Gene sequence-carbohydrate conjugates for enhanced liver- and kidney-specific targeting

Abstract

A gene sequence-carbohydrate conjugate having the formula: JPEG2025514180000018.jpg18170, wherein GS is a gene sequence, preferably a peptide nucleic acid or oligonucleotide, such as an mRNA sequence, an siRNA sequence, or a DNA sequence, and optionally each gene sequence is natural or modified, and the variables are as described herein. Also disclosed are uses of the gene sequence-carbohydrate conjugates, including as RNA therapeutics targeting the liver and kidney of mammals, including humans.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 335,174, filed April 26, 2022, the entire contents of which are incorporated herein in their entirety.

[0002] The present application is directed to gene sequence-carbohydrate conjugates, such as peptide nucleic acid-carbohydrate conjugates, methods for their preparation, compositions comprising the conjugates, and uses thereof. The conjugates are particularly useful for improved targeting of liver and kidney cells. [Background technology]

[0003] The liver and kidneys are important organs involved in vital bodily functions including metabolism, detoxification, excretion, protein and lipid synthesis, cytokine and growth factor secretion, and immune / inflammatory responses. Liver disorders such as hepatitis, alcoholic or nonalcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. It is estimated that more than one in seven U.S. adults, or about 37 million people, have chronic kidney disease, CKD. Renal fibrosis is the final sign of chronic kidney disease. Other kidney diseases include cancer, IgA nephropathy, membranous nephropathy, and acute kidney injury. Summary of the Invention [Problem to be solved by the invention]

[0004] Many therapeutic agents are delivered to body tissues where they are not intended to exert their effect, which can result in unintended or harmful side effects.Therefore, there is a need for safe and efficient delivery of therapeutic molecules (e.g., gene sequences and active agents such as drugs, genes, or proteins) to target sites in the body, including the liver and kidney.These unmet needs and long-standing problems are addressed by the new compositions and methods described below. [Means for solving the problem]

[0005] A gene sequence-carbohydrate conjugate having the formula:

[0006] [ka] wherein GS is a gene sequence, preferably a peptide nucleic acid or oligonucleotide, such as an mRNA sequence, an siRNA sequence, or a DNA sequence, optionally each gene sequence being natural or modified, for example, gamma-serine modified gamma peptide nucleic acid, alanine gamma peptide nucleic acid, clamp G modified peptide nucleic acid, locked nucleic acid (LNA), phosphorothioate (PS), phosphorodiamidate morpholino (PMO), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'F), 5'-methylcytosine, or combinations thereof, the gene sequence having a 3' end and a 5' end, R 1 and R 2 are each independently H or substituted or unsubstituted C 1 ~C 6 is alkyl, and X 1 But, O, NR 3 , C=O, or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 is alkyl, and X 2 But, O, NR 3 , or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 is alkyl, G 1is a direct bond or a group linking the PNA to the conjugate, and G 2 is H or a functional moiety, CL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a mono-, di-, tri-, or tetrasaccharide, preferably CL comprises carbohydrate residues derived from a mono- or disaccharide, and optionally the carbohydrate ligand comprises at its hydroxy or amino group, C 2 ~C 15 fully or partially acylated with acyl groups, preferably the carbohydrate ligand is fully or partially acetylated at its hydroxy or amino groups, 1 is 1 to 20, and n 2 is 0-20.

[0007] Methods are described for the generation of gene sequence-carbohydrate conjugates, in particular PNA-carbohydrate conjugates.

[0008] The pharmaceutical composition comprises a gene sequence-carbohydrate conjugate, in particular a PNA-carbohydrate conjugate, and a pharmaceutical excipient.

[0009] Methods are described for the use of gene sequence-carbohydrate conjugates, in particular PNA-carbohydrate conjugates.

[0010] A method for reducing expression of a targeted RNA involved in a health disorder in a subject comprises providing a gene sequence-lactobionic acid conjugate described herein to cells of the subject in vivo or ex vivo, wherein binding of the PNA of the conjugate to the targeted RNA reduces expression of the targeted RNA, in particular the targeted RNA is a microRNA.

[0011] A method for targeting and gene editing DNA in a health disorder in a subject comprises providing a gene sequence-carbohydrate conjugate according to any one of claims 1 to 20 to cells of the subject in vivo or ex vivo, wherein the DNA of the conjugate targeted to the cells modulates expression of the gene. [Brief description of the drawings]

