Genetic sequence-glycoconjugates for enhancing liver and kidney specific targeting

Abstract

Disclosed is a genetic sequence-glycoconjugate having the formula (I): # imgabs0 # wherein GS is a genetic sequence, preferably a peptide nucleic acid or oligonucleotide, such as an mRNA sequence, siRNA sequence or DNA sequence, optionally wherein each genetic sequence is natural or modified, and the variables are as described herein. Also disclosed are uses of the genetic sequence-glycoconjugates, including as RNA therapeutics that target the liver and kidney of mammals, including humans.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 335,174, filed on April 26, 2022, the entire contents of which are incorporated herein in their entirety. Background Art

[0003] The present patent application relates to a genetic sequence-sugar conjugate, such as a peptide nucleic acid-sugar conjugate, a method for preparing the conjugate, a composition containing the conjugate, and a use of the conjugate. The conjugate is particularly useful for improving the targeting of liver cells and kidney cells.

[0004] The liver and kidneys are important organs involved in critical body functions including metabolism, detoxification, excretion, synthesis of proteins and lipids, secretion of cytokines and growth factors, and immune / inflammatory responses. Liver disorders such as hepatitis, alcoholic or non-alcoholic 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 1 in 7 American adults, or about 37 million American adults, have chronic kidney disease (CKD). Renal fibrosis is the final manifestation of chronic kidney disease. Other kidney diseases include cancer, IgA nephropathy, membranous nephropathy, and acute kidney injury.

[0005] Many therapeutic agents are delivered to body tissues where they are not expected to play a role, which may lead to undesirable or harmful side effects. Therefore, it is necessary to safely and effectively deliver therapeutic molecules (e.g., genetic sequences and activating agents, such as drugs, genes or proteins) to their target sites (including liver and kidney) in the body. These unmet needs and long-standing unresolved problems are solved by the novel compositions and methods described below. Summary of the invention

[0006] Disclosed is a genetic sequence-sugar conjugate having the formula:

[0007]

[0008] wherein GS is a genetic sequence, preferably a peptide nucleic acid or oligonucleotide, such as an mRNA sequence, an siRNA sequence or a DNA sequence, optionally wherein each genetic sequence is natural or modified, for example, including γ-serine modified γ peptide nucleic acid, alanine γ 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 genetic sequence has a 3' end and a 5' end, R1 and R2 are each independently H or a substituted or unsubstituted C1 to C6 alkyl, X 1 Yes O, NR 3 , C=O or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, X 2 Yes O, NR 3 or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, G 1 is a direct bond or group connecting the PNA to the conjugate, G 2 is H or a functional moiety, CL is a sugar ligand comprising 2 to 16 sugar residues derived from a monosaccharide, a disaccharide, a trisaccharide or a tetrasaccharide, preferably wherein CL comprises sugar residues derived from a monosaccharide or a disaccharide, optionally wherein the sugar ligand is replaced by a C2 to C 15 The acyl group is fully acylated or partially acylated, preferably wherein the sugar ligand is fully acetylated or partially acetylated on its hydroxyl group or amino group, 1 is 1 to 20, and n 2 0 to 20.

[0009] Methods for producing genetic sequence-glycoconjugates, in particular PNA-glycoconjugates, are described.

[0010] The pharmaceutical composition comprises a genetic sequence-sugar conjugate, in particular a PNA-sugar conjugate, and a pharmaceutical excipient.

[0011] Methods of using genetic sequence-glycoconjugates, particularly PNA-glycoconjugates, are described.

[0012] A method for reducing the expression of a targeting RNA involved in a health condition in a subject comprises providing a genetic sequence-lactobionic acid conjugate as described herein to cells of the subject in vivo or ex vivo, wherein binding of the PNA of the conjugate to the targeting RNA reduces the expression of the targeting RNA, especially when the targeting RNA is a microRNA.

[0013] A method for DNA targeting and gene editing of a health condition in a subject comprises providing a genetic sequence-sugar 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 regulates expression of a gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings are exemplary embodiments and are provided to illustrate the present disclosure. The drawings are not intended to limit the compositions, methods, or articles prepared according to the present disclosure to the materials, conditions, or process parameters described herein.

[0015] Figure 1A The design and synthesis of the peptide nucleic acid of the linker ligand and the targeting miR-122 of lactobionic acid (LBA) are shown. Specifically, (A) is the synthesis scheme of lysine linker (1-4) and the schematic diagram of the conjugation of LBA and lysine linker (5-8). (B) shows the chemical structure of DNA and conventional PNA unit. (C) shows the nucleotide sequence of mature miR-122-5p (top) and seed region (underline). PNA oligomer (bottom) of targeting seed and full-length miR-122. PNA 1 and PNA 2 are designed to target seed region, while PNA 3 and PNA 4 can be bound to full-length miR-122. PNA2 and PNA 4 contain succinic acid (SA) for conjugation with LBA-lysine conjugate at the 5' end. Lysine (K) is added to the 3' end of PNA, and then the fluorescent probe 5-carboxytetramethylrhodamine (TAM) is added to each PNA. "OOO" indicates a trioxo-miniPEG linker.

[0016] FIG. 2 shows the conjugation of PNA 4 to LBA and tGalNAc and their quality control evaluation. Specifically, (A) shows the solution phase conjugation of PNA 4 (14) to LBA (8-16). (B) shows the solution phase conjugation of PNA 4 (14) to tGalNAc (13-18). (C) is the MALDI-MS of ligand-conjugated PNA, PNA4-LBA and PNA4-tGalNAc. The calculated mass and observed mass of the conjugate are plotted on the respective mass spectra. The inset shows the reverse phase-high performance liquid chromatography (RP-HPLC) trace of the specified conjugate.

[0017] Fig. 3 shows the results of biophysical target binding assessment of PNA-conjugated ligands. (A) shows the normalized thermal melting curves of short PNA and full-length PNA and conjugates with the target DNA sequence of miR-122 under low-salt physiological conditions. (B) shows the gel shift binding assay performed with a DNA:PNA ratio of 1:2 under low-salt physiological conditions. DNA and PNA-DNA heteroduplexes (blocking bands) were developed using SYBR Gold staining.

[0018] Fig. 4 shows the results of in vivo biodistribution studies of full-length PNA conjugates (PNA 3 and PNA 4). (A) shows IVIS imaging of organs harvested from C57BL6 / J mice treated with full-length PNA and ligand conjugates at different time points after 10 mg / kg subcutaneous administration. (B) shows a histogram depicting the absorption of full-length PNA and ligand conjugates in hepatocytes from C57BL6 / J mice after subcutaneous administration analyzed by flow cytometry at different time points. (C) shows confocal microscopy images of frozen sections of the liver from C57BL6 / J mice 1h after subcutaneous administration. Blue represents nuclei and red represents TAMRA. (D) shows the average luminous efficiency of TAMRA fluorescence in the liver of C57BL6 / J mice treated with full-length PNA and ligand conjugates at different time points after subcutaneous administration. The results are represented by the mean value of n=3, with the standard error mean as the error bar.

[0019] Fig. 5 shows the in vivo biodistribution study of anti-seed PNA conjugates (PNA 1 and PNA 2).(A) shows IVIS imaging of organs harvested from C57BL6 / J mice treated with anti-seed PNA and ligand conjugates at different time points after 5mg / kg subcutaneous administration.(B) shows a histogram depicting the absorption of anti-seed PNA and ligand conjugates in the hepatocytes of C57BL6 / J mice after subcutaneous administration analyzed by flow cytometry at 0.5h and 24h.(C) shows the average luminous efficiency of TAMRA fluorescence in the liver of C57BL6 / J mice treated with anti-seed PNA and ligand conjugates at different time points after subcutaneous administration.(D) shows a flow cytometry dot plot depicting the hepatocyte absorption of anti-seed PNA and ligand conjugates in the hepatocytes from C57BL6 / J mice after 1h after subcutaneous administration.BV786 channel represents hepatocytes stained with ASGPR antibodies for ASGPR receptors. The FITC channel represents hepatocytes stained with an antibody against HNF4α of hepatocytes, and the TAMRA channel represents PNA.

[0020] Figure 6 shows (A) the relative expression levels of miR-122, ALDOA and BCKDK (downstream targets of miR-122) in the hepatocytes of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates. The results are expressed as the mean value of n=6, with the standard error mean as the error bar. Statistical analysis was performed using a t-test. *p<0.05, **p<0.01. (B) Representative protein blots of AldoA protein and Bckdk protein (downstream targets of miR-122) in the hepatocytes of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates are shown. The results are expressed as n=2. (C) The relative protein levels of ALDOA and BCKDK (downstream targets of miR-122) in the hepatocytes of C57BL6 / J mice after subcutaneous administration of full-length PNA and ligand conjugates are shown. The results are expressed as the mean value of n≥3, with the standard error mean as the error bar. Statistical significance was analyzed using nonparametric one-way ANOVA on GraphPad Prism software. Uncorrected Dunn's test was performed for multiple comparisons. Significance levels relative to the indicated treatment groups *p<0.05, **p<0.01, ***p<0.001.

