytotoxic.

[0770] Example 9: Evaluation of off-target knockdown of ITFG1 expression in vivo

[0771] CG_200202, CG_200231, CG_200232, and CG_200242 were evaluated to determine whether they resulted in off-target knockdown of expression of genes other than ITFG1 in human Huh7 cells, or genes other than Itfg1 in mouse AML12 cells.

[0772] In further experiments, HuH7 or AML12 cells were transfected with CG_200201, CG_200202, CG_200231, CG_200232, CG_200242 as described in Example 4 and maintained in culture medium for 16 to 24 hours. RNA was purified by column purification (Favorgen) and used for library preparation and then sequenced on an Illumina sequencer. Differential expression analysis was performed using the NT sample as a reference. Off-target genes were defined as genes with a differential expression fold change of ≥-2 in the siRNA-treated samples compared to the NT sample.

[0773] The results are as follows:

[0774] siRNA molecules Cell lines #Off-target genes downregulated below the threshold CG_200201 Huh7 14 CG_200231 Huh7 8 CG_200202 Huh7 15 CG_200232 Huh7 11 CG_200242 Huh7 0 CG_200231 AML12 2 CG_200232 AML12 8 CG_200242 AML12 2

[0775] CG_200201 and CG_200202 without passenger strand mismatches were determined to result in significantly more off-target gene knockdown compared to equivalent molecules containing passenger strand mismatches (CG_200231, CG_200232).

[0776] Example 10: Evaluation of ITFG1 expression knockdown in vivo

[0777] The ability of GalNAc-conjugated CG_200277, CG_200278 and CG_200279 to knock down ITGF1 expression in vivo was evaluated.

[0778] On day 0, mice were subcutaneously injected with a single dose of 2 mg / kg body weight of CG_200277, CG_200278, CG_200279 or PBS conjugated to GalNAc. The livers of mice were harvested on days 4, 7, 10 or 14 after injection, placed in Trizol, and column-based RNA extraction was performed according to the manufacturer's protocol (Favorgen). cDNA synthesis was performed using the MaximaRT H-system, and qPCR was performed using SYBERGreen (Merck). The primers used for qPCR are shown in SEQ ID NOs: 244 and 245. The level of Itfg1 knockdown was calculated by 2^-ddct, normalized to the housekeeping gene Ppib, and further normalized to the PBS control. The fold change of Itfg1 mRNA level was calculated as: fold change = 2 -(样品(Itfg1 Ct-Ppib Ct)-PBS(Itfg1 Ct-Ppib Ct) .

[0779] The results are as follows Figure 7 Mice administered a single dose of 2 mg / kg of CG_200277, CG_200278, or CG_200279 conjugated to GalNAc showed significant knockdown of Itfg1 expression that lasted for at least 14 days.

[0780] In further experiments, the in vivo knockdown effect of CG_200277, CG_200278 and CG_200279 on Itfg1 expression was studied after administration of a single dose of siRNA conjugated to GalNAc at a concentration of 2 mg / kg, 6 mg / kg or 10 mg / kg. On day 0, mice were subcutaneously injected with a single dose of CG_200277, CG_200278 or CG_200279 conjugated to GalNAc or PBS. The livers of mice were harvested on day 7 and processed for qPCR as described above.

[0781] The results are as follows Figure 8 As shown. CG_200277, CG_200278 or CG_200279 conjugated with GalNAc showed dose-dependent knockdown of Itfg1 expression in vivo.

[0782] Example 11: In vivo toxicity evaluation

[0783] The in vivo toxicity of GalNAc-conjugated CG_200277, CG_200278, and CG_200279 was evaluated by analyzing serum AST and ALT levels.

[0784] Serum was collected from mice via the mandibular vein 11 days and 4 days before siRNA administration. On day 0, mice were subcutaneously injected with a single dose of 10 mg / kg body weight of CG_200277, CG_200278, CG_200279 conjugated to GalNAc or PBS, and serum was collected on days 2 and 4 after administration. Serum AST and ALT levels were determined using commercial kits (Abcam) according to the manufacturer's protocol.

[0785] The results are as follows Fig. 9A and 9B It was found that administration of CG_200277, CG_200278 or CG_200279 conjugated to GalNAc at a single dose of 10 mg / kg did not result in an increase in AST or ALT levels, and thus no significant toxicity of the molecules was detected.