[0012] The following figures are exemplary embodiments provided to illustrate the present disclosure. These figures are not intended to limit the compositions, methods, or articles made in accordance with the present disclosure to the materials, conditions, or process parameters described therein. [Figure 1A-1] Illustrates the design and synthesis of lactobionic acid (LBA)-appended linker ligands and peptide nucleic acids targeting miR-122. Specifically, (A) is a schematic diagram of the synthesis scheme of lysine linkers (1-4) and LBA conjugation with lysine linkers (5-8). [Figure 1A-2] Illustrates the design and synthesis of lactobionic acid (LBA)-appended linker ligands and peptide nucleic acids targeting miR-122. Specifically, (A) is a schematic diagram of the synthesis scheme of lysine linkers (1-4) and LBA conjugation with lysine linkers (5-8). [Figure 1B] (B) shows the chemical structures of DNA and normal PNA units. [Figure 1C] (C) shows the nucleotide sequence of mature miR-122-5p (top) and the seed region (underlined). PNA oligomers targeting seed and full-length miR-122 (bottom). PNA1 and 2 were designed to target the seed region, whereas PNA3 and 4 can bind full-length miR-122. PNA2 and 4 contain succinic acid (SA) at the 5' end for conjugation with LBA-lysine conjugate. Lysine (K) was added to the 3' end of the PNA, followed by the fluorescent probe 5-carboxytetramethylrhodamine (TAM) of each PNA. "OOO" represents the trioxo-miniPEG linker. [Figure 2A-1]Illustrates the conjugation of PNA4 with LBA and tGalNAc and their quality control assessment. Specifically, (A) shows the solution phase conjugation of PNA4(14) with LBA(8-16). [Figure 2A-2] Illustrates the conjugation of PNA4 with LBA and tGalNAc and their quality control assessment. Specifically, (A) shows the solution phase conjugation of PNA4(14) with LBA(8-16). [Figure 2B-1] (B) shows solution-phase conjugation of PNA4(14) with tGalNAc(13-18). [Figure 2B-2] (B) shows solution-phase conjugation of PNA4(14) with tGalNAc(13-18). [Figure 2C1] (C) MALDI-MS of ligand-conjugated PNA, PNA4-LBA, and PNA4-tGalNAc. The calculated and measured masses of the conjugates are plotted on each mass spectrum. The inset shows reverse phase-high performance liquid chromatography (RP-HPLC) traces of the indicated conjugates. [Figure 2C2] (C) MALDI-MS of ligand-conjugated PNA, PNA4-LBA, and PNA4-tGalNAc. The calculated and measured masses of the conjugates are plotted on each mass spectrum. The inset shows reverse phase-high performance liquid chromatography (RP-HPLC) traces of the indicated conjugates. [Figure 3A] 1 illustrates the results of biophysical target binding evaluation of PNA-conjugated ligands. (A) Normalized thermal melting curves of short and full-length PNAs and their conjugates with the target DNA sequence of miR-122 under low-salt physiological conditions. [Figure 3B](B) Gel shift binding assay under low salt physiological conditions with a DNA to PNA ratio of 1:2. DNA and PNA-DNA heteroduplexes (retarded bands) were visualized using SYBR Gold staining. [Figure 4A] 1 illustrates the results of an in vivo biodistribution study of full-length PNA conjugates (PNA3 and PNA4). (A) shows IVIS imaging of organs isolated from C57BL6 / J mice treated with full-length PNA and ligand conjugates at different time points after 10 mg / kg subcutaneous administration. [Figure 4B] (B) shows histograms depicting uptake of full-length PNA and ligand conjugates in liver cells from C57BL6 / J mice after subcutaneous administration at different time points analyzed by flow cytometry. [Figure 4C] (C) shows a confocal microscopy image of a liver cryosection from a C57BL6 / J mouse 1 hour after subcutaneous administration, with blue indicating nuclei and red indicating TAMRA. [Figure 4D] (D) shows the mean radiant efficiency for TAMRA fluorescence in the liver of C57BL6 / J mice treated with full-length PNA and ligand conjugates at different time points after subcutaneous administration. Results are presented as the mean of n=3, with standard error of the mean represented as error bars. [Figure 5A] Illustrating in vivo biodistribution studies of anti-seed PNA conjugates (PNA1 and PNA2), (A) shows IVIS imaging of organs isolated from C57BL6 / J mice treated with anti-seed PNA and ligand conjugates at different time points after 5 mg / kg subcutaneous administration. [Figure 5B] (B) shows histograms depicting uptake of anti-seed PNA and ligand conjugates in liver cells from C57BL6 / J mice 0.5 and 24 hours after subcutaneous administration by flow cytometry. [Figure 5C](C) shows the mean radiant efficiency for TAMRA fluorescence in the liver of C57BL6 / J mice treated with anti-seed PNA and ligand conjugates at different time points after subcutaneous administration. [Figure 5D] (D) Flow cytometry dot plot depicting hepatocyte uptake of anti-seed PNA and ligand conjugate in liver cells from C57BL6 / J mice after 1 hour subcutaneous administration. The BV786 channel represents hepatocytes stained with ASGPR antibody against ASGPR receptor. The FITC channel represents hepatocytes stained with HNF4α antibody against hepatocytes. The TAMRA channel represents PNA. [Figure 6A1] (A) Illustrates the relative miR-122, ALDOA and BCKDK (downstream target of miR-122) expression levels in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are presented as mean values ​​of n=6, with standard error of the mean values ​​shown as error bars. Statistical analysis was performed using t-test. *p<0.05, **p<0.01. [Figure 6A2] (A) Illustrates the relative miR-122, ALDOA and BCKDK (downstream target of miR-122) expression levels in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are presented as mean values ​​of n=6, with standard error of the mean values ​​shown as error bars. Statistical analysis was performed using t-test. *p<0.05, **p<0.01. [Figure 6A3] (A) Illustrates the relative miR-122, ALDOA and BCKDK (downstream target of miR-122) expression levels in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are presented as mean values ​​of n=6, with standard error of the mean values ​​shown as error bars. Statistical analysis was performed using t-test. *p<0.05, **p<0.01. [Figure 6B1](B) Representative Western blots of AldoA and Bckdk proteins (miR-122 downstream targets) in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are represented as n=2. [Figure 6B2] (B) Representative Western blots of AldoA and Bckdk proteins (miR-122 downstream targets) in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are represented as n=2. [Figure 6C1] (C) illustrates the relative ALDOA and BCKDK (downstream target of miR-122) protein levels in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are presented as mean values ​​of n>3, with standard error means shown as error bars. Statistical significance was analyzed with GraphPad Prism software using nonparametric one-way ANOVA. For multiple comparisons, an uncorrected Dunn's test was performed. Significance levels *p<0.05, **p<0.01, ***p<0.001 versus the indicated treatment groups. [Figure 6C2] (C) illustrates the relative ALDOA and BCKDK (downstream target of miR-122) protein levels in liver cells of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. Results are presented as mean values ​​of n>3, with standard error means shown as error bars. Statistical significance was analyzed with GraphPad Prism software using nonparametric one-way ANOVA. For multiple comparisons, an uncorrected Dunn's test was performed. Significance levels *p<0.05, **p<0.01, ***p<0.001 versus the indicated treatment groups. [Figure 7A] (A) Illustrates H&E staining of liver, kidney, and spleen from C57BL6 / J mice treated subcutaneously with full-length PNA and ligand conjugates at the end of the efficacy study. [Figure 7B](B) Cytokine panel assessment in C57BL6 / J mice treated subcutaneously with full length PNA and ligand conjugates at the end of the efficacy study. [Figure 7C] (C) shows the average body weight of C57BL6 / J mice during the efficacy study for different treatment groups. Results are presented as the mean of n=6 with standard error of the mean represented as error bars. [Figure 7D1] (D) shows CBC analysis including RBC, WBC, platelets, and hemoglobin (data not shown). [Figure 7D2] (D) shows CBC analysis including RBC, WBC, platelets, and hemoglobin (data not shown). [Figure 7D3] (D) shows CBC analysis including RBC, WBC, platelets, and hemoglobin (data not shown). [Figure 7E1] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E2](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E3] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E4](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E5] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E6](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E7] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E8](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E9] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E10](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E11] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E12](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E13] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E14](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E15] (E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 7E16](E) shows blood chemistry analysis including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes such as phosphorus, calcium, sodium, potassium, magnesium, chloride, and plasma glucose in C57BL6 / J mice at the end of the efficacy study for different treatment groups. Results are expressed as mean with n>3 and standard error of the mean is shown as error bars. [Figure 8A] 1 illustrates the biodistribution of PNA2-LBAAc in C57Bl6 / J mice at a subcutaneous dose of 5 mg / kg. (A) shows IVIS imaging of isolated organs from PNA1, PNA2-LBAAc, and saline-treated control mice. [Figure 8B] (B) shows the mean radiant efficiency for TAMRA fluorescence in the kidneys of C57BL6 / J mice. Results are expressed as the mean of n > 2, with standard error of the mean represented as error bars. [Figure 8C] (C) shows histograms depicting uptake of PNA and acetylated ligand conjugates in kidney cells from C57BL6 / J mice 24 and 72 hours after subcutaneous administration by flow cytometry. [Figure 8D] (D) Confocal microscopy image of a kidney cryosection from a C57BL6 / J mouse 24 hours after subcutaneous administration. Blue indicates nuclei. Red indicates TAMRA. [Figure 9A]Illustrates that LBAAc ligands efficiently delivered 22-mer long PNA sequences to the kidney. (A) shows the nucleotide sequences of miR-21 and anti-miR-21 PNAs and their ligand conjugates. PNA6 contains succinic acid (SA) at the 5' end for conjugation with LBA-lysine conjugates. Lysine (K) was added to the 3' end of the PNA, followed by the fluorescent probe 5-carboxytetramethylrhodamine (TAM) of each PNA. OOO represents a trioxo-miniPEG linker. [Figure 9B] (B) IVIS imaging of organs isolated from C57BL6 / J mice treated with PNA and ligand conjugates at 4 and 24 hours after 1.5 mg / kg subcutaneous administration. [Figure 9C] (C) shows histograms depicting uptake of PNA and acetylated ligand conjugates in kidney cells from C57BL6 / J mice 4 and 24 hours after subcutaneous administration by flow cytometry. [Figure 9D] (D) Mean radiant efficiency for TAMRA fluorescence in kidneys of C57BL6 / J mice. Results are expressed as the mean of n=2 with standard error of the mean represented as error bars. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Described herein are gene sequence-carbohydrate conjugates for targeted delivery of the conjugate to specific organs in mammals, particularly humans, more particularly the liver and kidney of mammals, such as humans. In one aspect, the gene sequence-carbohydrate conjugate is a peptide nucleic acid (PNA)-carbohydrate conjugate (PNAC). Specifically, the conjugate is a molecule that includes a carbohydrate ligand covalently linked to a gene sequence via a linker backbone that is covalently attached to both. The carbohydrate ligand can be selected to target the liver or kidney. Preferably, the conjugate selectively binds to a specific receptor on a cell to deliver the gene sequence or other therapeutic agent to the cell that has the receptor.

[0014] The conjugates used herein include a genetic sequence (GS) having a 3' end and a 5' end, which may be a PNA or an oligonucleotide, such as an mRNA sequence, an siRNA sequence, or a DNA sequence. Each genetic sequence may be natural or may be optionally modified, for example, in the order of nucleotides, or through modifications such as, for example, gamma-serine modified gamma peptide nucleic acid, alanine gamma peptide nucleic acid, clamp G modified peptide nucleic acid, locked nucleic acid (LNA), phosphorothioate (PS), phosphorodiamidate morpholino (PMO), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'F), 5'-methylcytosine, or combinations thereof. In one aspect, the genetic sequence is a PNA. The PNA may be modified as described below.

[0015] In one embodiment, the conjugate has 1-8, or 1-5, or 2-5, or 2-4 carbohydrate residues (ligands). The number and type of carbohydrate ligands are selected to target the liver or kidney, preferably to selectively target the liver and kidney. For example, the carbohydrate ligands can be selected to target the asialoglycoprotein receptor (ASPGR) expressed on cells. ASGPR is a C-type lectin expressed primarily on the sinusoidal surface of hepatocytes. In one embodiment, the conjugate has 1-8, or 1-5, or 2-5, or 2-4 galactose ligands to target ASPGR on liver cells and kidney cells. In one embodiment, the conjugate has 1-8, or 1-5, or 2-5, or 2-4 galactoseamine (GalNAc) ligands to target ASPGR on liver cells and kidney cells. In another embodiment, the conjugate has 1-8, or 1-5, or 2-5, or 2-4, or 2-3 lactobionic acid ligands for targeting ASPGR on liver and kidney cells.

[0016] The carbohydrate ligand of the conjugate can be fully or partially acylated at its hydroxy or amino groups with C2-C15 acyl groups. For example, the carbohydrate ligand can be fully or partially acetylated at its hydroxy or amino groups. Acetylation of the carbohydrate ligand can be carried out using an acetylation reagent such as acetic anhydride, acetyl chloride, mixed anhydrides, acids with coupling agents such as DCC, or similar reagents, and a base such as triethylamine, pyridine, DIEA, DMAP, or an organic, inorganic, or polymeric base as used in the art. In one aspect, the carbohydrate ligand is a GalNAc residue that is fully or partially acylated, preferably acetylated, preferably fully acetylated. Alternatively, the carbohydrate ligand is a lactobionic acid residue that is fully or partially acylated, preferably acetylated, preferably fully acetylated.

[0017] The carbohydrate ligand can be covalently attached to the genetic sequence by a backbone linker as shown in formula I. A variety of backbones can be used, but generally contain at least two functional groups, e.g., at least two amino groups, one or more for reaction with the carbohydrate ligand and one or more for reaction with the genetic sequence. The amino groups can be selectively protected as known in the art and as described in the Examples. The backbone can include moieties for modifying properties such as solubility. For example, lysine and arginine residues can be present in the backbone.

[0018] In the embodiment as shown in formula I, the group G 1 Or G 2 may be optionally present. 1 can be a linker from the backbone to the gene sequence, e.g., a linker having 1-20 carbon atoms and, optionally, one or more reactive groups, such as hydroxy, carboxy, thio, or amino. 1 Or G 2 can be a functional moiety. 1 , G 2 can provide the conjugate with structural features that can impart desired functionality, such as steric separation from the bound ligand, enhanced hydrophilicity or hydrophobicity, enhanced absorption of the conjugate, enhanced distribution of the conjugate in the body, or other functionality that is advantageous in medicinal chemistry and drug design. The functional moiety can be linked between the backbone and the gene sequence, or at the terminal end of the gene sequence, or both. In one aspect, the functional moiety G 1 , G 2 is, for example, a residue of polyethylene glycol, polypropylene glycol, or polyethylene-propylene glycol. 1 Or G 2, or both may be polyethylene glycol (PEG) groups. PEG groups consist of 1 to 25 ethylene glycol residues (-OCH 2 CH 2 O-), which may optionally be attached to the ligand, backbone or structure of the conjugate.