[0021] Fig. 7 shows (A) 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 efficacy study.(B) Cytokine panel evaluation in C57BL6 / J mice treated subcutaneously with full-length PNA and ligand conjugates at the end of efficacy study is shown.(C) Average body weight of C57BL6 / J mice during efficacy study for different treatment groups is shown.Results are represented by the mean value of n=6, with standard error mean value as error bar.(D) Blood chemistry analysis performed in C57BL6 / J mice at the end of efficacy study for different treatment groups is shown, including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK) and electrolytes (such as phosphate ions, calcium ions, sodium ions, potassium ions, magnesium ions, chloride ions) and plasma glucose. The results are presented as the mean of n ≥ 3, with standard error of the mean as error bars.

[0022] FIG8 shows the effect of PNA 2-LBA at a subcutaneous dose of 5 mg / kg AcBiodistribution in C57Bl6 / J mice. (A) shows IVIS imaging of organs harvested from PNA 1, PNA 2-LBAAc and saline-treated control mice. (B) shows the average luminous efficiency of TAMRA fluorescence in the kidneys of C57BL6 / J mice. The results are expressed as mean values ​​of n≥2, with standard error mean values ​​as error bars. (C) shows a histogram depicting the absorption of PNA and acetylated ligand conjugates in kidney cells from C57BL6 / J mice after subcutaneous administration analyzed by flow cytometry at 24h and 72h. (D) shows confocal microscopy images of frozen sections of kidneys from C57BL6 / J mice 24h after subcutaneous administration. Blue represents nuclei. Red represents TAMRA.

[0023] Figure 9 shows that LBA Ac The ligand effectively delivers 22-mer-long PNA sequences to the kidney. (A) shows the nucleotide sequences of miR-21 and anti-miR-21 PNA and their ligand conjugates. PNA 6 contains succinic acid (SA) for conjugation with LBA-lysine conjugates at the 5' end. Lysine (K) is added to the 3' end of PNA, and then the fluorescent probe 5-carboxytetramethylrhodamine (TAM) is added to each PNA. OOO represents trioxo-miniPEG joints. (B) shows IVIS imaging of organs harvested from C57BL6 / J mice treated with PNA and ligand conjugates at 4h and 24h time points after 1.5mg / kg subcutaneous administration. (C) shows a histogram depicting the absorption of PNA and acetylated ligand conjugates in the kidney cells of C57BL6 / J mice after subcutaneous administration analyzed by flow cytometry at 4h and 24h. (D) shows the average luminous efficiency of TAMRA fluorescence in the kidney of C57BL6 / J mice. The results are shown as the mean of n=2, with the standard error mean as the error bar. DETAILED DESCRIPTION

[0024] Described herein are genetic sequence-sugar conjugates for targeted delivery of conjugates to specific organs in mammals (especially humans), more specifically to the liver and kidneys of mammals (such as humans). In one aspect, the genetic sequence-sugar conjugate is a peptide nucleic acid (PNA)-sugar conjugate (PNAC). Specifically, the conjugate is a molecule comprising a sugar ligand and a genetic sequence, the sugar ligand being covalently linked to the genetic sequence via a linker backbone, and the linker backbone being covalently bonded to both the sugar ligand and the genetic sequence. Sugar ligands targeting the liver or kidneys can be selected. Preferably, the conjugate selectively binds to a specific receptor on a cell to deliver a genetic sequence or other therapeutic agent to a cell carrying the receptor.

[0025] The conjugate used herein comprises a genetic sequence (GS) with 3' end and 5' end, which can be a PNA or oligonucleotide, such as an mRNA sequence, a siRNA sequence or a DNA sequence. Each genetic sequence can be natural or optionally modified (e.g., with a certain nucleotide order or by modification), such as γ-serine-modified γ peptide nucleic acid, alanine γ peptide nucleic acid, clamp G-modified peptide nucleic acid, locked nucleic acid (LNA), thiophosphate (PS), phosphorodiamidate morpholino oligomer (PMO), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluorine (2'F), 5'-methylcytosine or a combination thereof. In one aspect, the genetic sequence is a PNA. PNA can be modified as described below.

[0026] In one aspect, the conjugate carries 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 sugar residues (ligands). The number and type of sugar ligands targeting the liver or kidney, preferably selectively targeting the liver and kidney, are selected. For example, sugar ligands targeting the asialoglycoprotein receptor (ASPGR) expressed on cells can be selected. ASGPR is a C-type lectin, mainly expressed on the sinusoidal surface of hepatocytes. In one aspect, the conjugate carries 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 galactose ligands targeting ASPGR on liver cells and kidney cells. In one aspect, the conjugate carries 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 galactosamine (GalNAc) ligands targeting ASPGR on liver cells and kidney cells. In another aspect, the conjugate carries 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4, or 2 to 3 lactobionic acid ligands that target ASPGR on liver cells and kidney cells.

[0027] The sugar ligand of the conjugate can be completely acylated or partially acylated by C2 to C15 acyl groups on its hydroxyl group or amino group. For example, the sugar ligand can be completely acetylated or partially acetylated on the hydroxyl group or amino group. The acetylation of the sugar ligand can be carried out using an acetylation agent (such as acetic anhydride, acetyl chloride, mixed anhydride), an acid and a coupling agent (reagents such as DCC) and a base (such as triethylamine, pyridine, DIEA, DMAP, etc., or an organic base, an inorganic base or a polymer base used in the art). In one aspect, the sugar ligand is a GalNAc residue that is completely acylated or partially acylated (preferably acetylated, preferably completely acetylated). Alternatively, the sugar ligand is a lactobionic acid residue that is completely acylated or partially acylated (preferably acetylated, preferably completely acetylated).

[0028] Sugar ligands 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 the backbone generally contains at least two functional groups, such as at least two amino groups, one or more of which are used to react with the sugar ligand, and one or more are used to react 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 a portion that changes properties (such as solubility). For example, lysine residues and arginine residues can be present in the backbone.

[0029] In one aspect as shown in Formula I, the group G 1 or group G 2 Can be present optionally. 1 It can be a linker from the backbone to the genetic sequence, for example a linker having 1 to 20 carbon atoms and optionally one or more reactive groups such as hydroxyl, carboxyl, thio or amino. 1 or G 2 It can be a functional part. Functional part G 1 Functional part G 2 The conjugate may be provided with structural features that may confer desired functions, such as separation from stearic acid of a binding ligand, enhancement of the hydrophilicity or hydrophobicity of the conjugate, promotion of absorption of the conjugate, promotion of distribution of the conjugate in vivo, or other functions that are advantageous in terms of medicinal chemistry and drug design. The functional moiety may be attached between the backbone and the genetic sequence or at the end of the genetic sequence, or both. In one aspect, the functional moiety G 1 Functional part G 2 G is, for example, a residue of polyethylene glycol, polypropylene glycol, or polyethylene glycol-propylene glycol. 1 or G 2 , or both of them can be polyethylene glycol (PEG) groups. The PEG group can contain 1 to 25 ethylene glycol residues (-OCH2CH2O-) that can terminate at free hydroxyl, amino, ether or similar functional parts. The PEG group is optionally bonded to the main chain or structure of the ligand, conjugate.

[0030] In another aspect, the functional moiety may include a therapeutic agent. For example, kielin, tolvaptan, nintedanib, paclitaxel, bleomycin, cyclosporin, cisplatin, romidepsin, doxorubicin, docetaxel, daunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, prednisolone, cytarabine, or pirfenidone. Other therapeutic agents can be found in the Merck Index published in print and online by the Royal Society of Chemistry (URL: https: / / www.rsc.org / merck-index). 1 may be a linker between the backbone and the genetic sequence and include a therapeutic agent covalently bound thereto. Alternatively, or additionally, the group G 2 The therapeutic agent may be covalently bound to the genetic sequence directly or through a linker.Although not shown in Formula I, a functional moiety such as a therapeutic agent may also be attached to the backbone using a linkage similar to that used to attach sugar residues.

[0031] In one aspect, the conjugate is a genetic 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. PNA-lactobionic acid conjugate of Formula Ia

[0032]

[0033] Where LBA is a lactobionic acid residue, X 1 YesNR 3 , O or C (R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, each X 3 O, NR independently 3 or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, n3 is 0 to 20, and n4 is 1 to 8. Preferably, G1 is the group that connects the PNA to the conjugate, R 1 and R 2 H, X respectively 1 , X 2 and X 3 Each is NH, and n1=6, n2=2 and n3=4. In one aspect, R 1 and R 2 Each is H, n3=4, and a PNA is attached to the conjugate at the 5' end.

[0034] For example, the PNA-lactobionic acid conjugate may have Formula Ia-1

[0035]

[0036] Among them G 1 and G 2 As defined above, preferably wherein G 1 is the functional moiety that connects the PNA to the conjugate, and G 2 Optionally, in any one of Formula Ia and Formula Ia-1, the hydroxyl group may be fully acylated or partially acylated, preferably acetylated, more preferably fully acetylated as described above, with an acyl group having 2 to 15 carbon atoms or 2 to 8 carbon atoms.