[0786] Example 12: Further in vivo characterization of siRNA molecules targeting ITFG1

[0787] The duration of knockdown of Itfg1 expression after CG_200277 treatment was investigated. Briefly, mice were injected with a single dose of CG_200277 at 6 mg / kg body weight, and livers were harvested at different time points from 1 to 6 weeks after injection. RNA was extracted from the liver and processed as described in Example 10 for qPCR analysis of Itfg1 mRNA levels.

[0788] The results are as follows Fig.10 A single dose of 6 mg / kg body weight of CG_200277 was found to induce at least -70% knockdown of Itfg1 expression that lasted for at least 6 weeks.

[0789] In further experiments, mice were injected with siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, CG_200279, or CG_200280) at doses ranging from 2 to 10 mg / kg body weight, and livers were harvested at various time points from 1 to 20 weeks after injection. RNA was extracted from the liver and processed as described in Example 10 for qPCR analysis of Itfg1 mRNA levels. It was found that the relevant siRNA induced a knockdown of ≥80% of Itfg1 expression for ≥2 weeks and maintained a knockdown of ≥50% of Itfg1 expression for up to 4 weeks, indicating favorable pharmacodynamic properties that can reduce dosing frequency.

[0790] In further experiments, the inventors investigated the dose of CG_200277 required to achieve 50% (ED50) and 80% (ED80) knockdown of Itfg1 expression 1 week after injection. Briefly, mice were injected with a single dose of CG_200277 or CG_200280 at 0.125, 0.25, 0.5, 1, 2 or 6 mg / kg body weight, and livers were harvested 1 week after injection. RNA was extracted from the liver and processed as described in Example 10 for qPCR analysis of Itfg1 mRNA levels.

[0791] The results are as follows Fig.11 It was found that CG_200277 and CG_200280 were highly potent in inducing knockdown of Itfg1 expression, with ED50 of both compounds being 0.8 mg / kg and ED80 being 3.4 mg / kg or 3.6 mg / kg, respectively.

[0792] In further experiments, the inventors studied the multi-dose toxicity of CG_200277. Briefly, mice were subcutaneously injected with CG_200277 at a dose of 20 mg / kg body weight three times in one week, on days 0, 3, and 7. Serum was collected from mice through the submandibular vein on days 1, 4, 8, 10, and 14, and analyzed for hepatotoxicity associations as described in Example 11. The liver was harvested on day 14 and processed for formalin paraffin embedding (FFPE) fixation, then sectioned and stained with hematoxylin and eosin (H&E) for subsequent pathological evaluation.

[0793] The results are as follows Fig. 12A and 12B As shown, no significant increase in serum ALT or AST levels was detected in the CG_200277-treated mice compared with the PBS-treated mice. Pathologists performed histological analysis of the liver, kidney, and spleen, confirming that no abnormalities were observed in the CG_200277-treated group.

[0794] In further experiments, the inventors studied the acute toxicity of CG_200277. Briefly, mice were subcutaneously injected with CG_200277 at doses up to 300 mg / kg body weight weekly for up to 6 weeks starting from day 0. Serum was collected from mice via the submandibular vein starting 7 days before the first dose and multiple times during the injection period and 4 weeks after the last injection. The serum was analyzed for hepatotoxicity relevance as described in Example 11. No significant increase in serum ALT or AST levels was detected in mice treated with CG_200277 compared to PBS-treated control mice.

[0795] Example 13: Evaluation of in vitro stability of siRNA targeting ITFG1 comprising nucleotide modifications

[0796] To evaluate the stability of siRNA containing nucleotide modifications, CG_200280 was incubated in human serum or liver lysosomal extracts at 37°C for up to 7 days. Subsequently, siRNA and its degradation products were purified from the reaction mixture using a silica column and analyzed by LC-MS. It was found that siRNA was very stable and nuclease-resistant, with ≥70% of the full-length nucleotide sequence of siRNA remaining after 3 days of incubation in human serum or liver lysosomal extracts, and ≥50% of the full-length nucleotide sequence of siRNA remaining after 7 days of incubation in human serum or liver lysosomal extracts. Specifically, after incubating CG_200280 in human serum for 24 hours, 93% of SEQ ID NO:215 and 91% of SEQ ID NO:241 were still retained. The results show that siRNA can remain stable under physiological conditions to prolong the pharmacodynamics.