[0019] In another embodiment, the functional moiety can include a therapeutic agent, such as kielin, tolvaptan, nintedanib, paclitaxel, bleomycin, cyclosporine, cisplatin, romidepsin, doxorubicin, docetaxel, danunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, prednisolone, cytarabine, or pirfenidone. Other therapeutic agents can be found in the Merck Index, published by the Royal Society of Chemistry, which is published in print and online at https: / / www.rsc.org / merck-index. For example, G 1 is a linker between the scaffold and the genetic sequence, which may contain a therapeutic agent covalently attached thereto. Alternatively, or in addition, the group G 2 can be a therapeutic agent covalently attached to the genetic sequence, either directly or through a linker. Although not shown in Formula I, linkages similar to those that link carbohydrate residues can also be used to link functional moieties, such as therapeutic agents, to the backbone.

[0020] In one aspect, the conjugate is a gene sequence-lactobionic acid conjugate. Lactobionic acid (LBA) is a disaccharide formed from gluconic acid and galactose. In some embodiments, lactobionic acid is derivatized as part of the conjugate. A PNA-lactobionic acid conjugate of formula Ia,

[0021] [ka] wherein LBA is a lactobionic acid residue X, and X 1 But NR 3 , O, or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 alkyl, and each X 3 However, independently, O, NR 3 , or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 is alkyl, n 3 is 0 to 20, and n 4 is 1 to 8. 1 is a group that links the PNA to the conjugate, R 1 and R 2 are H and X 1 , X 2 , and X 3 are each NH, and n 1 =6, n 2 = 2 and n 3 = 4. In one embodiment, R 1 and R 2 are each H, and n 3 =4, and the PNA is linked to the conjugate at the 5' end.

[0022] For example, the PNA-lactobionic acid conjugate can be a conjugate of formula Ia-1:

[0023] [ka] In the formula, G 1 and G 2 is as defined above, preferably G 1 is the functional moiety that links the PNA to the conjugate, and G 2is the functional moiety. Optionally, in any of formulas Ia and Ia-1, the hydroxyl groups can be fully or partially acylated with acyl groups having 2 to 15 carbon atoms or 2 to 8 carbon atoms, preferably acetylated, and more preferably fully acetylated as described above.

[0024] In another embodiment, the conjugate may be of formula Ib:

[0025] [ka] In the formula, X 1 is C=O or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 is alkyl, and X 4 But, O, NR 3 , or C(R 3 ) 2 and R 3 is H or substituted or unsubstituted C 1 ~C 6 alkyl, and CL is substituted or unsubstituted C 1 ~C 12 C containing alkyl, amide, ester, or ether groups 6 ~C 12 A carbohydrate residue linked to CH by a linker chain of 1-30 atoms containing an aryl, 4 is 2 or 3. The carbohydrate residue in formula Ib can be derived from N-acetylgalactosamine and can be a fully or partially acylated carbohydrate residue in which the acyl group has 2 to 15 carbon atoms or 2 to 8 carbon atoms, e.g., fully acetylated.

[0026] A method is described for conjugating a genetic sequence to a carbohydrate ligand to provide a genetic sequence-carbohydrate conjugate. The method comprises functionalizing the genetic sequence to provide a free -COOH functional group, and coupling the free -COOH functional group of the modified genetic sequence with the Y of a compound of formula II. 2 and forming a bond between

[0027] [ka] In the formula, Y 2 But -NHR 3 or -OH. The method can be carried out by solution phase or solid phase synthesis, or a combination thereof. The gene sequence, e.g. PNA, can be obtained by solution phase or solid phase synthesis, or a combination thereof, as known in the art. The gene sequence can be modified as described below. In addition, the method can include the step of adding a G 1 , G 2 , or a combination thereof.

[0028] In one embodiment, the method of conjugating a genetic sequence to a lactobionic acid-backbone ligand to provide a genetic sequence-lactobionic acid conjugate comprises functionalizing the genetic sequence to provide a free -COOH functional group, and coupling the free -COOH functional group of the modified genetic sequence with a Y 2 and forming a bond between

[0029] [ka] In the formula, Y 2 But -NHR 3 or -OH. Again, the gene sequence is preferably a PNA. In one embodiment, the method may further comprise reacting lactobionic acid with the backbone of formula IV,

[0030] [ka] In the formula, X 3 is -OH or NHR 3 and X 2 protected O or protected NHR 3 It is.

[0031] In one embodiment, as described in the Examples and shown in Figures 1 and 2, a lactobionic acid residue can be coupled by its alpha and epsilon amino groups to a backbone containing lysine residues. The carboxyl group of the lysine is coupled to an amino group on an alkyldiamine, and the other amino group is coupled to a succinyl COOH group linked to a peptide nucleic acid. In another embodiment, the alkyldiamine can be replaced by an alkanediol to form a backbone with an ester linkage. Alternatively, the succinic acid at the 5' end can be replaced by a substituted or unsubstituted C-C 20 The PNA can be replaced by a dicarboxylic acid. The PNA is modified with a functional moiety, such as a trioxo-miniPEG spacer and a succinic acid at the 5' end, to provide a free -COOH functional group after cleavage. Some PNAs modified in this way are commercially available. The free COOH group can then react with an amino group, a hydroxy group, an alkyl halide, or other suitable functional group on the ligand backbone, such as lactobionic acid or GalNAc.

[0032] In particular, when GalNAc is used, GalNAc can be linked to the backbone by a group bearing an alkyl ether, amide, or ester residue to provide a carbohydrate ligand. Some of these ligands are commercially available or can be synthesized using chemical synthesis methods familiar to those skilled in the art. General methods for chemical synthesis can be found, among other sources, in "Comprehensive Organic Transformations: A Guide to Functional Group Preparations," Richard C. Larock, Wiley-VCH: 1999 and "March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure," Jerry March & Michael Smith, John Wiley & Sons Inc.: 2001. Of course, other carbohydrate residues can be similarly linked to the backbone by a group, e.g., a chain bearing an alkyl ether, amide, or ester residue to form a ligand.

[0033] The method for the use of the conjugate is further described.For example, the conjugate can be used to treat cancer in the liver and kidney.In one aspect, the conjugate can be used to treat renal fibrosis.In another aspect, the conjugate can be used to treat renal cancer.In another aspect, the conjugate can be used to treat renal disease.The formulation can be directly administered to a subject for in vivo gene therapy.

[0034] The conjugates, particularly PNAC, can be used as RNA therapeutics. Specifically, the conjugates, particularly PNAC, can target microRNA (miRNA) sequences. The conjugates can be used to control gene expression at the post-transcriptional level. miRNAs play an important role in maintaining physiological processes by controlling gene expression through regulating messenger RNA (mRNA) stability and translation. The use of conjugates to target RNA in cells, such as mRNA or miRNA, can affect gene expression by inhibiting the expression of the RNA at the translation stage, in the case of mRNA, and / or down-regulating or up-regulating the expression of miRNA and its downstream effects on its target genes. The conjugates can be used to control the abnormal expression of miRNAs that cause several destructive diseases. The conjugates can be used to treat cancers where atypical miRNA levels lead to altered processes including differentiation, proliferation, and apoptosis. In a preferred embodiment, the conjugates are used to treat cancers in the liver and kidney. In one aspect, the conjugates can be used to treat renal fibrosis. In one embodiment, the conjugates can be used to treat renal cancer. In one embodiment, the conjugates can be used to treat renal disease.

[0035] Thus, in one aspect, a method for reducing the expression of a target RNA involved in a health disorder in a subject comprises providing a gene sequence-lactobionic acid conjugate as described herein to a cell of the subject in vivo or ex vivo, and the binding of the PNA of the conjugate to the target RNA reduces the expression of the target RNA, and in particular, the target RNA is a microRNA.In one aspect, the RNA therapeutic is used in targeting liver cells or kidney cells, or a combination thereof, to regulate the expression of cellular nucleic acid function of a cancer cell, particularly including liver cancer cells or kidney cancer cells, or a combination thereof, of a subject in need thereof.In one aspect, the PNA comprises a kidney-specific microRNA, even more specifically, miR-21.The condition for treatment (requiring treatment) can be renal fibrosis and polycystic kidney disease.

[0036] In another aspect, a method for targeting and gene editing DNA in a health disorder in a subject comprises providing a gene sequence-carbohydrate conjugate according to any one of claims 1 to 20 to a cell of the subject in vivo or ex vivo, wherein the DNA of the conjugate targeted to the cell modulates expression of the gene.

[0037] The gene sequence-carbohydrate conjugates can be used for ex vivo or in vivo treatment of a subject in need thereof. The method typically involves contacting cells, ex vivo or in vivo, with an effective amount of the conjugate, optionally in combination with an enhancing agent, to deliver a therapeutic agent, e.g., modify the expression of an RNA. In one aspect, the method involves contacting a population of target cells with an effective amount of the conjugate to modify the expression of an RNA to achieve a therapeutic result.

[0038] Gene sequence-carbohydrate conjugates are generally provided as formulations that contain an effective amount of the conjugate and a polymer, lipid, protein, or other pharmaceutical excipient for organ-specific delivery. Pharmaceutically acceptable carriers (also referred to in the art as excipients) are selected so that the formulation is suitable for the mode of administration. Pharmaceutically acceptable carriers are determined in part by the particular conjugate to be administered and by the particular method used to administer the conjugate. For example, the formulation may be for topical, localized, or systemic administration in a suitable pharmaceutical carrier. Thus, there is a wide variety of suitable formulations for conjugates. Remington's Pharmaceutical Sciences, 15th Edition by EW Martin (Mark Publishing Company, 1975) discloses typical carriers and preparation methods. For example, the formulation may include pharma- ceutically acceptable carriers such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, bulking agents, desiccants, antioxidants, antimicrobial agents, preservatives, binders, bulking agents, silica, solubilizers, or stabilizers. The conjugates may also be encapsulated in suitable biocompatible microcapsules, microparticles, nanoparticles, or microspheres formed from biodegradable or non-biodegradable polymers or proteins or liposomes for targeting to cells. The particles may allow for controlled release of the active agent. The particles may be microparticles and / or nanoparticles. The particles may include one or more polymers. One or more of the polymers may be synthetic polymers. The particles may be formed, for example, by single or double emulsion techniques, or nanoprecipitation. Such systems are well known to those skilled in the art and may be optimized for use with the appropriate nucleic acid.