[0037] In another aspect, the conjugate may have Formula Ib

[0038]

[0039] Where X 1 is C=O or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, X 4 Yes O, NR 3 or C(R 3 )2, where R 3 is H or a substituted or unsubstituted C1 to C6 alkyl group, CL is a sugar residue connected to CH via a linker chain having 1 to 30 atoms, the linker chain comprising a substituted or unsubstituted C1 to C6 alkyl group containing an amide group, an ester group or an ether group. 12 Alkyl or C6 to C 12 aryl, and n4 is 2 or 3. The sugar residue in Formula Ib may be derived from N-acetylgalactosamine, and may be a fully acylated or partially acylated sugar residue, wherein the acyl group has 2 to 15 carbon atoms or 2 to 8 carbon atoms, for example a fully acetylated or partially acetylated sugar residue, such as a fully acetylated sugar residue.

[0040] A method for conjugating a genetic sequence to a sugar ligand to provide a genetic sequence-sugar conjugate is described. The method comprises functionalizing the genetic sequence to provide a free -COOH functional group; and reacting the free -COOH functional group of the modified genetic sequence with Y of the compound of formula II. 2 Bond

[0041]

[0042] where Y 2 Yes - NHR 3 or -OH. The method can be performed by solution phase synthesis or solid phase synthesis or a combination thereof. The genetic sequence, such as PNA, can be obtained by solution phase synthesis or solid phase synthesis or a combination thereof as known in the art. The genetic sequence can be modified as described below. In addition, the method can also include using G before functionalizing the genetic sequence. 1 The precursor of G 2 precursors or a combination thereof to modify the genetic sequence.

[0043] In one aspect, 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 reacting the free -COOH functional group of the modified genetic sequence with Y of Formula III. 2 Bond

[0044]

[0045] where Y 2 Yes - NHR 3 Or -OH. In addition, the genetic sequence is preferably PNA. In one aspect, the method can further include reacting lactobionic acid with the backbone of formula IV

[0046]

[0047] Where X 3 Is -OH or NHR 3 , and X 2 is protected O or protected NHR 3 .

[0048] In one aspect, as described in the Examples and as shown in Figures 1 and 2, the lactobionic acid residue can be coupled to a backbone comprising a lysine residue via its α and ε amino groups. The lysine carboxyl group is then coupled to an amino group on an alkyl diamine, while another amino group is coupled to a succinyl COOH group attached to a peptide nucleic acid. In other aspects, the alkyl diamine can be substituted with an alkane diol to form a backbone with an ester bond. Alternatively, the succinic acid at the 5' end can be substituted with a substituted or unsubstituted C to C20 Dicarboxylic acid substitution. PNA is modified at the 5' end by a functional moiety (e.g., trioxo-miniPEG spacer and succinic acid) 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 hydroxyl group, an alkyl halide or other suitable functional group (e.g., lactobionic acid or GalNAc) on the ligand backbone.

[0049] Especially when using GalNAc, GalNAc can be connected to the main chain by the group carrying alkyl ether residue, amide residue, ester residue, to provide sugar ligand. Some of these parts are commercially available or can be synthesized using the chemical synthesis method familiar to those of ordinary skill in the art. The general method of chemical synthesis is particularly found 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 and Michael Smith, John Wiley & Sons Inc.: 2001. Of course, other sugar residues can be similarly connected to the main chain by the group (such as chain) carrying alkyl ether residue, amide residue or ester residue, to form a part.

[0050] Methods of using the conjugates are also described. For example, the conjugates can be used to treat cancers in the liver and kidneys. In one aspect, the conjugates can be used to treat renal fibrosis. In another aspect, the conjugates can be used to treat renal cancer. In another aspect, the conjugates can be used to treat kidney disease. The formulations can be directly administered to a subject for in vivo gene therapy.

[0051] Conjugates, especially PNAC, can be used as RNA therapeutics. Specifically, conjugates, especially PNAC, can target microRNA (miRNA) sequences. Conjugates can be used to control gene expression at the post-transcriptional level. MiRNA controls gene expression by regulating messenger RNA (mRNA) stability and translation, thereby playing a key role in maintaining physiological processes. Conjugates are used to target RNA in cells, such as mRNA or miRNA, and can inhibit RNA expression at the translation stage with respect to mRNA, and / or affect gene expression by down-regulating or up-regulating miRNA expression and its downstream effects on its target gene. Conjugates can be used to control abnormal expression of miRNA that causes several fatal diseases. Conjugates can be used to treat cancer, where atypical miRNA levels cause changes in processes including differentiation, proliferation and apoptosis. In a preferred embodiment, conjugates are used to treat cancer in the liver and kidneys. In one aspect, conjugates can be used to treat renal fibrosis. In one aspect, conjugates can be used to treat renal cancer. In one aspect, conjugates can be used to treat kidney disease.

[0052] Therefore, in one aspect, the method for reducing the expression of the targeting RNA involved in the health condition of the subject includes providing the genetic sequence-lactobionic acid conjugate as described herein to the cells of the subject in vivo or in vitro, wherein the PNA of the conjugate combines with the targeting RNA to reduce the expression of the targeting RNA, specifically, the targeting RNA is a microRNA. In one aspect, RNA therapeutic agents are used to target liver cells or kidney cells or a combination thereof to regulate the expression of the cell nucleic acid function of the subject in need, and the cell is especially a cancer cell, including a liver cancer cell or a renal cancer cell or a combination thereof. In one aspect, PNA includes kidney-specific microRNA, more specifically miR-21. The condition (demand) for treatment can be renal fibrosis and polycystic kidney disease.

[0053] In another aspect, a method for DNA targeting and gene editing for a health condition in a subject comprises providing a genetic sequence-sugar 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 regulates expression of a gene.

[0054] Genetic sequence-sugar conjugates can be used to treat subjects in need ex vivo or in vivo. The method generally includes contacting cells with an effective amount of the conjugate (optionally in combination with a potentiator) ex vivo or in vivo to deliver the therapeutic agent, such as to alter the expression of RNA. In one aspect, the method includes contacting a target cell population with an effective amount of the conjugate to alter the expression of RNA, thereby achieving a therapeutic outcome.

[0055] Genetic sequence-sugar conjugates are usually provided in a preparation comprising an effective amount of the conjugate and a polymer, lipid, protein or other pharmaceutical excipient for organ-specific delivery. A pharmaceutically acceptable carrier (also referred to as an excipient in the art), wherein a preparation suitable for the mode of administration is selected. A pharmaceutically acceptable carrier is determined in part by the specific conjugate administered and by a specific method for administering the conjugate. For example, the preparation can be externally administered, topically administered or systemically administered in a suitable pharmaceutical carrier. Therefore, there are a variety of preparations suitable for conjugates. Remington's Pharmaceutical Sciences by EWMartin, 15th edition (Mark Publishing Company, 1975) discloses typical carriers and preparation methods. For example, the preparation may include a pharmaceutically acceptable carrier (such as a salt), a carrier, a buffer, an emulsifier, a diluent, an excipient, a chelating agent, a filler, a desiccant, an antioxidant, an antimicrobial agent, a preservative, a bonding agent, a filler, silicon dioxide, a solubilizing agent or a stabilizer. The conjugate can also be encapsulated in suitable biocompatible microcapsules, microparticles, nanoparticles or microspheres formed by biodegradable or non-biodegradable polymers or proteins or liposomes to target cells. The particles can control the release of the active agent. The particles can be microparticles and / or nanoparticles. The particles can contain one or more polymers. One or more of the polymers can be synthetic polymers. One or more particles can be formed by, for example, single emulsion technology or double emulsion technology or nanoprecipitation. Such systems are well known to those skilled in the art and can be optimized for use with appropriate nucleic acids.

[0056] Formulations suitable for parenteral administration (e.g., administration by intraarticular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and nonaqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, solutions, or emulsions, which may contain suspending agents, solubilizers, thickening agents, dispersants, stabilizers, and preservatives. Formulations for injection may be present in unit dosage form, for example, in ampoules or multidose containers, optionally with the addition of preservatives. The conjugates may take forms such as sterile aqueous or nonaqueous solutions, suspensions, and emulsions, which in some aspects 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, groundnut oil), mineral oil, organic esters for injection (such as ethyl oleate), or fixed oils (including synthetic monoglycerides or diglycerides). Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butylene glycol, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutritional supplements, and electrolyte supplements (such as those based on Ringer's dextrose). Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases may also be present. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oils may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (such as oleic acid) may be used to prepare injections. The various parameters for preparing and formulating the conjugates can be readily determined by one skilled in the art without resorting to undue experimentation.

[0057] The conjugates, alone or in combination with other suitable components, can also be prepared as aerosol formulations for administration by inhalation (i.e., they can be "nebulized"). Aerosol formulations can be placed in acceptable pressurized propellants (such as dichlorodifluoromethane, propane, nitrogen and air). For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant.

[0058] An effective amount or therapeutically effective amount of a conjugate may be a dose sufficient to treat, inhibit or alleviate one or more symptoms of a disease or condition, or to provide a desired pharmacological and / or physiological effect (e.g., to reduce, inhibit or reverse one or more of the underlying pathophysiological mechanisms of a disease or condition). The exact dose will vary according to a variety of factors, such as formulation and subject-dependent variables (e.g., age, immune system health, clinical symptoms, etc.).