[0797] Example 14: In vitro evaluation of the immunogenicity of siRNA targeting ITFG1 comprising nucleotide modifications

[0798] In order to evaluate the immunogenicity of siRNA containing nucleotide modifications, the siRNA in Table 2 of Example 1 was incubated with human peripheral blood mononuclear cells (PBMC) in vitro for up to 24 hours, and the cell culture supernatant was collected and analyzed for the expression of proinflammatory cytokines including IL-6, IL-10 and TNFα. It was found that siRNA did not significantly upregulate the expression of these cytokines compared to the expression of control PBMC incubated with PBS.

[0799] Example 15: Evaluation of the ability of siRNA targeting ITFG1 to induce hepatocyte proliferation in vivo

[0800] Mice were administered 6 mg / kg body weight of siRNA targeting ITFG1 (e.g., CG_200277 or CG_200280) or PBS (control). One week after injection of siRNA or PBS, mice underwent a two-thirds partial hepatectomy (see, e.g., Mitchell and Willenbring Nat. Protoc. (2008) 3(7): 1167-70), which induces hepatocyte proliferation. Liver was harvested from mice 42 hours after partial hepatectomy and hepatocyte proliferation was evaluated by immunostaining analysis of the expression of the classic proliferation marker Ki67. Briefly, liver was fixed in 4% paraformaldehyde and equilibrated in 30% sucrose solution before being embedded in optimal cutting temperature embedding medium. The liver was sectioned and routinely immunostained with anti-Ki67 antibody (Abcam) (secondary antibody recognizing Ki67 antibody (Life Technologies)) and counterstained with DAPI to mark the nucleus. Images of stained sections were acquired on an automated imaging system, and the proportion of cells expressing Ki67 was automatically counted using ImageJ macro.

[0801] like Fig.13A As shown, it was found that the proportion of proliferating hepatocytes after partial hepatectomy was increased by more than 50% in mice administered with siRNA targeting ITFG1 (eg, CG_200277 or CG_200280) compared with mice administered with PBS.

[0802] To evaluate the cessation of accelerated proliferation induced by siRNA targeting ITFG1 (e.g., CG_200280), mice were administered 6 mg / kg body weight of siRNA targeting ITFG1 (e.g., CG_200280) or PBS (control). Ten days after injection of siRNA or PBS, mice were subjected to two-thirds partial hepatectomy as described above. Fourteen days after partial hepatectomy, livers were harvested from mice and weighed. Fig. 13B showed that there was no significant difference in the weight of the regenerating liver between PBS- or CG_200280-treated mice, indicating that CG_200280-induced Itfg1 knockdown did not lead to uncontrolled proliferation and hepatomegaly.

[0803] Example 16: Evaluation of the ability of CG_200277 to improve liver disease in vivo

[0804] 16.1 Chemically induced liver fibrosis

[0805] The ability of siRNA targeting ITFG1 (eg, CG_200277, CG_200278, CG_200279, or CG_200280) to inhibit liver fibrosis or slow the progression of fibrosis in a chemically induced liver fibrosis model was evaluated.

[0806] Briefly, mice were administered thioacetamide three times a week for 4 to 12 weeks to induce liver fibrosis (see, e.g., Delire et al., J. Clin. Transl. Hepatol. (2015) 3(1):53-66), followed by subcutaneous injections of siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, CG_200279, or CG_200280) or PBS (control) at doses ranging from 2 to 10 mg / kg body weight every 2 to 4 weeks for various time periods up to 12 weeks. Liver was harvested from mice at different time points, and the extent of liver fibrosis was evaluated by histology. Mice treated with siRNA targeting ITFG1 showed significantly less liver fibrosis compared to control mice administered PBS.

[0807] In further experiments, mice were first injected subcutaneously with siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, CG_200279, or CG_200280) at a dose of 2 to 10 mg / kg body weight. Then, starting from one week after the first injection of siRNA, mice were administered thioacetamide three times a week for 4 to 12 weeks to induce liver fibrosis. During the entire thioacetamide treatment period, siRNA was administered once every 2 to 4 weeks at a dose of 2 to 10 mg / kg body weight, or PBS (control) was administered. Liver was harvested from mice at different time points, and the degree of liver fibrosis was evaluated by histology. Compared with the control mice administered PBS, mice treated with siRNA targeting ITFG1 showed significantly less liver fibrosis.