[0039] For example, suitable formulations for parenteral administration by intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, solutions, or emulsions that may contain suspending agents, solubilizing agents, thickening agents, dispersing agents, stabilizers, and preservatives. Preparations for injection are presented in unit dosage form, for example, in ampoules or multi-dose containers, and preservatives may be added, if desired. The conjugates may take the form of sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which in certain embodiments may be isotonic with the blood of the subject. Examples of non-aqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, and the like, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono- or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases. In addition, sterile fixed oils are routinely employed as solvents or suspending media. For this purpose, any sterile fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.Those skilled in the art can easily determine the various parameters for preparing and formulating the conjugates without undue experimentation.

[0040] The conjugates, alone or in combination with other suitable components, can also be made into aerosol formulations to be administered via inhalation (i.e., the conjugates can be "nebulized"). The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and air. For administration by inhalation, the compounds are delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers, using a suitable propellant.

[0041] An effective or therapeutically effective amount of a conjugate may be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or otherwise produce a desired pharmacological and / or physiological effect, e.g., reduce, inhibit, or negate one or more of the underlying pathophysiological mechanisms underlying a disease or disorder. The exact dosage will vary according to a variety of factors, such as formulation- and subject-dependent variables (e.g., age, immune health, clinical symptoms, etc.).

[0042] The conjugates, particularly formulations containing the conjugates, can be administered to or otherwise contacted with target cells once, twice, or three times daily, once, twice, three, four, five, six, seven times weekly, or once, twice, three, four, five, six, seven, or eight times monthly. For example, in some embodiments, the compositions are administered every 2 or 3 days, or on average about 2 to about 4 times per week. EXAMPLES

[0043] The present disclosure is illustrated by the following examples, which are not intended to limit the scope of the claims.

[0044] Unless otherwise specified, materials and reagents were obtained from Sigma-Aldrich or Thermo Fisher Scientific and used as received. tGalNAc ligand was purchased from Sussex Research (Ottawa, Canada).

[0045] Example 1. Synthesis of PNA-Carbohydrate Conjugates PNA-LBA conjugates were synthesized as shown in FIG. 1A and FIGS. 2A and 2B.

[0046] 1,6-Diaminohexane-lysine backbone: The 1,6-diaminohexane-lysine scaffold was synthesized by an amide coupling reaction (FIG. 1A). The synthesis was initiated by coupling FMOC-protected lysine with 6-aminoBoc-protected 1,6-diaminohexane in DMF solvent with HBTU and DIEA. The two FMOC groups on the lysine were then deprotected using 20% ​​piperidine in DMF solution to liberate the alpha and epsilon amino groups on the lysine to provide the scaffold for coupling to two lactobionic acid molecules.

[0047] Carbohydrate Ligands: LBA-Based Ligands In the second step shown in Figure 1A, the scaffold was functionalized with LBA. First, LBA was converted to the lactone form by refluxing in methanol with a catalytic amount of TFA, and then the solution was basified with DIPEA to react the lactone form to the scaffold. After the reaction, the hydroxyl group on LBA was acetylated for purification. Mass spectrometry, as well as 1 H and 13 C NMR spectroscopy confirmed the identity of the reaction products.

[0048] Functionalized Peptide Nucleic Acids: PNA oligomers targeting miR-122 (Figure 1C) were synthesized on an MBHA resin solid support implementing known standard BOC deprotection and synthesis protocols. Using normal PNA monomers, short (8mer) and full-length (22mer) PNAs targeting the seed region and the complete sequence of miR-122 were synthesized.

[0049] Lysine was added to the 3' end of PNA using standard methods. For solution-phase conjugation, PNA oligomers were then modified with a trioxo-miniPEG™ spacer at the 3' end (using 11-(boc-amino)-3,6,9-trioxaundecanoic acid) and succinic acid at the 5' end to generate a free -COOH functional group after cleavage. Solid-phase synthesis was performed using a 0.2 M solution of PNA monomer / lysine / trioxo-miniPEG / succinic acid / TAMRA in NMP, 0.52 M DIEA in DMF, and 0.39 M HBTU in DMF. For cellular uptake and biodistribution studies, TAMRA fluorescent dye was coupled onto the 3' end of PNA through a trioxo-miniPEG spacer. Detailed procedures for these materials and methods for PNA synthesis on MBHA resin have been reported previously (Malik, Shipra, Frank J. Slack, and Raman Bahal. "Formulation of PLGA nanoparticles containing short cationic peptide nucleic acids." MethodsX 7 (2020). 101115).

[0050] Carbohydrate-PNA conjugates: As shown in Figure 2A, functionalized PNA (PNA4) was coupled to the amino group of the LBA backbone ligand via the succinic acid at the 5' end. The reaction was carried out in a DMF / DMSO solvent mixture using HATU as the coupling reagent and DIEA as the base. The resulting amide bearing acetyl protection on the hydroxy group of LBA was treated with sodium methoxide in methanol to give the PNA4-LBA conjugate.

[0051] As shown in FIG. 2B, GalNAc-PNA4 conjugates were similarly prepared by reacting functionalized PNA containing a 5′-terminal succinic acid with a trioxo-miniPEG spacer and a free —COOH with the amino group of the GalNAc link backbone using HATU as a coupling reagent catalyzed by DIEA in DMF / DMSO solvent.

[0052] For quality control purposes, HPLC purity profiles and MALDI mass spectra of the carbohydrate ligand-conjugated PNAs were recorded at each step (Figure 2C). The PNA, PNA4-LBA, and PNA4-tGalNAc products were analyzed by MALDI-MS, demonstrating product formation. The products were also analyzed by reverse-phase HPLC, as shown in the inset of Figure 2C.

[0053] Example 2. In vitro binding studies Modifications on the PNA strand must not interfere with its target binding. To address whether modifications of the PNA strand by conjugation interfere with target binding, PNAs and their carbohydrate-PNA conjugates were assessed for their affinity to their targets by gel shift assays and thermal melting curve analysis.

[0054] Gel shift assay: PNAs were incubated with target DNA at a ratio of 2:1 (PNA:DNA) overnight at 37°C, and then samples were evaluated for separation on an 8% polyacrylamide gel. PNA1, PNA2-LBA, and PNA2-tGalNAc showed limited target binding due to their shorter sequences designed to target the seed region of miR-122 (Figure 3B). Full-length PNAs and conjugates showed complete binding to target DNA as seen by their retarded bands (Figure 3B). Gel images also showed more retardation in the bands for PNA4-LBA and PNA4-tGalNAc due to the increased molecular weight of PNA after carbohydrate ligand conjugation. Melting curve analysis supported these results, with an increase in melting temperature observed for carbohydrate ligand-conjugated PNA-DNA dimers (Figure 3A).

[0055] Example 3. Biodistribution studies in C57BL / 6J mice. Biodistribution of PNA and Ligand-PNA Conjugates A 24-hour biodistribution study after a single 125 μM, 10 mL / kg subcutaneous dose was studied in C57BL / 6J mice. All PNAs were tagged with a fluorescent TAMRA (rhodamine dye) probe. Three mice each at 0.5, 1, 2, 4, 8, and 24 hours were imaged for TAMRA fluorescence on an IVIS imaging device and sacrificed to remove major internal organs. Fluorescence images of different organs, including liver, lung, heart, kidney, and spleen, were recorded to examine the relative biodistribution (Figure 4A and Figure 5A). After subcutaneous administration, full-length PNA (PNA3) showed minimal concentrations in the liver only for up to 1 hour. From the early time points, a substantial portion of PNA3 in the kidney indicated its rapid elimination through renal excretion. Both PNA4-LBA and PNA4-tGalNAc targeted the liver very efficiently and were uniformly distributed in the liver for up to 24 hours (Figure 4A). The time-dependent PNA-TAMRA fluorescence curves from isolated liver were plotted and used to calculate the relative amount of PNA in the liver (Figure 4D). The area under the curve (AUC) of time liver fluorescence was calculated using GraphPad Prism software. Overall, the AUC0-24 fold changes of the PNA conjugates were significantly higher than PNA3. The AUC0-24 fold changes for PNA4-LBA and PNA4-tGalNAc were 20.34 ± 2.53 (p < 0.01), respectively. ** ) and 25.88 ± 4.52 (p ** ). PNA4-tGalNAc showed high initial concentrations in the liver, but the change in AUC0-24 was not significant compared to PNA4-LBA. Next, we performed FACS analysis of liver cells after passing liver tissue through a cell strainer followed by RBC lysis. The FACS data again confirmed comparable liver targeting and retention of PNA4-LBA and PNA4-tGalNAc (Figure 4B). Also, the visible fluorescence differences in IVIS images at early time points were not as pronounced in the FACS analysis. Next, cryosections were performed from 1 hour liver samples to image TAMRA fluorescence from tissue. Fluorescence images confirmed homogenous distribution for PNA4-LBA and PNA4-tGalNAc in liver sections (Figure 4C).