[0059] The conjugate, in particular a formulation comprising the conjugate, can be administered to or contacted with a target cell once a day, twice a day, or three times a day; once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once a month, twice a month, three times a month, four times a month, five times a month, six times a month, seven times a month, or eight times a month. For example, in some embodiments, the composition is administered once every two days or once every three days, or an average of about 2 to about 4 times a week.

[0060] Example

[0061] The present disclosure is illustrated by the following examples, which are not intended to limit the claims.

[0062] Unless otherwise indicated, materials and reagents were obtained from Sigma-Aldrich or Thermo Fisher Scientific and used as original samples. tGalNAc ligand was purchased from Sussex Research, Ottawa, Canada.

[0063] Example 1. Synthesis of PNA-glycoconjugates

[0064] PNA-LBA conjugates such as Figure 1A and Figure 2A and Figure 2B Synthesis shown.

[0065] 1,6-Diaminohexane-lysine backbone:

[0066] The 1,6-diaminohexane-lysine backbone was synthesized by amide coupling reaction ( Figure 1A The synthesis was started by coupling FMOC protected lysine with 6-aminoBoc protected 1,6-diaminohexane using HBTU and DIEA in DMF solvent. The two FMOC groups on lysine were then deprotected using 20% ​​piperidine in DMF to release the alpha and epsilon amino groups on lysine, providing a backbone for coupling to two lactobionic acid molecules.

[0067] Sugar ligand: LBA backbone ligand

[0068] exist Figure 1A In the second step shown, the backbone is functionalized with LBA. LBA is first converted to the lactone form by refluxing in methanol with a catalytic amount of TFA, and the solution is then basified with DIPEA to react the lactone form to give the backbone. After the reaction, the hydroxyl groups on the LBA are acetylated for purification. The identity of the reaction product is determined by mass spectrometry and by 1 H and 13C NMR spectroscopy to confirm.

[0069] Functionalized Peptide Nucleic Acids:

[0070] PNA oligomer targeting miR-122 ( Figure 1C ) were synthesized on MBHA resin solid support by implementing known and standard BOC deprotection and synthesis protocols. Short (8-mer) and full-length (22-mer) PNAs targeting the seed region and the complete sequence of miR-122 were synthesized using conventional PNA monomers.

[0071] Lysine was added to the 3' end of the PNA using standard methods. For solution phase conjugation, the PNA oligomer was then conjugated at the 3' end with trioxo-miniPEG TM The spacer was modified (using 11-(boc-amino)-3,6,9-trioxaundecanoic acid) and modified with succinic acid at the 5' end to produce a free -COOH functional group after cleavage. Solid phase synthesis was performed using a 0.2M solution of PNA monomer / lysine / trioxo-miniPEG / succinic acid / TAMRA in NMP, a 0.52M solution of DIEA in DMF, and a 0.39M solution of HBTU in DMF. TAMRA fluorescent dye was coupled to the 3' end of PNA via a trioxo-miniPEG spacer for cellular uptake and biodistribution studies. Detailed procedures for these materials and methods for synthesizing PNA on MBHA resin have been previously reported (Malik, Shipra, Frank J. Slack, and Raman Bahal. "Formulation of PLGA nanoparticles containing short cationicpeptide nucleic acids." Methods X 7 (2020). 101115).

[0072] Sugar-PNA conjugates:

[0073] As shown in Figure 2A, the functionalized PNA (PNA 4) was coupled to the amino group of the LBA backbone ligand via the succinic acid at the 5' end. HATU was used as a coupling agent and DIEA was used as a base in a DMF / DMSO solvent mixture. The resulting amide carrying acetyl protection on the hydroxyl group of LBA was treated with a solution of sodium methoxide in methanol to obtain the PNA 4-LBA conjugate.

[0074] like Figure 2BAs shown, GalNAc-PNA 4 conjugates were similarly prepared by reacting a functionalized PNA containing a trioxo-miniPEG spacer and succinic acid with a free -COOH at the 5' end with the amino group of the GalNAc-linked backbone in DMF / DMSO solvent using HATU as a coupling agent catalyzed by DIEA.

[0075] For quality control purposes, the HPLC purity spectra and MALDI mass spectra of the sugar ligand-conjugated PNA were recorded at each step ( FIG. 2C ). PNA, PNA4-LBA, and PNA4-tGalNAc products were analyzed by MALDI-MS, and the results showed that the products had formed. The products were also analyzed by reverse phase HPLC, as shown in the inset in FIG. 2C .

[0076] Example 2. In vitro binding studies

[0077] Modifications on the PNA chain should not hinder its target binding. To address the question of whether modification of the PNA chain by conjugation would hinder target binding, the affinity of PNAs and their sugar-PNA conjugates for their targets was evaluated by gel shift assays and thermal melting curve analysis.

[0078] Gel shift assay:

[0079] PNAs were incubated with target DNA at a ratio of 2:1 (PNA:DNA) at 37°C overnight, and the samples were then evaluated for separation on an 8% polyacrylamide gel. PNA 1, PNA 2-LBA, and PNA2-tGalNAc showed limited target binding due to the shorter sequences ( Figure 3B ). The full-length PNA and the conjugate showed complete binding to the target DNA, as indicated by their retardation bands ( Figure 3B The gel images also showed more retardation of the PNA 4-LBA and PNA 4-tGalNAc bands due to the increase in molecular weight of PNA after sugar ligand conjugation. These results were confirmed by melting curve analysis, where an increase in the melting temperature of sugar ligand-conjugated PNA-DNA dimers was observed ( Figure 3A ).

[0080] Example 3. Biodistribution studies in C57BL / 6J mice

[0081] Biodistribution of PNA and ligand-PNA conjugates

[0082] A 24h biodistribution study after a single 125μM, 10ml / kg subcutaneous dose was performed in C57BL / 6J mice. All PNAs were labeled with fluorescent TAMRA (rhodamine dye) probes. TAMRA fluorescence was imaged at 0.5h, 1h, 2h, 4h, 8h and 24h for three mice each on an IVIS imager, and the mice were euthanized to harvest major internal organs. Fluorescence images of different organs (including liver, lungs, heart, kidneys and spleen) were recorded to observe relative biodistribution ( Figure 4A and Figure 5A ). After subcutaneous administration, full-length PNA (PNA 3) showed minimal concentration in the liver, only up to 1 h. From the initial time point onwards, the majority of PNA 3 in the kidney indicated its rapid elimination via renal excretion. Both PNA 4-LBA and PNA 4-tGalNAc were very effectively targeted to the liver and were evenly distributed in the liver for up to 24 h ( Figure 4A ). The time-dependent PNA-TAMRA fluorescence curve of the isolated liver was plotted and used to calculate the relative amount of PNA in the liver ( Figure 4D ). GraphPad Prism software was used to calculate the area under the curve (AUC) of time-liver fluorescence. In summary, the AUC0-24 fold change of the PNA conjugate was significantly higher than that of PNA 3. The AUC0-24 fold changes of PNA 4-LBA and PNA 4-tGalNAc were 20.34±2.53 (p**) and 25.88±4.52 (p**), respectively. PNA 4-tGalNAc showed an initial high concentration in the liver, but the change in AUC0-24 was not significant compared to PNA 4-LBA. Then, after passing the liver tissue through a cell filter followed by RBC lysis, FACS analysis of the hepatocytes was performed. The FACS data once again confirmed that the liver targeting and retention of PNA 4-LBA and PNA 4-tGalNAc were comparable ( Figure 4B ). In addition, the fluorescence differences observed in the IVIS images at early time points were not as significant in the FACS analysis. The 1 h liver samples were then cryosectioned to image TAMRA fluorescence from the tissue. The fluorescence images confirmed the uniform distribution of PNA 4-LBA and PNA 4-tGalNAc in the liver sections ( Figure 4C ).

[0083] Biodistribution of anti-seed ligand-PNA conjugates:

[0084] Similar to the full-length PNA (PNA 3), it was also observed that the unconjugated short PNA 1 was eliminated by the kidney 1 h after administration, thereby reducing liver accumulation ( Figure 5A). Both PNA 2-LBA and PNA2-tGalNAc delivered PNA to the liver. Both conjugates accumulated in the liver for up to 24 h, with maximum concentrations at one hour; however, concentrations decreased significantly over time ( Figure 5A ). Throughout the study, PNA 2-tGalNAc produced higher liver accumulation than PNA 2-LBA. GraphPadPrism software was used to calculate the AUC of liver fluorescence, and the results showed that PNA-2tGalNAc was significantly higher than PNA 1 and PNA 2-LBA ( Figure 5C ). The AUCs of PNA 2-LBA and PNA 2-tGalNAc were 9 (p*) and 28 (p****) times that of unconjugated PNA 1, respectively. The high liver accumulation of PNA 2-tGalNAc can be attributed to two key factors: one factor is that the ASGPR affinity of tGalNAc in lactose acid is higher than that of galactose, and the other factor is the rapid elimination of PNA from the body. Apparently, for shorter PNAs, it seems that the higher ASGPR affinity of tGalNAc determines liver uptake and elimination. The organ distribution at 0.5h also showed that the concentration of PNA2-LBA in the kidney was higher than that of PNA 2-tGalNAc ( Figure 5A ).