[0808] 16.2 WDF Model of Liver Fibrosis and Steatosis

[0809] The ability of siRNA targeting ITFG1 (eg, CG_200277 or CG_200280) to inhibit or slow the progression of liver fibrosis and steatosis in a mouse model of non-alcoholic fatty liver disease (NAFLD) was evaluated.

[0810] In brief, mice were fed a Western diet with fructose (WDF) for 6 months to establish NAFLD (see, e.g., Baena et al., Sci Rep (2016) 6:26149, Machado et al., PloS One (2015) 10:e0127991). After 6 months of WDF, livers were harvested from mice to determine baseline characteristics. The remaining mice were then subcutaneously injected with CG_200277 (6 mg / kg body weight) or PBS (control) once a week for 12 weeks. Liver was harvested from mice, and the degree of ITFG1 knockdown efficacy, liver fibrosis, steatosis, proliferation and hepatic stellate cell activity were evaluated. Part of the liver was fixed formalin paraffin embedded (FFPE), then sliced ​​and stained with hematoxylin and eosin (H&E) and Sirius red for subsequent pathological evaluation, as described in Example 12. RNA was extracted from the liver and qPCR analysis was performed as described in Example 10. The markers evaluated by qPCR were Itfg1, Ki67 and Acta2. The primers used for qPCR are shown in SEQ ID NO: 246 to 249. At 4, 8 and 12 weeks of CG_200277 treatment, serum was collected from mice via the submandibular vein and analyzed for albumin levels using a commercial kit (Abcam). Parts of the liver were subjected to Western blot analysis as described in Example 6.

[0811] like Figures 14A to 14IAs shown, compared with PBS controls, mice treated with CG_200277 had 85% knockdown of ITFG1 at the mRNA level and 90% knockdown at the protein level, reduced fibrosis (some mice in the CG_200277 group had lower fibrosis scores than the baseline group), reduced steatosis (some mice in the CG_200277 group had lower steatosis scores than the baseline group), increased proliferation (increased Ki67 mRNA expression), reduced hepatic stellate cell activity (reduced Acta2 mRNA and smooth muscle actin expression) and comparable liver function (similar albumin levels).

[0812] 16.3 CDHFD Model of Hepatic Fibrosis and Steatosis

[0813] The ability of siRNA targeting ITFG1 (CG_200277 or CG_200280) to inhibit liver fibrosis and steatosis in a mouse model of acute liver injury and fat accumulation was evaluated.

[0814] Briefly, mice were fed a choline-deficient high-fat diet (CDHFD) for 8 weeks to establish acute steatosis and fibrosis (see, e.g., Raubenheimer et al., Diabetes (2006) 55(7):2015-0-2020, Suzuki-Kemuriyama et al., FEBS Open Bio (2021) 11:2950-2965). Mice were then subcutaneously injected with 6 mg / kg body weight of siRNA targeting ITFG1 (CG_200280) or PBS (control) once a week for 4 weeks. As described in Example 16.2, the liver was stained with H&E and PSR for image quantification and pathological evaluation. Sections stained with Sirius red were imaged and the positive staining areas were quantified by ImageJ. Mice treated with siRNA targeting ITFG1 exhibited significantly less liver fibrosis and / or steatosis compared to control mice administered PBS ( Fig.15 ).

[0815] 16.4 Primary bile duct ligation model of liver fibrosis and cholestasis

[0816] The ability of siRNA targeting ITFG1 (CG_200277) to inhibit liver fibrosis in a mouse model of acute cholestasis-induced liver injury was evaluated.