[0056] Biodistribution of anti-seed ligand-PNA conjugates: Similar to full-length PNA (PNA3), unconjugated short chain PNA1 was also found to be excreted by the kidney 1 hour after administration, reducing liver accumulation (Figure 5A). Both PNA2-LBA and PNA2-tGalNAc delivered PNA to the liver. Both conjugates accumulated in the liver for up to 24 hours, with maximum concentrations at 1 hour, however, concentrations declined significantly with time (Figure 5A). PNA2-tGalNAc resulted in higher liver accumulation than PNA2-LBA throughout the study. GraphPad Prism software was used to calculate the AUC of liver fluorescence, which was significantly higher for PNA-2 tGalNAc than PNA1 and PNA2-LBA (Figure 5C). The AUC of PNA2-LBA and PNA2-tGalNAc was 9 (p * ) and 28(p **** ) times higher. The high liver accumulation of PNA2-tGalNAc may be due to two main factors: one is the higher ASGPR affinity of tGalNAc than galactose in lactobionic acid, and the other is the rapid excretion of PNA from the body. Apparently, for shorter PNAs, the higher ASGPR affinity of tGalNAc seems to determine the hepatic uptake and excretion. Organ distribution at 0.5 h also showed that PNA2-LBA concentration was higher in the kidney than PNA2-tGalNAc (Figure 5A).

[0057] Confirmation of these observations by flow cytometry analysis was performed (Figure 5B). Histogram plots representing PNA concentrations in hepatocytes showed good overlap at 0.5 hours for PNA2-LBA and PNA2-tGalNAc, yet PNA2-tGalNAc showed higher fluorescence intensity. Conjugated PNAs showed long-term liver retention despite no significant changes in miR-122 levels in liver samples.

[0058] Hepatocyte targeting with LBA-PNA and tGalNAc-PNA conjugates: In an in vitro study using HepG2 cells, LBA and tGalNAc ligands were found to exhibit ASGPR-mediated cellular uptake. These results were confirmed after in vivo treatment in mice. One hour after subcutaneous administration of PNA and carbohydrate ligand-PNA conjugates, mouse livers were perfused and digested in situ. Liver cells were isolated using collagenase-mediated liver digestion, and the resulting liver cell suspension was enriched for hepatocytes using a Percoll gradient. The resulting hepatocyte fraction was stained with ASGPR and HNF-4α (hepatocyte-specific marker) fluorescent antibodies and analyzed by flow cytometry. Quadrant 2 represents TAMRA fluorescence of PNA, and double-positive quadrant 3 represents hepatocytes containing PNA. The higher Q3 / Q2 ratio for both PNA2-LBA and PNA2-tGalNAc over PNA1 confirmed the preferential accumulation of carbohydrate ligand-PNA conjugates in hepatocytes (Figure 5D).

[0059] Example 5. Efficacy Study miR-122 expression is liver specific and constitutes 60-70% of the hepatocyte miRNA pool. In healthy liver, miR-122 expression has a critical role in cholesterol and fatty acid metabolism. In biodistribution studies, both LBA and tGalNAc were found to exhibit excellent liver-targeted delivery of PNA. From the gel shift and melting curve analysis described above, it was found that the presence of carbohydrate ligands on the 5' end of PNA did not affect its target binding ability. It was the primary objective of this study to determine these findings for in vivo efficacy and safety. miR-122 levels were tested in liver samples from the biodistribution study and excellent knockdown of miR-122 levels was found by PNA4-LBA and PNA4-tGalNAc at 4, 8, and 24 hours. PNA3 also showed some degree of miR-122 knockdown. However, the levels of downstream targets of miR-122 (ALDOA, BCKDK, GYS1, NDRG3, and CUX-1) did not change significantly (data not provided). PNA for 24 hours or / and single dose was not sufficient to knockdown downstream targets of miR-122 highly expressed in liver. Short PNA and conjugates did not show significant knockdown of miR-122 due to poor binding of short PNA at the target site.

[0060] A comparison of the efficacy of PNA3, PNA4-LBA, and PNA4-tGalNAc in a multiple dose study with a saline treatment group serving as a control was performed. To determine efficacy, the levels of miR-122 and its downstream targets were measured in liver samples by RT-PCR and Western blot analysis. Both PNA4-LBA and PNA4-tGalNAc showed a more significant knockdown of miR-122, up to 75%, than PNA3 (p ** ) (FIG. 6A). Analysis of mRNA expression of downstream targets ALDOA, BCKDK, GYS1, NDRG3, and CUX-1 showed a significant increase in mRNA levels for PNA4-LBA and PNA4-tGalNAc treatments (≧p *) (FIG. 6A). Next, Western blot analysis for AlDoA and Bckdk proteins showed that PNA4-LBA (p * ) and PNA4-tGalNAc treatments (Figures 6B and 6C). PNA3 treatment did not show significant changes in the levels of downstream targets. Because miR-122 is a crucial regulator of cholesterol and fatty acid metabolism, we compared plasma levels of cholesterol and triglycerides. Plasma cholesterol and triglyceride levels in the PNA4-LBA and PNA4-tGalNAc groups were lower than those in the PNA3 and saline-treated groups. Cholesterol and triglyceride levels were significantly lower after PNA4-LBA treatment (p * ). PNA4-LBA and PNA4-tGalNAc treated groups showed lower plasma glucose levels, but the differences were not statistically significant (FIG. 7E). Furthermore, changes in liver weight (% of body weight) were also significantly less for PNA4-LBA and PNA4-tGalNAc, which correlates with reduced fat storage in the liver. Overall, the changes in all physiological parameters were modest, but considering the short duration of treatment in healthy mice, these findings are meaningful and support the efficacy of PNA after targeted delivery to the liver.

[0061] Example 6. Safety Study Safety evaluation was performed for PNA3, PNA4-LBA, and PNA4-tGalNAc in C57BL / 6J mice. For safety evaluation after three 5 mg / kg doses, mouse body weight and vital organ weights, histopathology by H&E staining (liver, kidney, and spleen), CBC analysis, comprehensive blood chemistry, and plasma levels of electrolytes and cytokine panel were determined. H&E staining performed on tissue sections of liver, kidney, and spleen did not show any significant histological differences between treatment groups (Figure 7A). No significant differences in mouse body weight were observed between different treatment groups during the study (Figure 7C). Similarly, during organ harvest (liver, kidney, spleen, heart, and lung), we did not find any obvious signs of toxicity. Organ weights (% of body weight) did not vary significantly, except for reduced liver weight in PNA4-LBA and PNA4-tGalNAc treatment groups. The observed reduction in fat accumulation by miR-122 knockdown may be one possible explanation for the context of the present invention, since the liver is a fat-storing organ. Elevated cytokine levels are a major indicator for any immune response elicited from oligonucleotide treatment. Blood was collected 24 hours after the last dose to estimate plasma levels of cytokines TNFα, IL-12p70, MCP-1, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IL-3, MIP-1α, MIP-1β, IL-25 using Luminex-based assays. None of these cytokines were found to be upregulated, and there was no significant difference in cytokine levels in saline, PNA3, PNA4-LBA, and PNA4-tGalNAc treatments (Figure 7B). The intrinsic levels of cytokines in our samples were lower than the calibration range of the kit.CBC analysis (Figure 7D), including RBC, WBC, platelets, and hemoglobin (data not shown), as well as comprehensive blood chemistry, including aspartate transaminase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK), and electrolytes, did not show any significant differences between the various treatment groups (Figure 7E). Electrolytes included phosphorus, calcium, sodium, potassium, magnesium, and chloride. Overall, the adjudication concluded that the tested dose regimens of PNA4-LBA and PNA4-tGalNAc were highly safe and effective.

[0062] Example 7. Kidney-targeted delivery of short PNA During the investigation of functional enhancement of LBA ligands, acetylated LBA ligand (LBAAc) showed good potential for targeting kidney. PNA2-LBAAc distribution was found to be mainly restricted to kidney according to our biodistribution study in C57BL / 6J mice (5 mg / kg, subcutaneous). Initially, at 1 h, both PNA1 and PNA2-LBAAc were distributed mainly in kidney and to some extent in liver (Figure 8A). In general, naked / unformulated PNAs exhibited rapid renal excretion, and we found maximum fluorescence intensity for short-chain PNA (PNA1) in kidney at 30 min, indicating its excretion peak time. Compared with PNA-1, PNA2-LBAAc after the initial distribution phase showed maximum accumulation in kidney (Figure 8A). The average emission efficiency of TAMRA-labeled PNA from kidney was determined at different time points (1, 4, 24, 48, and 72 h) for quantitative analysis (Figure 8B). Interestingly, the concentration of PNA2-LBAAc in the kidney at 48 hours was comparable to the excretory phase (1 hour) concentration of PNA1. These findings demonstrate the targeting and accumulation of PNA2-LBAAc in the kidney.

[0063] Flow cytometry analysis confirms PNA2-LBAAc conjugate distribution in the kidney: Kidney samples from 24 and 72 hours were passed through a 40 μm strainer, and after RBC lysis, kidney cell suspensions were fixed with 4% PFA and analyzed by flow cytometer. Histogram plots showing TAMRA fluorescent signal in kidney cells confirmed the IVIS study results showing higher retention up to 72 hours for PNA2-LBAAc in the kidney compared to PNA-1 (Figure 8C). Both IVIS and flow cytometry analysis confirmed kidney-targeted delivery of PNAs by LBAAc ligands. Those kidney sections were then imaged to examine the distribution of PNAs in the kidney. 10 μm thick sections from 24 hour kidney samples were fixed, permeabilized, and stained with DAPI for nuclear visualization. Fluorescence imaging observed a uniform distribution of PNA2-LBAAc within the kidney (Figure 8D). TAMRA signal intensity represents higher tubular accumulation of PNA2-LBAAc.