[0085] These observations were confirmed by flow cytometric analysis ( Figure 5B ). The histogram representing the concentration of PNA in hepatocytes shows that at 0.5 h PNA 2-LBA and PNA 2-tGalNAc have good overlap, but PNA 2-tGalNAc still shows higher fluorescence intensity. The conjugated PNA showed prolonged liver retention, even though the miR-122 levels in the liver samples were not significantly changed.

[0086] Hepatocyte targeting by LBA-PNA and tGalNAc-PNA conjugates:

[0087] In in vitro studies using HepG2 cells, it was found that LBA and tGalNAc ligands showed ASGPR-mediated cellular uptake. These results were confirmed after in vivo treatment of mice. After subcutaneous administration of PNA and sugar ligand-PNA conjugates for 1 h, the mouse liver was perfused and digested in situ. Collagenase-mediated liver digestion was used to separate hepatocytes, and Percoll gradients were used to enrich the hepatocytes in the resulting hepatocyte suspension. The resulting hepatocyte fractions were stained with ASGPR and HNF-4α (hepatocyte-specific marker) fluorescent antibodies and analyzed by flow cytometry. Quadrant 2 represents the TAMRA fluorescence of PNA, and the double-positive quadrant 3 represents hepatocytes containing PNA. The Q3 / Q2 ratios of both PNA 2-LBA and PNA 2-tGalNAc were higher than those of PNA 1, confirming that sugar ligand-PNA conjugates preferentially accumulate in hepatocytes ( Figure 5D ).

[0088] Example 5. Efficacy Study

[0089] miR-122 expression is specific to the liver, and this expression accounts for 60% to 70% of the hepatocyte miRNA pool. In a healthy liver, this expression plays a decisive role in cholesterol and fatty acid metabolism. In a biodistribution study, it was found that both LBA and tGalNAc showed excellent liver-targeted delivery of PNA. From the gel migration and melting curve analysis described above, it can be found that the presence of sugar ligands at the 5' end of PNA does not affect its target binding ability. Evaluating the in vivo efficacy and safety of these findings is the primary goal of this study. The level of miR-122 was tested in liver samples from a biodistribution study, and it was found that PNA 4-LBA and PNA 4-tGalNAc could knock down miR-122 levels extremely well at 4h, 8h and 24h. PNA 3 also showed a certain 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). 24 h time or / and a single dose of PNA were not sufficient to knock down the downstream targets of miR-122 that were highly expressed in the liver. Due to the weak binding of short PNA at the target site, short PNA and conjugates did not show significant knockdown of miR-122.

[0090] The efficacy of PNA 3, PNA 4-LBA, and PNA 4-tGalNAc was compared in a multiple dose study, with the saline treatment group being used as a control. For efficacy evaluation, the levels of miR-122 and its downstream targets in liver samples were measured by RT-PCR and Western blot analysis. Compared with PNA 3, both PNA 4-LBA and PNA 4-tGalNAc showed a significant knockdown of up to 75% of miR-122 (p**) (Fig. 6A). The mRNA expression analysis of downstream targets ALDOA, BCKDK, GYS1, NDRG3, and CUX-1 showed that the mRNA levels treated with PNA 4-LBA and PNA 4-tGalNAc increased significantly (≥p*) (Fig. 6A). Then, after PNA 4-LBA treatment (p*) and PNA 4-tGalNAc treatment, Western blot analysis of AldoA and Bckdk proteins confirmed protein upregulation (Fig. 6B and Fig. 6C). PNA 3 treatment did not show significant changes in downstream target levels. Since miR-122 is a key regulator of cholesterol and fatty acid metabolism, we compared the plasma levels of cholesterol and triglycerides. The plasma cholesterol and triglyceride levels of PNA 4-LBA and PNA 4-tGalNAc groups were lower than those of the PNA 3 treatment group and the saline treatment group. After PNA 4-LBA treatment (p*), cholesterol and triglyceride levels were significantly lower. The PNA 4-LBA treatment group and the PNA 4-tGalNAc treatment group showed lower plasma glucose levels, but the difference was not statistically significant (Figure 7E). In addition, for PNA 4-LBA and PNA 4-tGalNAc, liver weight changes (percentage of body weight) were also significantly smaller, which was related to reduced fat storage in the liver. In short, the changes in all physiological parameters were not great, but considering that the treatment duration of healthy mice was shorter, these findings were meaningful and confirmed the efficacy of PNA after targeted delivery to the liver.

[0091] Example 6. Safety Study

[0092] Safety evaluation of PNA 3, PNA 4-LBA and PNA 4-tGalNAc was performed in C57BL / 6J mice. For the safety evaluation after three 5 mg / kg doses, mouse body weight and vital organ weights, histopathological analysis (by H&E staining) (liver, kidney and spleen), CBC analysis, comprehensive blood chemistry and electrolyte levels and plasma levels of a panel of cytokines were evaluated. H&E staining of liver, kidney and spleen tissue sections did not show any significant histological differences between the treatment groups ( Fig. 7A No significant differences in the body weight of mice were observed between the different treatment groups during the study period ( Figure 7C). Similarly, during the harvest of organs (liver, kidney, spleen, heart and lung), we did not find any obvious signs of toxicity. Except for the reduction of liver weight in the PNA 4-LBA and PNA 4-tGalNAc treatment groups, there was no significant change in organ weight (percentage of body weight%). Because the liver is a fat storage organ, the observed reduction in fat accumulation due to miR-122 knockdown can be a possible explanation for the context of the present invention. Elevated cytokine levels are the main indicators of any immune response triggered by oligonucleotide treatment. Blood was collected at 24h of the last dose for estimating the 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 by Luminex-based assays. No cytokine upregulation was found, and there were no significant differences in cytokine levels among saline, PNA 3, PNA 4-LBA, and PNA 4-tGalNAc treatments ( Figure 7B ). The intrinsic levels of cytokines in our samples were below the calibration range of the kit. CBC analysis (including RBC, WBC, platelets and hemoglobin (data not shown)) (Figure 7D) and comprehensive blood chemistry analysis (including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), albumin, globulin, total bilirubin, creatinine, blood urea nitrogen (BUN), creatine phosphokinase (CPK) and electrolytes) did not show any significant differences between the different treatment groups (Figure 7E). Electrolytes include phosphate ions, calcium ions, sodium ions, potassium ions, magnesium ions and chloride ions. In summary, the evaluation concluded that the test dosage regimen of PNA 4-LBA and PNA4-tGalNAc is highly safe and effective.

[0093] Example 7. Kidney-targeted delivery of short PNA

[0094] During the functional enhancement study of LBA ligands, acetylated LBA ligands (LBAAc) showed good potential for targeting the kidney. According to the biodistribution study we conducted in C57BL / 6J mice (5mg / kg, subcutaneous), it was found that PNA 2-LBAAc distribution was mainly confined to the kidney. Initially, at 1h, both PNA 1 and PNA 2-LBAAc were mainly distributed in the kidney, and to a certain extent in the liver (Fig. 8). Generally, naked / unformulated PNAs show rapid kidney elimination, and we found that short PNA (PNA 1) had maximum fluorescence intensity in the kidney at 30 minutes, which means that it eliminated the peak time. Compared with PNA-1, PNA 2-LBAAc showed maximum accumulation in the kidney after the initial distribution period ( Fig. 8A The average luminescence efficiency of TAMRA-labeled PNA from kidneys at different time points (1h, 4h, 24h, 48h and 72h) was determined for quantitative analysis ( Figure 8B Interestingly, the PNA 2-LBAAc concentration in the kidney at 48 h was comparable to that of PNA 1 during the elimination phase (1 h). These findings demonstrate the targeting and accumulation of PNA 2-LBAAc in the kidney.

[0095] Confirmation of PNA by flow cytometry analysis Distribution of 2-LBAAc conjugate in kidney:

[0096] Kidney samples from 24h and 72h were passed through a 40μm filter, followed by RBC lysis, and kidney cell suspensions were fixed with 4% PFA and analyzed on a flow cytometer. Histograms showing TAMRA fluorescence signal in kidney cells confirm the IVIS findings, which demonstrate that PNA 2-LBAAc has higher retention in the kidney compared to PNA-1 for up to 72h ( Figure 8C ). Both IVIS and flow cytometry analysis confirmed the kidney-targeted delivery of PNA by LBAAc ligand. The kidney sections were then imaged to observe the distribution of PNA in the kidney. 10 μm thick sections from 24h kidney samples were fixed, permeabilized and stained with DAPI for nucleus visualization. In fluorescent imaging, PNA 2-LBAAc was observed to be evenly distributed in the kidney ( Fig.8D ). The TAMRA signal intensity indicated that PNA 2-LBAAc had a higher tubular accumulation.