[0817] In brief, mice received a single 6 mg / kg body weight dose of CG_200277 or PBS. Seven days after administration, they underwent primary bile duct ligation surgery (e.g., Tag et al., J Vis Exp (2015) 96:52438) to induce acute obstructive cholestasis. On days 1, 3, 5, and 7 after surgery, additional doses of CG_200277 or PBS were administered to mice at 6 mg / kg body weight. Mice were weighed on the day of surgery and 3, 5, 7, 10, and 13 days after surgery. Serum and liver were harvested from mice 14 days after surgery. The relevance of hepatotoxicity in serum, such as AST, ALT, and total bilirubin, was evaluated on a Roche Cedex bioanalyzer. As described in Example 12, FFPE and histopathological evaluation of the liver were performed.

[0818] Compared with the PBS control group, mice receiving CG_200277 had higher body weight within 2 weeks after surgery ( Fig.16A ), AST, ALT and total bilirubin were lower ( Fig. 16B ), and lower fibrosis scores ( Fig. 16C ).

[0819] Example 17: Evaluation of long-term in vivo toxicity

[0820] The long-term in vivo toxicity of siRNA targeting ITFG1 was evaluated.

[0821] Mice were administered a dose of siRNA targeting ITFG1 (CG_200277) monthly for one year to maintain different levels of Itfg1 knockdown. The body weight and wet liver weight of the mice were recorded. Serum was collected from the mice via the submandibular vein, and liver, kidney, spleen, bone marrow and other organs were obtained from mice 6 and 12 months after the first injection and evaluated as described in Example 12. There was no difference in AST, ALT and ALB in mice receiving a 6-month dose of CG_200277 compared to PBS controls ( Fig.17A ), and there were no histopathological differences between the tissues (liver, kidney, spleen and lymph nodes) of the CG_200277-treated and control groups. It was also found that the wet liver weight and liver: body weight ratio of the CG_200277-treated and control mice were similar ( Fig. 17B ). Long-term administration of siRNA targeting ITFG1 did not cause significant toxicity. Similar results were observed in mice treated with PBS or CG_200277 for 12 months ( Fig. 17C and 17D ).

[0822] Example 18: CG_200280-induced ITFG1 expression in rat and non-human primate (cynomolgus monkey) liver The lowered evaluation

[0823] The knockdown efficacy of siRNA (CG_200280) targeting ITFG1 was evaluated in rats and cynomolgus monkeys.

[0824] Rats were subcutaneously administered PBS or a single dose of one of two doses (2 mg / kg or 6 mg / kg body weight) of siRNA targeting ITFG1 (CG_200280), and liver and kidney were harvested 7 days after injection, and ITFG1 expression was quantified by qPCR using primers of SEQ ID NOs: 250 and 251 as described in Example 10. Despite the presence of a single mismatch between the siRNA and the rat ortholog of Itfg1 outside the seed region, a dose-dependent knockdown of ITFG1 expression was observed in rat liver ( Fig. 20A No changes in ITFG1 expression were observed in rat kidneys, emphasizing the specificity of the GalNAc conjugate for the liver ( Fig. 20B ).

[0825] The cynomolgus monkeys were subcutaneously administered PBS or a single dose of siRNA targeting ITFG1 (CG_200280) at 2 mg / kg or 6 mg / kg body weight on day 0. Two biopsy samples were taken from the liver of each cynomolgus monkey on days 14, 28, 56, and 84 and processed by qPCR as described in Example 10 to quantify ITFG1 expression using primers of SEQ ID NO: 252 and 253. Dose-dependent knockdown of ITFG1 expression was observed in the liver of cynomolgus monkeys, with 6 mg / kg producing 80% knockdown on day 14. Both doses maintained approximately 50% knockdown 84 days after siRNA administration ( Fig. 20C ).

[0826] Example 19: Evaluation of serum toxicity-related responses in rats administered a single high-dose siRNA

[0827] The toxicity of ITFG1-targeting siRNA was evaluated in rats.

[0828] Rats were administered PBS or a single dose of siRNA targeting ITFG1 (CG_200280) at different doses (30 mg / kg, 100 mg / kg, or 400 mg / kg body weight). Serum was collected from rats immediately before injection and 48 hours after injection. Serum was analyzed for serum toxicity correlates such as AST and ALT. No significant differences in AST and ALT levels were observed between the two time points in all treatment groups (400 mg / kg, Fig.21 ).

[0829] Example 20: Evaluation of the pharmacokinetics of CG_200280 in rat and cynomolgus monkey plasma The pharmacokinetics of CG_200280 were evaluated in rat and cynomolgus monkey plasma.