[0064] Kidney-targeted delivery of full-length (22mer) PNA: miR-21 is a kidney-specific microRNA found to be upregulated in renal fibrosis and a proven target for fibrosis treatment. Full-length (22mer) PNA oligomers, namely PNA5, PNA6, and LBAAc conjugate (PNA6-LBAAc) targeting miR-21 were designed and synthesized (Figure 9A). PNA6 was conjugated with LBAAc as described in Figure 2A.

[0065] Biodistribution assessment of PNA5 and PNA6-LBAAc in C57BL / 6J mice at a low subcutaneous dose of 1.5 mg / kg (1 / 10 molar equivalent of short-chain PNA1 and 2): LBA for kidney targeting AcFor comprehensive validation of the biodistribution of lower doses, we chose to use the IVIS imaging. From the IVIS imaging, the TAMRA fluorescence signal was restricted to the kidney for both PNA5 and PNA6-LBAAc (Figure 9B). At 4 hours, both PNA and conjugate showed similar distribution in the kidney, and their mean fluorescence intensities were comparable. At 24 hours, PNA6-LBAAc concentrations increased in the kidney, whereas PNA5 disappeared. Overall, a four-fold increase in kidney retention was observed for PNA6-LBAAc when compared to unconjugated PNA5 at 24 hours (Figure 9D). The results were supported by flow cytometry analysis of kidney cells. Kidney cells obtained after RBC lysis were fixed with 4% PFA and analyzed for TAMRA fluorescence. Histogram plots representing TAMRA fluorescence from 4-hour and 24-hour samples supported the excretion of PNA5 and accumulation of PNA6-LBAAc in kidney cells (Figure 9C). Overall, PNA5 was rapidly excreted without any tissue distribution, following the normal pharmacokinetics of PNA. On the other hand, PNA6-LBAAc showed slower biodistribution, likely due to the increased hydrophobicity of the conjugate. The Tmax for kidney accumulation of PNA6-LBAAc is >4 hours. Critically, the higher concentration of PNA6-LBAAc in the kidney at 24 hours compared to the 4 hour time point for PNA5 was due to the increased concentration of LBAAc in the kidney. Ac The renal targeting and potency of the ligand was confirmed.

[0066] The article Dhuri K, Bechtold C, Quijano E, Pham H, Gupta A, Vikram A, Bahal R. “Antisense Oligonucleotides: An Emerging area in Drug Discovery and Development.” Journal of Clinical Medicine, 2020, is incorporated by reference in its entirety.

[0067] The following abbreviations are used herein:

[0068] [Table 1]

[0069] Terms in this application have the following definitions.

[0070] As used herein, the compositions, methods, and articles may alternatively comprise, consist of, or consist essentially of any suitable components or steps disclosed herein. The compositions, methods, and articles may additionally or alternatively be formulated to be devoid of, or substantially free of, any steps, ingredients, materials, ingredients, adjuvants, or species that are not otherwise necessary to accomplish the function or purpose of the compositions, methods, and articles. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "Or" means "and / or" unless the context clearly dictates otherwise. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event occurs and instances when it does not occur.

[0071] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0072] As used herein, the terms "hydrocarbyl" and "hydrocarbon" refer to substituents containing carbon and hydrogen, and optionally having one to three heteroatoms, such as oxygen, nitrogen, halogen, silicon, sulfur, or combinations thereof. "Alkyl" refers to a linear or branched chain saturated monovalent hydrocarbon group. "Alkylene" refers to a linear or branched chain saturated divalent hydrocarbon group. "Alkylidene" refers to a linear or branched chain saturated divalent hydrocarbon group, with both valencies on a single common carbon atom. "Alkenyl" refers to a linear or branched chain monovalent hydrocarbon group having at least two carbons joined by a carbon-carbon double bond. "Cycloalkyl" refers to a non-aromatic monovalent monocyclic or polycyclic hydrocarbon group having at least three carbon atoms, and "cycloalkenyl" refers to a non-aromatic cyclic divalent hydrocarbon group having at least three carbon atoms, with at least one degree of unsaturation. "Aryl" refers to an aromatic monovalent group containing only carbon in the aromatic ring. "Arylene" refers to an aromatic divalent group containing only carbon in the aromatic ring. "Alkylaryl" refers to an aryl group substituted with an alkyl group as defined above, with 4-methylphenyl being an exemplary alkylaryl group. "Arylalkyl" refers to an alkyl group substituted with an aryl group as defined above, with benzyl being an exemplary arylalkyl group. "Acyl" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a carbonyl carbon bridge (-C(=O)-). "Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge (-O-). "Aryloxy" refers to an aryl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge (-O-).

[0073] Unless otherwise indicated, each of the foregoing groups may be unsubstituted or substituted, provided that the substitution does not significantly adversely affect the synthesis, stability, or use of the compound. As used herein, the term "substituted" means that at least one hydrogen on the specified atom or group is replaced with another group, provided that the normal valence of the specified atom is not exceeded. If the substituent is oxo (i.e., =O), then two hydrogens on the atom are replaced. Combinations of substituents or variables are permissible, provided that the substitution does not significantly adversely affect the synthesis or use of the compound. Exemplary groups that may be present in the "substituted" position include C-substituted aryl groups such as cyano, hydroxyl, nitro, azido, alkanoyl (acyl, etc. 2~6 alkanoyl groups), carboxamide, C 1~6 Or C 1~3 Alkyl, cycloalkyl, alkenyl, and alkynyl (including groups having at least one unsaturated linkage and 2 to 8 or 2 to 6 carbon atoms), C 1~6 Or C 1~3 Alkoxy, phenoxy, etc. 6~10 Aryloxy, C 1~6 Alkylthio, C 1~6 Or C 1~3 Alkylsulfinyl, C 1~6 Or C 1~3 Alkyl sulfonyl, amino di(C 1~6 Or C 1~3 ) alkyl, C having at least one aromatic ring 6~12 Aryl (e.g., phenyl, biphenyl, naphthyl, etc., in which each ring is either substituted or unsubstituted aromatic), C having 1 to 3 separated or fused rings and 6 to 18 ring carbon atoms 7~19 Examples of arylalkoxy include, but are not limited to, arylalkyl or arylalkoxy having 1 to 3 separated or fused rings and 6 to 18 ring carbon atoms (benzyloxy is an exemplary arylalkoxy). The indicated number of carbon atoms in a group does not include any substituents.

[0074] As used herein, peptide nucleic acid (PNA) is an artificially synthesized polymer with a backbone containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. As shown in Figure 1, the various purine and pyrimidine bases are linked to the backbone by methylene bridges (-CH2-) and carbonyl groups (-(C=O)-) to nitrogens on the backbone. By convention, PNAs are represented like peptides, with the N-terminus at the top (or left) and the C-terminus at the bottom (or right). PNAs are not formally peptides or nucleic acids, but rather a hybrid of the two.

[0075] In some embodiments, the PNA monomers forming the PNA oligomer are modified at the gamma position in the polyamide backbone (γPNA), as illustrated below, where "B" is the nucleobase and "R" is a substitution at the gamma position.

[0076] [ka]

[0077] Substitution at the gamma position creates chirality and provides helical preorganization to the PNA oligomer, resulting in substantially increased binding affinity to the target RNA. Other advantageous properties can be imparted depending on the chemical nature of the specific substitution at the gamma position (the "R" group in chiral gamma PNA described above). The synthesis of gamma PNA is described in U.S. Patent No. 10,221,216, which is incorporated herein by reference, for disclosure of gamma PNA and methods of synthesizing gamma PNA.

[0078] Examples of gamma substitutions with other side chains include gamma substitutions of alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, and derivatives thereof, where "derivatives thereof" are defined herein as those chemical moieties that are covalently attached to these amino acid side chains, e.g., to the amino acid side chains of serine, cysteine, threonine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, and arginine.

[0079] In one embodiment, the PNA oligomer that forms the PNA / RNA / PNA triplex is a gamma PNA with a tail clamp, or a gamma tc PNA.

[0080] Chemical modifications of the basic PNA structure are known and can be used. For example, fluorine-modified, cyclopentyl-modified, mini-PEG-modified, guanidinium-modified, pyrrolidinyl-modified, and 2-aminopyridinyl-modified PNA are known in the art and can be selected for the preparation of PNA oligomers to improve cell permeability or increase RNA binding affinity. Mini-PEG-containing γ-PNAs and their synthesis methods are described in U.S. Patent No. 10,793,605.

[0081] PNA oligomers can also contain other positively charged moieties to increase the solubility of PNA to increase cell permeability and / or to increase the affinity of PNA to target RNA.Commonly used positively charged moieties include the amino acids lysine and arginine, but other positively charged moieties can also be useful.Lysine and arginine residues can be added to the tcPNA linker or to the carboxy or N-terminus of the PNA oligomer chain.

[0082] Exemplary modifications to PNAs include, but are not limited to, the incorporation of charged amino acid residues such as lysine at the termini or internal portions of the oligomer, the inclusion of polar groups in the backbone, carboxymethylene bridges, and nucleobases, chiral PNAs with substitutions on the original N-(2-aminoethyl)glycine backbone, replacement of the original aminoethylglycyl backbone with a negatively charged scaffold, conjugation of high molecular weight polyethylene glycol (PEG) to one of the termini, fusion of PNAs to RNA, redesign of the backbone structure, and conjugation of PNAs to DNA or RNA to generate chimeric oligomers. These modifications improve solubility, but often result in reduced binding affinity and / or sequence specificity.

[0083] Gamma-PNA modifications include serine, lysine, glutamic acid, or alanine modifications. In particular, when the PNA is serine gamma modified, gamma PNA targets RNA more efficiently compared to conventional full-length PNAs based on their binding affinity.

[0084] Phosphorothioate analogs of DNA, RNA, and OMe-RNA have sulfur instead of oxygen as one of the non-bridging ligands attached to phosphorus.