[0097] Kidney-targeted delivery of full-length (22-mer) PNA:

[0098] miR-21 is a kidney-specific microRNA that has been found to be upregulated in renal fibrosis and is a proven target for fibrosis treatment. Full-length (22-mer) PNA oligomers, namely PNA 5, PNA 6, and LBAAc conjugates (PNA 6-LBAAc) targeting miR-21 were designed and synthesized. Fig. 9A ). As described in FIG2A , PNA 6 was conjugated with LBAAc.

[0099] Evaluation of the biodistribution of PNA 5 and PNA 6-LBAAc in C57BL / 6J mice at a low subcutaneous dose of 1.5 mg / kg (1 / 10 of the molar equivalent of short PNA 1 and PNA 2): Biodistribution of a lower dose was selected for kidney-targeted LBAs Ac According to IVIS imaging, the TAMRA fluorescence signals of both PNA5 and PNA 6-LBAAc were confined to the kidney ( Fig. 9B). At 4 h, both PNA and the conjugate showed similar distribution in the kidney, and their mean fluorescence intensities were comparable. At 24 h, the PNA 6-LBAAc concentration in the kidney increased, while PNA 5 disappeared. In summary, a 4-fold increase in renal retention of PNA 6-LBAAc was observed at 24 h when compared to unconjugated PNA 5. ( Fig.9D ). The results were confirmed by flow cytometric analysis of kidney cells. Kidney cells obtained after RBC lysis were fixed with 4% PFA and analyzed for TAMRA fluorescence. Histograms showing TAMRA fluorescence from 4h and 24h samples confirm the elimination of PNA 5 and accumulation of PNA 6-LBAAc in kidney cells ( Fig. 9C ). In conclusion, PNA 5 follows the conventional pharmacokinetics of PNA and is rapidly eliminated without any tissue distribution. Whereas PNA 6-LBAAc shows a slower biodistribution due to the increased hydrophobicity of the conjugate. The Tmax of kidney accumulation of PNA 6-LBAAc is likely >4h. Crucially, the higher concentration of PNA 6-LBAAc in the kidney at 24h compared to the 4h time point for PNA 5 confirms the LBA Ac Renal targeting and potential of ligands.

[0100] The paper 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 herein by reference in its entirety.

[0101] The following abbreviations are used herein.

[0102]

[0103]

[0104] The terms used in this patent application have the following definitions.

[0105] As used herein, compositions, methods and articles may alternatively include, consist of or consist essentially of any suitable component or step disclosed herein. Compositions, methods and articles may additionally or alternatively be formulated to be free of or substantially free of any step, component, material, ingredient, adjuvant or substance, in other words, these steps, components, materials, ingredients, adjuvants or substances are not necessary for realizing the function or target of compositions, methods and articles. Unless the context clearly indicates otherwise, the singular forms "one", "a kind of" and "said" include plural referents. Unless the context clearly indicates otherwise, "or" means "and / or". "Optional" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes the situation where the event occurs and the situation where the event does not occur.

[0106] Unless defined otherwise, 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.

[0107] As used herein, the terms "hydrocarbyl" and "hydrocarbon" refer to substituents comprising carbon and hydrogen, optionally with 1 to 3 heteroatoms such as oxygen, nitrogen, halogen, silicon, sulfur, or combinations thereof; "alkyl" refers to a straight or branched saturated monovalent hydrocarbon group; "alkylene" refers to a straight or branched saturated divalent hydrocarbon group; "alkylene" refers to a straight or branched saturated divalent hydrocarbon group in which both valence bonds are on a single common carbon atom; "alkenyl" refers to a straight or branched monovalent hydrocarbon group having at least two carbons connected by a carbon-carbon double bond; "cycloalkyl" refers to a non-aromatic monovalent monocyclic or polycyclic hydrocarbon group having at least three carbon atoms, "cycloalkenyl" refers to a non-aromatic cyclic divalent hydrocarbon group having at least three carbon atoms and having at least one degree of unsaturation; "aryl" refers to a cycloalkyl group having at least three carbon atoms and having at least one degree of unsaturation in one or more aromatic rings. an aromatic monovalent group containing only carbon; “arylene” refers to an aromatic divalent group containing only carbon in one or more aromatic rings; “alkylaryl” refers to an aryl group substituted by an alkyl group as defined above, wherein 4-methylphenyl is an exemplary alkylaryl group; “arylalkyl” refers to an alkyl group substituted by an aryl group as defined above, wherein benzyl is an exemplary arylalkyl group; “acyl” refers to an alkyl group as defined above with the specified number of carbon atoms attached through a carbonyl carbon bridge (—C(═O)—); “alkoxy” refers to an alkyl group as defined above with the specified number of carbon atoms attached through an oxygen bridge (—O—); and “aryloxy” refers to an aryl group as defined above with the specified number of carbon atoms attached through an oxygen bridge (—O—).

[0108] Unless otherwise indicated, each of the aforementioned multiple 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 a specified atom or group is replaced by another group, provided that the normal valence of the specified atom is not exceeded. When the substituent is an oxo group (i.e., =O), then two hydrogens on the atom are replaced. Combinations of substituents or variables are allowed, provided that the substitution does not significantly adversely affect the synthesis or use of the compound. Exemplary groups that may be present in a "substituted" position include, but are not limited to, cyano; hydroxyl; nitro; azido; alkanoyl (such as C 2-6 Alkanoyl groups, such as acyl); carboxamide; C 1-6 or C 1-3 Alkyl, cycloalkyl, alkenyl and alkynyl (including groups having at least one unsaturated bond and 2 to 8 or 2 to 6 carbon atoms); C 1-6 or C 1-3 Alkoxy; C 6-10 Aryloxy, such as phenoxy; C 1-6 Alkylthio; C 1-6 or C 1-3 Alkylsulfinyl; C 1-6 or C 1-3 Alkylsulfonyl; aminodi(C 1-6 or C 1-3 ) alkyl; C having at least one aromatic ring 6-12 Aryl (e.g., phenyl, biphenyl, naphthyl, etc., each ring is a substituted or unsubstituted aromatic ring); C having 1 to 3 separate rings or condensed rings and 6 to 18 ring carbon atoms 7-19 or an arylalkoxy group having 1 to 3 separate or fused rings and 6 to 18 ring carbon atoms, wherein benzyloxy is an exemplary arylalkoxy group. The specified number of carbon atoms of the group does not include any substituents.

[0109] As used herein, peptide nucleic acid (PNA) is an artificially synthesized polymer in which the backbone comprises repeated N-(2-aminoethyl)-glycine units linked by peptide bonds. Various purine bases and pyrimidine bases are linked to the backbone by methylene bridges (-CH2-) and are linked to nitrogen on the backbone by carbonyl groups (-(C=O)-), as shown in Figure 1. By convention, PNA is represented with the N-terminus up (or on the left) and the C-terminus down (or on the right), as in a peptide. PNA is not a peptide or nucleic acid in the formal sense, but a hybrid of the two.

[0110] In some aspects, the PNA monomers forming the PNA oligomers are modified at the γ position of the polyamide backbone (γPNA) as shown below (wherein "B" is a nucleobase and "R" is a substitution at the γ position).

[0111]

[0112]

[0113] Substitution at the γ position produces chirality and provides helical pre-organization for the PNA oligomer, resulting in significantly increased binding affinity to the target RNA. Depending on the chemical nature of the specific substitution at the γ position (the "R" group in the chiral γPNA described above), other favorable properties may be conferred. The synthesis of γPNA is described in U.S. Patent No. 10,221,216, which is incorporated herein by reference for its disclosure of γPNA and methods for synthesizing γPNA.

[0114] Examples of gamma substitutions with other side chains include alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, and derivatives thereof. "Derivatives thereof" are defined herein as chemical moieties covalently attached to the side chains of these amino acids, e.g., chemical moieties covalently attached to the side chains of serine, cysteine, threonine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, and arginine.

[0115] In one aspect, the PNA oligomer forming the PNA / RNA / PNA triplex is γtcPNA or γPNA with a tail clamp.

[0116] Chemical modifications of the basic PNA structure are known and can be used. For example, fluorine-modified PNA, cyclopentyl-modified PNA, mini-PEG-modified PNA, guanidinium-modified PNA, pyrrolidinyl-modified PNA, and 2-aminopyridine-modified PNA are known in the art and can be selected to prepare PNA oligomers to improve cell permeability or increase RNA binding affinity. γ-PNA containing mini-PEG and its synthesis method are described in U.S. Patent No. 10,793,605,

[0117] PNA oligomers can also contain other positively charged parts, thereby increasing the solubility of PNA, increasing cell permeability and / or increasing the affinity of PNA to target RNA. Commonly used positively charged parts include amino acids lysine and arginine, but other positively charged parts can also be useful. Lysine residues and arginine residues can be added to tcPNA joints, or can be added to the carboxyl or N-terminal of the PNA oligomer chain.