[0830] At 0 hours, rats were subcutaneously injected with a single dose of 0, 30, 100 or 400 mg / kg of CG_200280, while cynomolgus monkeys were subcutaneously injected with a single dose of 0, 2 or 6 mg / kg body weight of CG_200280. Plasma was collected from animals before injection (0 minutes), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours after injection. The amount of full-length SEQ ID NO: 215 and 241 present in each plasma sample was analyzed by LC-MS to calculate the half-life (t 1 / 2 ), the time it takes to reach maximum plasma concentration (T max ), the maximum concentration of each sequence detected in plasma at all time points (C max ) and the amount of body exposure to the sequence (AUC). The results are summarized in the table below.

[0831] Rat

[0832]

[0833] Cynomolgus monkey

[0834]

[0835] Example 21: Knockdown of ITFG1 expression in primary human hepatocytes by free uptake and evaluation of toxicity

[0836] 21.1 Efficacy of siRNA Knockdown of ITFG1 Expression

[0837] Freshly thawed primary human hepatocytes were incubated in vitro for 24 hours with siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, or CG_200279) at concentrations ranging from 0.1 nM to 10 μM, followed by RNA harvesting from the cells using a column-based kit (Favorgen) according to the manufacturer's instructions. ITFG1 mRNA expression was evaluated by qPCR as described in Example 2 above. siRNA targeting ITFG1 was found to inhibit ITFG1 expression in primary human hepatocytes by free uptake with IC50 values ​​between 1 nM and 500 nM.

[0838] 21.2 Toxicity

[0839] Freshly thawed primary human hepatocytes were incubated in vitro for 72 hours with siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, or CG_200279) at concentrations ranging from 1 nM to 10 μM. Cell viability was then evaluated using the CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer's instructions. It was found that siRNA targeting ITFG1 did not show significant toxicity to primary human hepatocytes at concentrations <10 μM.

[0840] Example 22: siRNA-mediated knockdown of ITFG1 expression in non-hepatic cell lines and evaluation of its functional consequences

[0841] 22.1 Efficacy of siRNA Knockdown of ITFG1 Expression

[0842] Non-hepatic cell lines of human and / or mouse origin, such as CCL206 (mouse lung) and CRL1722 (mouse myoblasts), were transfected with siRNA targeting ITFG1 (e.g., CG_200277, CG_200278, CG_200279, or CG_200280) at concentrations ranging from 80 fM to 100 nM and maintained in culture medium for 3 days as described above. The fold change in the level of mRNA encoding ITFG1 was determined as described in Example 2 (for human cell lines) or Example 5 (for mouse cell lines). It was found that ...

Claims

1. An inhibitory nucleic acid for reducing gene and / or protein expression of ITFG1, characterized in that: The inhibitory nucleic acid comprises or encodes an antisense nucleic acid targeting a nucleotide sequence comprising or consisting of one of SEQ ID NO: 26, 33, 13 to 25, 27 to 32, 33 to 44, 128, 135, 115 to 127, 129 to 134, or 136 to 146.

2. The inhibitory nucleic acid according to claim 1, characterized in that The inhibitory nucleic acid comprises or encodes an antisense nucleic acid targeting a nucleotide sequence comprising or consisting of SEQ ID NO: 26, 33, 128 or 135.

3. The inhibitory nucleic acid according to claim 1 or claim 2, characterized in that 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 one of SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178.

4. The inhibitory nucleic acid according to any one of claims 1 to 3, characterized in that 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 SEQ ID NO:58, 65, 160 or 167.

5. The inhibitory nucleic acid according to any one of claims 1 to 4, characterized in that The inhibitory nucleic acid comprises: (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178, or a nucleotide sequence having at least 75% sequence identity with the following: SEQ ID NO: 58, 65, 45 to 57, 59 to 64, 66 to 76, 160, 167, 147 to 159, 161 to 166, or 168 to 178; and (ii) a nucleic acid comprising a nucleotide sequence having the reverse complement of the nucleotide sequence of (i), or a nucleotide sequence having at least 75% sequence identity with the reverse complement of the nucleotide sequence of (i).

6. The inhibitory nucleic acid according to any one of claims 1 to 5, characterized in that The inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table I, and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table I, wherein the sequences in columns A and B are selected from the same row of Table I.