[0085] Morpholinos, also known as morpholino oligomers and phosphorodiamidate morpholino oligomers (PMOs), are a type of oligomer used in molecular biology to modify gene expression. Their molecular structure contains DNA bases attached to a backbone of methylene morpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (~25 bases) specific sequences on the base-pairing surface of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function.

[0086] Locked nucleic acids are RNA derivatives in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformational restriction increases the binding affinity to complementary sequences and provides a chemical approach for the control of gene expression and the optimization of microarrays.

[0087] 2'-O-methylation is an RNA nucleoside modification in which a methyl group is added to the 2' hydroxyl of the ribose moiety of the nucleoside to generate a methoxy group. 2'-O-methylated nucleosides are found predominantly in ribosomal RNA and small nuclear RNAs and occur in functionally essential regions of the ribosome and spliceosome.

[0088] Similar to 2'-OMe nucleoside modifications of RNA, the 2'-O-methoxyethyl-RNA (2'-MOE) backbone provides enhanced duplex stability and significant nuclease resistance.

[0089] 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation.

[0090] 5'-methylcytosine is a methylated form of the DNA base cytosine (C), which regulates gene transcription and plays several other biological roles.[1] When cytosine is methylated, the DNA maintains the same sequence, but the expression of the methylated gene can change.

[0091] G-clamp heterocyclic modifications, which are cytosine analogs that clamp onto guanine by forming additional hydrogen bonds, have been rationally designed to enhance oligonucleotide / RNA hybrid affinity. PNAs containing internally-linked guanidinium moieties (GPNAs) are readily taken up by mammalian cells and bind to DNA and RNA with high affinity and sequence specificity.

[0092] A protecting group is a functional group that converts a reactive functional group in an organic molecule so that it does not undergo reaction for another functional group in the structure. Protecting groups are widely used in various forms in organic synthesis. The need for protection and deprotection, and the selection of appropriate protecting groups can be easily determined by those skilled in the art. The chemical nature of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 4d.Ed., Wiley&Sons, 2007. The adjustments to the protecting groups described herein, as well as the formation and cleavage methods, can be adjusted as necessary to take into account various substituents.

[0093] Residue is used to represent any of the moieties that are integrated to make up a larger molecule such as a conjugate. For example, lysine residue refers to the essential lysine amino acid structure of the conjugate, which is covalently linked to an alkyldiamine via the lysine carboxyl group (by an amide functional group) and covalently linked to a lactobionic acid molecule via its alpha amino group and epsilon amino group (by an amide bond), as shown in the drawings of this specification. Residue can also be referred to as moiety.

[0094] RNA as used herein includes different types of RNA that perform different functions, including messenger RNA, transfer RNA, ribosomal RNA, and microRNA. MicroRNA (miRNA) is involved in gene expression. miRNA is a non-coding region of mRNA that is believed to be important in either promoting or inhibiting gene expression. They can involve small sequences of about 25 nucleotides.

[0095] A sequence of bases is a run of bases represented by a series of five different sets of letters that represent the order of nucleotides that form alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequences are usually presented from the 5' to the 3' end. For DNA, the sense strand is used. Since nucleic acids are usually linear (unbranched) polymers, specifying a sequence is equivalent to defining the covalent structure of the entire molecule.

[0096] Gene expression is the process by which genes that code for information are converted into structures that exist and operate within a cell. Expressed genes include those that are transcribed into mRNA and then translated into proteins, and those that are transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA). miRNAs are non-coding regions of mRNA that are believed to be important in either promoting or inhibiting gene expression.

[0097] Below are brief descriptions of some gene names and microRNAs known in the art. Additional information can be found at the website https: / / www.genecards.org / , which is incorporated herein by reference.

[0098] PKD1 gene is polycystin 1 (interacting transient receptor potential channel or polycystic kidney disease 1); PKD2 gene is polycystin 2 (transient receptor potential cation channel or polycystic kidney disease 2); β-catenin means catenin β1 and junction plakoglobin; glutamine synthetase means lengusin (lens protein with glutamine synthetase domain); c-Myc means MYC binding protein, TTR means transthyretin also known as prealbumin (amyloidosis type I, carpal tunnel syndrome type 1, factor VII blood clotting factor VII, or blood clotting factor VII (serum prothrombin promoting factor)), Eg5 means neuronatin, PCSK9 means proprotein convertase subtilisin / kexin type 9, AAT means apoptosis antagonistic transcription factor, TPX2 means TPX2, apoB means apolipoprotein B, SAA means serum amyloid A1 cluster (RSV), PDGF means platelet-derived growth factor subunit A, miR-122 means microRNA 122, miR-223 means microRNA 223, and miR-21 was one of the first microRNAs identified and is located at the Vacuole Membrane Protein 1 (VMP1) locus on chromosome 17. It has been implicated in both neoplastic and non-neoplastic pathologies through many of its gene targets. Three of the major targets of miR-21 are phosphatase and tensin homolog (PTEN), Tropomyosin 1 (TPM1), and Programmed Cell Death 4 (PDCD4), and miR-155 is a microRNA in humans. miR-155 plays a role in various physiological and pathological processes involving the progression of malignancies, viral infections, and cardiovascular diseases.

[0099] miR-132 is a microRNA with described targets that include mediators of neurodevelopment, synaptic transmission, inflammation, and angiogenesis.

[0100] miR-125b is a microRNA that is involved in the regulation of NF-κB, p53, PI3K / Akt / mTOR, ErbB2, Wnt, and other signaling pathways, thereby controlling cell proliferation, differentiation, metabolism, apoptosis, drug resistance, and tumor immunity.

[0101] miR-146a is a microRNA whose target genes are thought to be involved in regulating pathophysiological processes in neurological diseases, particularly in neuroinflammatory responses, and is thought to play a critical role in neuroinflammation during the progression of neurological diseases.

[0102] The microRNA precursor miR-181 is a small non-coding RNA molecule that is transcribed as a ~70 nucleotide precursor and then processed by the RNase-III enzyme Dicer to give a ~22 nucleotide mature product. They target and regulate protein expression by inhibiting translation and / or inducing degradation of target messenger RNA. This new class of genes has recently been shown to play a central role in malignant transformation. miRNAs are downregulated in many tumors and therefore appear to function as tumor suppressor genes. The mature products miR-181a, miR-181b, miR-181c, or miR-181d are believed to have a regulatory role at the post-transcriptional level through complementarity to target mRNAs.

[0103] let-7The lethal-7 (let-7) gene was first discovered in C. elegans as a key developmental regulator and became one of the first two microRNAs known (the other being lin-4). Expression of let-7 members is controlled by MYC binding to their promoters. let-7 has been demonstrated to be a direct regulator of RAS expression in human cells. Many reports have shown that the expression levels of let-7 are frequently low and that the chromosomal cluster of let-7 is often deleted in many cancers. let-7 is expressed at higher levels in more differentiated tumors, which also have lower levels of activated oncogenes such as RAS and HMGA2. Thus, the expression level of let-7 may be a prognostic marker in some cancers related to the differentiation stage.

[0104] miR-34a is a microRNA that has been found to be dysregulated in various cancers and is also the first miRNA demonstrated to be directly regulated by the tumor suppressor p53. The miR-34 family is known to inhibit tumorigenesis. Expression of the miR-34 family relies on endogenous expression or mimics transfection.

[0105] miR-805 is a microRNA that has been reported to be downregulated in LPS-treated macrophages. The microRNA miR-690 has been reported to be highly expressed in M2-polarized bone marrow-derived macrophage exosomes and function as an insulin sensitizer both in vivo and in vitro.

[0106] miR-134 is a brain-specific microRNA that has been reported to be specifically localized in hippocampal neurons and may indirectly regulate synapse development through antisense pairing with LIMK1 mRNA. In the human brain, SIRT1 is thought to mediate CREB protein through miR-134, giving microRNA a role in higher brain functions such as memory formation.

[0107] Polycystic kidney disease (PKD) is an inherited disorder in which clusters of cysts develop primarily in the kidneys, causing them to enlarge and lose function over time. Cysts are non-cancerous round sacs that contain fluid. Cysts vary in size and can grow very large. Having many cysts or large cysts can damage the kidneys.

[0108] All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in this application contradicts or conflicts with a term in an incorporated reference, the term from this application will take precedence over the conflicting term from the incorporated reference.

[0109] While typical embodiments have been described for purposes of illustration, the foregoing description should not be deemed a limitation on the scope of the present specification. Accordingly, various modifications, adaptations, and alternatives may occur to those skilled in the art without departing from the spirit and scope of the present specification.

Claims

1. The gene sequence-carbohydrate conjugate is given by the following formula: 【Chemistry 1】 During the ceremony, GS is a gene sequence, and each gene sequence is either natural or modified, and the gene sequence has a 3' end and a 5' end. R １ and R ２ However, each independently, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, X １ is O, NR ３ , C=O, or C(R ３ ) ２ and R ３ is H, or substituted or unsubstituted C １ to C ６ alkyl, X ２ However, O, NR ３ , or C(R ３ ) ２ And R ３ However, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, G １ However, it is a group that directly bonds or links PNA to the conjugate, G ２ However, H is the sensual part, CL is a carbohydrate ligand containing 2 to 16 carbohydrate residues derived from a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. Optionally, the carbohydrate ligand has a hydroxyl group or an amino group with C ２ ~C １５ It is completely or partially acylated with an acyl group. n １ However, the range is 1 to 20. n ２ However, these are gene sequence-carbohydrate conjugates ranging from 0 to 20.