[0118] Exemplary modifications to PNA include, but are not limited to, incorporation of charged amino acid residues, such as lysine at the end or inside of the oligomer; inclusion of polar groups in the backbone, carboxymethylene bridges, and nucleobases; carrying substituents on the original N-(2-aminoethyl)glycine backbone of chiral PNA; replacing the original aminoethylglycyl backbone with a negatively charged scaffold; conjugating high molecular weight polyethylene glycol (PEG) to one of the multiple ends; fusing PNA to RNA to produce chimeric oligomers, redesigning the backbone architecture, and conjugating PNA to DNA or RNA. These modifications improve solubility, but generally result in reduced binding affinity and / or sequence specificity.

[0119] γ-PNA modifications include serine modification, lysine modification, glutamic acid modification or alanine modification. In particular, when PNA is modified with serine γ, γPNA can target RNA more effectively than conventional full-length PNA based on binding affinity.

[0120] Phosphorothioate analogs of DNA, RNA, and OMe-RNA replace oxygen with sulfur as one of a variety of non-bridging ligands bonded to phosphorus.

[0121] Morpholino oligomers (morpholino), also known as morpholino oligomers and phosphorodiamidate morpholino oligomers (PMO), are a class of oligomers used to modify gene expression in molecular biology. Its molecular structure contains a DNA base attached to the main chain of a methylene morpholine ring connected by a phosphorodiamidate group. Morpholino oligomers block the approach of small (about 25 bases) specific sequences on the base pairing surface of other molecules and ribonucleic acid (RNA). Morpholino oligomers are used as a research tool for reverse genetics by knocking down gene function.

[0122] Locked nucleic acids are RNA derivatives in which the ribose ring is constrained by a methylene bond between the 2'-oxygen and 4'-carbon. This conformational restriction increases the binding affinity of complementary sequences and provides a chemical approach to control gene expression and optimize microarrays.

[0123] 2'-O-methylation is a nucleoside modification of RNA in which a methyl group is added to the 2' hydroxyl group of the ribose moiety of the nucleoside to produce a methoxy group. 2'-O-methylated nucleosides are mainly found in ribosomal RNA and small nuclear RNA, and are present in regions essential for the function of the ribosome and spliceosome.

[0124] Like the 2'-OMe nucleoside modification of RNA, the 2'-O-methoxyethyl-RNA (2'-MOE) backbone provides enhanced duplex stability and significant nuclease resistance.

[0125] 2'-Fluoro (2'-F) is a potent RNA analog with high RNA binding affinity and resistance to nuclease degradation.

[0126] 5'-Methylcytosine is the methylated form of the DNA base cytosine (C), which regulates gene transcription and has several other biological roles. [1] When cytosine is methylated, the DNA maintains the same sequence, but the expression of the methylated gene can be altered.

[0127] G-clamp heterocyclic modifications, cytosine analogs that clamp onto guanine by forming additional hydrogen bonds, are rationally designed to enhance oligonucleotide / RNA hybrid affinity. PNAs containing an internally linked guanidinium moiety (GPNA) are readily taken up by mammalian cells and bind to DNA and RNA with high affinity and sequence specificity.

[0128] Protective groups are functional groups that transform reactive functional groups in organic molecules so that they do not react with another functional group in the structure. Protective groups are widely used in organic synthesis in various forms. The need for protection and deprotection and the selection of appropriate protective groups can be easily determined by those skilled in the art. The chemical properties of protective groups can be found in, for example, Greene, et al., Protective Groups in Organic Synthesis, 4th edition, Wiley & Sons, 2007. The adjustment of protective groups as described herein and the formation and cleavage methods can be adjusted according to the needs of various substituents.

[0129] Residue is used to describe any part that is incorporated to constitute a larger molecule (such as a conjugate). For example, a lysine residue refers to a lysine amino acid structure that is part of a conjugate that is covalently bonded to an alkyl diamine (via an amide functional group) through a lysine carboxyl group and to a lactobionic acid molecule (via an amide bond) through an alpha amino group and an epsilon amino group, as shown in the accompanying drawings herein. A residue may also be referred to as a part.

[0130] As used herein, RNA includes different types of RNA that provide different functions, including messenger RNA, transfer RNA, ribosomal RNA and microRNA. MicroRNA (miRNA) participates in gene expression. MiRNA is a non-coding region of mRNA, which is considered to be important in promoting or inhibiting gene expression. These can involve small sequences of about 25 nucleotides.

[0131] A base sequence is a string 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) molecule or an RNA (GACU) molecule. By convention, sequences are usually presented from the 5' end to the 3' end. For DNA, the positive strand is used. Because nucleic acids are usually linear (non-branched) polymers, specifying a sequence is equivalent to defining the covalent structure of the entire molecule.

[0132] Gene expression is the process of converting genes that encode information into structures that exist and function in cells. The genes expressed include those that are transcribed into mRNA and then translated into protein, as well as those that are transcribed into RNA but not translated into protein (e.g., transfer RNA and ribosomal RNA). miRNA is a non-coding region of mRNA that is thought to be important in promoting or inhibiting gene expression.

[0133] Below is a brief description of some gene names and microRNAs known in the art. Additional information can be found on the website https: / / www.genecards.org / , which is incorporated herein by reference.

[0134] The PKD1 gene is polycystin 1, transient receptor potential channel-interacting or polycystic kidney disease 1. The PKD2 gene is polycystin 2, transient receptor potential cation channel or polycystic kidney disease 2, beta-catenin for catenin beta 1 and plakoglobin, glutamine synthetase for lengsin protein (crystallin with glutamine synthetase domain),

[0135] c-Myc for MYC binding protein, TTR for transthyretin, also known as prealbumin, amyloidosis type I, carpal tunnel syndrome 1, factor VII coagulation factor VII or coagulation factor VII (serum prothrombin conversion accelerating factor), Eg5 for neurogenin, PCSK9 for proprotein convertase subtilisin / kexin type 9, AAT for apoptosis antagonist transcription factor, TPX2 for TPX2 microtubule nucleating factor, apoB for apolipoprotein B, SAA for serum amyloid A1 cluster, RSV, PDGF for platelet-derived growth factor subunit A, miR-122 for microRNA 122, miR-223 for microRNA 223, miR-21 is one of the first microRNAs discovered and is localized within the vacuole membrane protein 1 (VMP1) locus on chromosome 17. It is involved in neoplastic and non-neoplastic pathological processes through multiple gene targets. The three main 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 involved in malignant growth, viral infection, and cardiovascular disease progression.

[0136] miR-132 is a microRNA with described targets that include mediators of neural development, synaptic transmission, inflammation, and angiogenesis.

[0137] miR-125b is a microRNA involved in regulating NF-κB, p53, PI3K / Akt / mTOR, ErbB2, Wnt and other signaling pathways, thereby controlling cell proliferation, differentiation, metabolism, apoptosis, drug resistance and tumor immunity.

[0138] miR-146a is a microRNA whose target genes are thought to be involved in regulating the pathophysiological processes of neurological diseases, especially neuroinflammatory responses. It is believed to play a key role in neuroinflammation during the progression of neurological diseases.

[0139] miR-181 (microRNA precursor) is a small non-coding RNA molecule that is transcribed as a precursor of approximately 70 nucleotides and subsequently processed by the RNase-III type enzyme Dicer to produce a mature product of approximately 22 nucleotides. They target and regulate protein expression by inhibiting the translation of target messenger RNA and / or inducing the degradation of target messenger RNA. Recent studies have shown that this new class of genes plays 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 play a regulatory role at the post-transcriptional level by complementing the target mRNA.

[0140] The let-7 lethal-7 (let-7) gene was first discovered in nematodes as a key developmental regulator and has become one of the first two known microRNAs (the other is lin-4). The expression of let-7 members is controlled by the binding of MYC to its promoter. Let-7 has been shown to be a direct regulator of RAS expression in human cells. Many reports show that the expression level of let-7 is generally low, and 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). Therefore, the expression level of let-7 can be a prognostic marker for several cancers associated with differentiation stage.

[0141] miR-34a is a microRNA that has been detected to be dysregulated in a variety of cancers and is also the first miRNA to be shown to be directly regulated by the tumor suppressor gene p53. The miR-34 family is known to suppress tumorigenesis. The expression of the miR-34 family depends on endogenous expression or simulated transfection.

[0142] miR-805 is reported to be a microRNA downregulated in LPS-treated macrophages. miR-690 (microRNA) is highly expressed in exosomes of M2-polarized bone marrow-derived macrophages and is reported to function as an insulin sensitizer both in vivo and in vitro.

[0143] miR-134 is a brain-specific microRNA; it is reported to be specifically localized in hippocampal neurons and can indirectly regulate synaptic development through antisense pairing with LIMK1 mRNA. In the human brain, SIRT1 is thought to mediate CREB protein through miR-134, conferring a role for the microRNA in higher brain functions such as memory formation.

[0144] Polycystic kidney disease (PKD) is an inherited condition in which clusters of cysts develop primarily within the kidneys, causing the kidneys to enlarge and lose function over time. Cysts are noncancerous, round sacs that contain fluid. Cysts vary in size, and they can grow very large. Too many cysts or cysts that are too large can damage the kidneys.

[0145] All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in this patent application contradicts or conflicts with a term in the incorporated reference, the term from this patent application takes precedence over the conflicting term from the incorporated reference.