7. The inhibitory nucleic acid according to any one of claims 1 to 5, characterized in that The inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising the nucleotide sequence shown in column A of Table II, and (ii) a nucleic acid comprising the nucleotide sequence shown in column B of Table II, wherein the sequences in columns A and B are selected from the same row of Table II.

8. The inhibitory nucleic acid according to any one of claims 1 to 7, characterized in that The inhibitory nucleic acid comprises one or more modified nucleotides selected from the group consisting of 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'-phosphate, thioate, 2'-O-methylcytidine-3'-phosphorothioate, 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.

9. The inhibitory nucleic acid according to any one of claims 1 to 8, characterized in that The inhibitory nucleic acid comprises or consists of: (i) a nucleic acid comprising a nucleotide sequence (including modifications thereof) as shown in column A of Table III, and (ii) a nucleic acid comprising a nucleotide sequence (including modifications thereof) as shown in column B of Table III, wherein the sequences in columns A and B are selected from the same row of Table III; Optionally, wherein the inhibitory nucleic acid comprises or consists of: (a) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 235; (b) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 241; (c) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 254; (d) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 255; (e) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 256; (f) (i) SEQ ID NO: 215, and (ii) SEQ ID NO: 257; (g) (i) SEQ ID NO:216, and (ii) SEQ ID NO:235; or (h) (i) SEQ ID NO:226, and (ii) SEQ ID NO:

240.

10. The inhibitory nucleic acid according to any one of claims 1 to 9, characterized in that The inhibitory nucleic acid comprises a moiety that promotes uptake of the inhibitory nucleic acid by hepatocytes, optionally wherein the moiety that promotes uptake of the inhibitory nucleic acid by hepatocytes is or comprises an N-acetylgalactosamine (GalNAc) moiety.

11. The inhibitory nucleic acid according to any one of claims 1 to 10, characterized in that The inhibitory nucleic acid is siRNA.

12. A nucleic acid, optionally isolated, encoding an inhibitory nucleic acid according to any one of claims 1 to 11.

13. An expression vector comprising the nucleic acid according to claim 12.

14. A composition comprising the inhibitory nucleic acid according to any one of claims 1 to 11, the nucleic acid according to claim 12, or the expression vector according to claim 13, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

15. A cell comprising the inhibitory nucleic acid of any one of claims 1 to 11, the nucleic acid of claim 12, or the expression vector of claim 13.

16. A method for reducing the expression of ITFG1 gene and / or protein in cells in vitro or in vivo, comprising introducing the inhibitory nucleic acid according to any one of claims 1 to 11, the nucleic acid according to claim 12, or the expression vector according to claim 13 into the cells.

17. Use of the inhibitory nucleic acid according to any one of claims 1 to 11, the nucleic acid according to claim 12, the expression vector according to claim 13, or the composition according to claim 14 for reducing gene and / or protein expression of ITFG1 in vitro or in vivo.

18. An inhibitory nucleic acid according to any one of claims 1 to 11, a nucleic acid according to claim 12, an expression vector according to claim 13, or a composition according to claim 14, for use in a method of medical treatment or prevention.

19. An inhibitory nucleic acid according to any one of claims 1 to 11, a nucleic acid according to claim 12, an expression vector according to claim 13, or a composition according to claim 14, for use in a method of treating or preventing a disease / disorder selected from the group consisting of a disease / disorder characterized by fibrosis, a disease / disorder characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis , alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small-for-size syndrome, split liver transplantation liver grafts), cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy right ventricular cardiomyopathy,ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture), skin keloid, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental insults, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, Peyronie's disease disease), renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

20. Use of an inhibitory nucleic acid according to any one of claims 1 to 11, a nucleic acid according to claim 12, an expression vector according to claim 13, or a composition according to claim 14 for the preparation of a medicament for treating or preventing a disease / disorder selected from the group consisting of a disease / disorder characterized by fibrosis, a disease / disorder characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), Schistosoma japonicum liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, hepatic ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis Lung, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy , polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)),Diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after glaucoma trabeculectomy), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental damage, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, longitudinal Septal fibrosis, retroperitoneal fibrosis, myelofibrosis, Peyronie's disease, renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative disease, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