2. The gene sequence is a peptide nucleic acid or oligonucleotide comprising an mRNA sequence, a siRNA sequence, or a DNA sequence, Each of the aforementioned gene sequences includes gamma-serine modified gamma peptide nucleic acid, alanine gamma peptide nucleic acid, clamp G modified peptide nucleic acid, locked nucleic acid (LNA), phosphorothioate (PS), phosphorodiamidate morpholino (PMO), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'F), 5'-methylcytosine, or a combination thereof. The CL comprises a carbohydrate residue derived from a monosaccharide or disaccharide, The conjugate according to claim 1, wherein the carbohydrate ligand is completely or partially acetylated at its hydroxyl group or amino group.

3. The conjugate according to claim 1, wherein the gene sequence comprises a chemically modified nucleotide including PNA, mRNA, or siRNA.

4. The conjugate according to claim 3, wherein the chemically modified nucleotide comprises locked nucleic acid (LNA), phosphorothioate (PS), phosphorodiamidate morpholino (PMO), 2'-O-methyl (2'-O-Me), G-clamp, 2'-O-methoxyethyl (2'-O-MOE), siRNA, 2'-fluoro (2'F), 5'-methylcytosine, or a combination thereof.

5. The conjugate according to claim 1, wherein the gene sequence is effective in targeting asialoclycoprotein receptors (ASGPR) on hepatocytes, or the PNA is effective in targeting kidney cells.

6. A conjugate according to claim 1 for regulating a target gene, target mRNA, microRNA, or non-coding RNA.

7. The conjugate according to claim 6, wherein the target gene or RNA is PKD1, PKD2, beta-catenin, glutamine synthetase, c-Myc, TTR, factor VII, Eg5, PCSK9, AAT, TPX2, apoB, SAA, RSV, PDGF, miR-122, miR-223, miR-21, miR-155, miR-132, miR-125b, miR-146a, miR-181, let-7, miR-34a, miR-805, miR-690, miR-134, miR-494, miR-202-5p, or miR-192.

8. G １ However, it has 1 to 20 carbon atoms, or G １ The conjugate according to claim 1, wherein the PNA is a functional portion that connects to the conjugate.

9. The conjugate according to claim 8, wherein G1 is a residue of polyethylene glycol, polypropylene glycol, or polyethylene-propylene glycol, or such a residue of a trioxo-minipolyethylene glycol (PEG) chain.

10. G ２ However, it has 1 to 20 carbon atoms, or G ２ The conjugate according to claim 1, wherein the PNA is a functional portion that connects to the conjugate.

11. The conjugate according to claim 10, wherein G2 is a residue of polyethylene glycol such as a trioxo-miniPEG chain, polypropylene glycol, or polyethylene-propylene glycol.

12. The carbohydrate ligand is X １ The conjugate according to claim 1, further comprising a linker for adhesion to a surface.

13. The conjugate according to claim 1, wherein the carbohydrate residue is a fully or partially acylated carbohydrate residue having 2 to 15 carbon atoms in the acyl group.

14. The conjugate according to claim 13, wherein the carbohydrate residue is a completely or partially acetylated carbohydrate residue.

15. The conjugate according to claim 1, wherein the functional portion on the conjugate further comprises a linker for attachment to kyerin, tolvaptan, nintedanib, paclitaxel, bleomycin, cyclosporine, cisplatin, romidepsin, doxorubicin, docetaxel, danunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, prednisolone, cytarabine, or pirfenidone.

16. The conjugate according to claim 1 for regulating a target gene, mRNA, microRNA, non-coding RNA, DNA, hormone, cellular protein, or enzyme.

17. The conjugate according to claim 1, wherein the target gene or RNA is PKD1, PKD2, GPX1, GPX4, CYP11B2, ERCC4, ERCC2, GSTO1, GSTO2, UMOD, MGP, GLO1, SLC7A9, SHROOM3, VEGFA, APOL1, MYH9, miR-21, miR-17, MiR-10, miR-192, miR-216a and miR-217, miR-192, miR-377, miR-200c, miR-141, miR-205, and miR-192.

18. The conjugate according to claim 1, wherein the carbohydrate residue is derived from N-acetylgalactosamine (GalNAc), galactose, lactobionic acid, or an acetylated ester thereof.

19. The conjugate according to claim 18, wherein the carbohydrate residue is a completely or partially acetylated product of N-acetylgalactosamine (GalNAc), galactose, or lactobionic acid. 【Request Item 20】 【Chemistry 2】 During the ceremony, LBA is a lactobionic acid residue, X １ However, NR ３ , O, or C(R ３ ) ２ And R ３ However, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, each X ３ However, independently, O, NR ３ , or C(R ３ ) ２ And R ３ However, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, n ３ However, the range is 0 to 20. n ４ The conjugate according to claim 1, wherein the formula is 1 to 8.

21. G １ However, it is a base that connects the PNA to the conjugate, R １ and R ２ However, each of them is H, X １ , X ２ , and X ３ However, each of them is NH, n １ = 6, n ２ = 2, and n ３ The conjugate according to claim 20, wherein the value is 4.

22. R １ and R ２ However, each of them is H, n ３ = 4, The conjugate according to claim 20, wherein the PNA is connected to the conjugate at its 5' end. 【Request Item 23】 【Chemistry 3】 The conjugate according to claim 20, which is the formula.

24. The conjugate according to claim 23, wherein G1 is a functional portion that connects PNA to the conjugate, and G2 is a functional portion.

25. The conjugate according to claim 20, wherein the lactobionic acid residue is completely or partially acylated with an acyl group having 2 to 15 carbon atoms.

26. The conjugate according to claim 25, wherein the lactobionic acid residue is completely or partially acetylated. 【Request Item 27】 【Chemistry 4】 During the ceremony, X １ However, C = O or C(R ３ ) ２ And R ３ However, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, X ４ However, O, NR ３ , or C(R ３ ) ２ And R ３ However, H, or substituted or unsubstituted C １ ~C ６ It is alkyl, CL is a substituted or non-substituted C １ ~C １２ C containing alkyl, amide, ester, or ether groups ６ ~C １２ A carbohydrate residue linked to CH by a linker chain of 1 to 30 atoms including aryl atoms. n ４ The conjugate according to claim 1, wherein the formula is 2 or 3.

28. The conjugate according to claim 27, wherein the carbohydrate residue is a fully or partially acylated carbohydrate residue having 2 to 15 carbon atoms in the acyl group.

29. The conjugate according to claim 28, wherein the carbohydrate residue is a completely or partially acetylated carbohydrate residue.

30. The conjugate according to claim 27, wherein the carbohydrate residue is derived from N-acetylgalactosamine (GalNAc) or from fully or partially acylated acetylgalactosamine.

31. The conjugate according to claim 1, formulated with a polymer, lipid, protein, or other pharmaceutical excipient for organ-specific delivery.

32. The gene sequence is conjugated to a carbohydrate ligand to obtain the gene sequence-carbohydrate conjugate described in claim 1 (wherein X ２ However, O or NR ３ A method to provide the following: The objective is to provide a free -COOH functional group by functionalizing the aforementioned gene sequence, The free -COOH functional group of the modified gene sequence and Y of the compound of the following formula ２ This includes forming a bond between and 【Transformation 5】 In the formula, Y ２ However, -NHR ３ Alternatively, a method involving -OH.

33. The method according to claim 32, wherein the conjugation is performed by solution-phase synthesis, solid-phase synthesis, or a combination thereof.

34. Before functionalizing the aforementioned gene sequence, G １ G ２ The method according to claim 32, further comprising modifying the gene sequence with a precursor of a combination thereof.

35. The gene sequence is conjugated to a lactobionic acid-backbone ligand to obtain the gene sequence-lactobionic acid conjugate described in claim 20 (wherein X １ and X ２ However, each is independent of O or NR ３ A method to provide the following: The objective is to provide a free -COOH functional group by functionalizing the aforementioned gene sequence, The free -COOH functional group of the modified gene sequence and Y of the following formula ２ This includes forming a bond between and 【Transformation 6】 In the formula, Y ２ However, -NHR ３ Alternatively, a method involving -OH.

36. This further includes reacting lactobionic acid with the skeleton of the following formula, 【Transformation 7】 During the ceremony, X ３ is -OH or NHR ３ and X ２ However, protected O or protected NHR ３ The method according to claim 35.

37. A gene sequence-carbohydrate conjugate according to any one of claims 1 to 31 for use in a method for reducing the expression of targeted RNA involved in health hazards in a subject, wherein the method is A gene sequence-carbohydrate conjugate comprising providing a gene sequence-lactobionic acid conjugate according to any one of claims 1 to 31 to the target cells in vivo or ex vivo, wherein the binding of the PNA of the conjugate to the target RNA reduces the expression of the target RNA.

38. The gene sequence-carbohydrate conjugate according to claim 37, wherein the targeted RNA is a microRNA.

39. A gene sequence-carbohydrate conjugate according to any one of claims 1 to 31 for use in a method for targeting DNA and gene editing in a health disorder in a subject, wherein the method is A gene sequence-carbohydrate conjugate comprising providing the gene sequence-carbohydrate conjugate according to any one of claims 1 to 31 to the target cells in vivo or ex vivo, wherein the DNA of the conjugate targeted to the cells modulates gene expression.

40. The gene sequence-carbohydrate conjugate according to claim 37, comprising targeting liver cells or kidney cells and performing the procedure on a subject that requires the regulation of the expression of cellular nucleic acid function, and administering the gene sequence-carbohydrate conjugate according to any one of claims 1 to 31 to the subject.

41. The gene sequence-carbohydrate conjugate according to claim 37, wherein the cell is a cancer cell.

42. The gene sequence-carbohydrate conjugate according to claim 37, wherein the PNA comprises the kidney-specific microRNA, miR-21.

43. The gene sequence-carbohydrate conjugate according to claim 40 for the treatment of renal fibrosis and polycystic kidney disease.

44. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 31 and a pharmaceutical excipient.

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