[0146] Although typical aspects have been described for the purpose of illustration, the foregoing description should not be considered to limit the scope of this document. Accordingly, various modifications, adaptations and substitutions may be made by those skilled in the art without departing from the spirit and scope of this document.

Claims

1. A genetic sequence-sugar conjugate having the formula: in GS is a genetic sequence, preferably a peptide nucleic acid or oligonucleotide, such as an mRNA sequence, an siRNA sequence or a DNA sequence, optionally wherein each genetic sequence is natural or modified, for example including γ-serine modified γ peptide nucleic acid, alanine γ peptide nucleic acid, clamp G modified peptide nucleic acid, locked nucleic acid (LNA), phosphorothioate (PS), phosphorodiamidate morpholino oligomer (PMO), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'F), 5'-methylcytosine or a combination thereof, the genetic sequence having a 3' end and a 5' end, R1 and R2 are each independently H or a substituted or unsubstituted C1 to C6 alkyl group, X 1 Yes O, NR 3 , C=O or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, X 2 Yes O, NR 3 or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, G 1 is a direct bond or group connecting the PNA to the conjugate, G 2 is H or the functional part, CL is a sugar ligand comprising 2 to 16 sugar residues derived from a monosaccharide, a disaccharide, a trisaccharide or a tetrasaccharide, preferably wherein CL comprises sugar residues derived from a monosaccharide or a disaccharide, Optionally, wherein the sugar ligand is substituted with C2 to C 15 The acyl group is fully acylated or partially acylated, preferably wherein the sugar ligand is fully acetylated or partially acetylated at its hydroxyl group or amino group, n 1 is 1 to 20, and n 2 0 to 20.

2. The conjugate of claim 1, wherein the genetic sequence comprises a PNA, mRNA or siRNA containing chemically modified nucleotides, such as locked nucleic acids (LNA), phosphorothioates (PS), phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methyl (2'-O-Me), G-clamps, 2'-O-methoxyethyl (2'-O-MOE), siRNA, 2'-fluoro (2'F), 5'-methylcytosine, or a combination thereof.

3. The conjugate of claim 1, wherein the genetic sequence effectively targets the asialoglycoprotein receptor (ASGPR) on hepatocytes, or wherein the PNA effectively targets kidney cells.

4. The conjugate according to any one of claims 1 to 3, which is used to regulate a target gene, a target mRNA, a microRNA or a non-coding RNA.

5. The conjugate of claim 4, wherein the target gene or RNA is PKD1, PKD2, β-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.

6. The conjugate according to any one of claims 1 to 5, wherein G 1 has 1 to 20 carbon atoms, or wherein G 1 is a functional moiety that connects the PNA to the conjugate, for example, wherein G 1 is the residue of polyethylene glycol, polypropylene glycol or polyethylene glycol-propylene glycol, such as the residue of a trioxo-short polyethylene glycol (PEG) chain.

7. The conjugate according to any one of claims 1 to 6, wherein G 2 has 1 to 20 carbon atoms, or wherein G 2 is a functional moiety that connects the PNA to the conjugate, for example, wherein G 2 is a residue of polyethylene glycol, polypropylene glycol or polyethylene glycol-propylene glycol, such as a trioxo-miniPEG chain.

8. The conjugate according to any one of claims 1 to 7, wherein the sugar ligand further comprises a 1 of the connector.

9. The conjugate according to any one of claims 1 to 8, wherein the sugar residue is a fully acylated or partially acylated sugar residue, wherein the acyl group has 2 to 15 carbon atoms, such as a fully acetylated or partially acetylated sugar residue.

10. The conjugate of any one of claims 1 to 9, wherein the functional moiety on the conjugate further comprises a linker for attachment to kielin, tolvaptan, nintedanib, paclitaxel, bleomycin, cyclosporine, cisplatin, romidepsin, doxorubicin, docetaxel, daunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, prednisolone, cytarabine or pirfenidone.

11. The conjugate according to any one of claims 1 to 10, for use in regulating a target gene, mRNA, microRNA, non-coding RNA, DNA, hormone, cellular protein or enzyme.

12. The conjugate of any one of claims 1 to 11, 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.

13. The conjugate according to any one of claims 1 to 12, wherein the sugar residue is derived from N-acetylgalactosamine (GalNAc), galactose, lactobionic acid or their acetylated esters, preferably wherein the sugar residue is a fully acetylated or partially acetylated product of N-acetylgalactosamine (GalNAc), galactose or lactobionic acid.

14. The conjugate according to any one of claims 1 to 13, which has the formula in LBA is a lactobionic acid residue, X 1 YesNR 3 , O or C (R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, Each X 3 O, NR independently 3 or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, n3 is 0 to 20, and n4 is 1 to 8.

15. The conjugate according to claim 13, wherein G 1 is a group that connects the PNA to the conjugate, R 1 and R 2 Each is H, X 1 , X 2 and X 3 Each is NH, and n1=6, n2=2 and n3=4.

16. The conjugate according to claim 13, wherein R 1 and R 2 Each is H, n3=4, and The PNA is linked to the conjugate at the 5' end.

17. The conjugate of claim 13, which has the formula: Preferably, G 1 is the functional moiety that connects the PNA to the conjugate, and G 2 It's the functional part.

18. The conjugate according to any one of claims 13 to 16, wherein the lactobionic acid residue is fully or partially acylated with an acyl group having 2 to 15 carbon atoms, preferably wherein the lactobionic acid residue is fully or partially acetylated.

19. The conjugate according to any one of claims 1 to 12, which has the formula in X 1 is C=O or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, X 4 Yes O, NR 3 or C(R 3 )2, where R 3 is H or substituted or unsubstituted C1 to C6 alkyl, CL is a sugar residue connected to CH via a linker chain having 1 to 30 atoms, the linker chain comprising a substituted or unsubstituted C1 to C2 containing an amide group, an ester group or an ether group. 12 Alkyl or C6 to C 12 Aryl, and n4 is 2 or 3.

20. The conjugate according to claim 18, wherein the sugar residue is a fully acylated or partially acylated sugar residue, wherein the acyl group has 2 to 15 carbon atoms, such as a fully acetylated or partially acetylated sugar residue.

21. The conjugate of claim 19, wherein the sugar residue is derived from N-acetylgalactosamine (GalNAc) or from fully acylated or partially acylated acetylgalactosamine.

22. The conjugate of any one of claims 1 to 20, formulated with a polymer, lipid, protein or other pharmaceutical excipient for organ-specific delivery.

23. A method of conjugating a genetic sequence to a sugar ligand to provide a genetic sequence-sugar conjugate according to any one of claims 1 to 12, wherein X 2 Is O or NR 3 , the method comprising: functionalizing the genetic sequence to provide a free -COOH functional group; as well as The free -COOH functional group of the modified genetic sequence and the compound of the formula Y 2 Bond where Y 2 Yes - NHR 3 or -OH.

24. The method of claim 23, wherein the conjugation is performed by solution phase synthesis, solid phase synthesis, or a combination thereof.

25. The method of claim 22 or claim 23, further comprising: 1 The precursor of G 2 precursors or a combination thereof to modify the genetic sequence.

26. A method of conjugating a genetic sequence to a lactobionic acid-backbone ligand to provide a genetic sequence-lactobionic acid conjugate according to any one of claims 13 to 17, wherein X 1 and X 2 Each independently is O or NR 3 , the method comprising: functionalizing the genetic sequence to provide a free -COOH functional group; as well as The free -COOH functional group of the modified genetic sequence and Y of the following formula 2 Bond where Y 2 Yes - NHR 3 or -OH.

27. The method according to claim 25, further comprising: Let lactobionic acid react with the main chain of the following formula in X 3 Is -OH or NHR 3 ,and X 2 is protected O or protected NHR 3 .

28. A method for reducing expression of a targeted RNA involved in a health condition in a subject, the method comprising: The genetic sequence-lactobionic acid conjugate according to any one of claims 1 to 20 is provided to cells of the subject in vivo or ex vivo, wherein binding of the PNA of the conjugate to the targeting RNA reduces expression of the targeting RNA.

29. The method of claim 27, wherein the targeting RNA is a microRNA.

30. A method for DNA targeting and gene editing of a health condition in a subject, the method comprising: The genetic sequence-sugar conjugate according to any one of claims 1 to 20 is provided to cells of the subject in vivo or ex vivo, wherein DNA of the conjugate targeted to the cells regulates expression of a gene.

31. The method of claim 27 or claim 28, targeting liver cells or kidney cells to modulate expression of a cellular nucleic acid function in a subject in need thereof, comprising administering to the subject a genetic sequence-sugar conjugate according to any one of claims 1 to 20.

32. The method of any one of claims 27 to 29, wherein the cell is a cancer cell.

33. The method of any one of claims 1 to 32, wherein the PNA comprises a kidney-specific microRNA, miR-21.

34. The method of claim 31 for treating renal fibrosis and polycystic kidney disease.

35. A pharmaceutical composition comprising a pharmaceutical excipient and the conjugate according to any one of claims 1 to 20.

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