21. A method for treating or preventing a disease or condition in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of an inhibitory nucleic acid according to any one of claims 1 to 11, a nucleic acid according to claim 12, an expression vector according to claim 13, or a composition according to claim 14, characterized in that: The disease or condition is selected from the group consisting of a disease / condition characterized by fibrosis, a disease / condition characterized by damage and / or death of liver cells, chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation, cholestatic liver disease, pulmonary fibrosis, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, bronchiolitis obliterans, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, pneumonia, pulmonary edema, chronic bronchitis, asthma, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial Fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne Muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis,Progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors, fibrotic tumor diseases, fibrosis induced by chemical or environmental damage, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, Peyronie's disease, renal tubular disease, renal tubular acidosis, hypokalemia, hyperkalemia, acute tubular necrosis, Fanconi syndrome, Liddle syndrome, nephrogenic diabetes insipidus, pseudohypoaldosteronism type I, chronic kidney disease, acute kidney injury, neurodegenerative diseases, amyotrophic lateral sclerosis, epilepsy, frontotemporal dementia, Parkinson's disease, Huntington's disease, multiple system atrophy, prion disease, ulcerative colitis, inflammatory bowel syndrome, chronic inflammation of the gastrointestinal tract, or celiac disease.

22. The inhibitory nucleic acid for use according to claim 19, the use according to claim 20, or the method according to claim 21, characterized in that The disease / disorder is a disease / disorder characterized by fibrosis, or a disease / disorder characterized by damage and / or death of liver cells.

23. The inhibitory nucleic acid for use, use or method according to claim 22, characterized in that The disease / disorder characterized by fibrosis is selected from the group consisting of chronic liver disease, liver fibrosis, liver cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced Liver injury, Autoimmune liver injury, Liver transplantation, Extended hepatectomy, Small liver syndrome, Split liver transplantation, Cholestatic liver disease, Pulmonary fibrosis, Interstitial lung disease (ILD), Idiopathic interstitial pneumonia (IIP), Idiopathic pulmonary fibrosis (IPF), Cystic fibrosis, Progressive massive fibrosis, Scleroderma, Bronchiolitis obliterans, Hermansky-Pudlak syndrome, Asbestosis, Silicosis, Sarcoidosis, Tumor stroma in lung disease, Chronic obstructive pulmonary disease (COPD), Emphysema, Pneumonia, Pulmonary edema, Chronic bronchitis, Asthma, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM), Atrial fibrosis, Atrial fibrillation, Ventricular fibrosis, Ventricular fibrillation, Myocardial fibrosis, Brugada syndrome, Myocarditis, Heart Endomyocardial fibrosis, myocardial infarction, fibrosing vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, chronic pulmonary hypertension, AIDS-related pulmonary hypertension, varicose veins, cerebral infarction, tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport syndrome, HIV-related nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, pancreatic fibrosis, cystic fibrosis, chronic pancreatitis, gliosis, Alzheimer's disease, multiple sclerosis, muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), fibrosing myopathy, inflammation Inflammatory bowel disease (IBD), Crohn's disease, microscopic colitis, primary sclerosing cholangitis (PSC), scleroderma, nephrogenic systemic fibrosis, Dupuytren's contracture, cutaneous keloids, Graves' ophthalmopathy, preretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, postoperative fibrosis (e.g., posterior capsule after cataract surgery, filtering blebs after trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, arthrofibrosis, arthritis, adhesive capsulitis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease (GVHD), prefibrotic tumors,Fibrotic tumor diseases, fibrosis induced by chemical or environmental insults, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, vulvar cancer, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis and Peyronie's disease.

24. The inhibitory nucleic acid for use, use or method according to claim 23, characterized in that The disease / disorder characterized by damage and / or death of liver cells is selected from the group consisting of: chronic liver disease, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), hepatitis, steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), schistosomiasis liver disease, congenital liver disease, liver cancer, hepatocellular carcinoma (HCC), acute liver injury (ALI), acute liver failure, acute liver disease, viral hepatitis, liver ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), acetaminophen-induced liver injury, autoimmune liver injury, liver transplantation, extended hepatectomy, small liver syndrome, split liver transplantation and cholestatic liver disease.

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