Rnai agent targeting angptl3 and PCSK9 and use thereof

Abstract

A dual-targeting RNAi molecule targeting ANGPTL3 and PCSK9. Antisense strands of two RNAi molecules are covalently linked, and sense strands respectively comprise two ligands. The dual-targeting RNAi molecule can yield a synergistic effect, thereby enhancing therapeutic efficacy, or reducing the drug dosage and minimizing side effects.

Description

RNAi agents targeting ANGPTL3 and PCSK9 and their applications Technical Field

[0001] This invention relates to RNAi molecules targeting ANGPTL3 and PCSK9 and their applications. The invention also relates to pharmaceutical compositions comprising these RNAi agents and their use in the prevention or treatment of diseases. Background Technology

[0002] Small nucleic acid drugs differ from traditional small molecule drugs in that their advantages, such as specific targeting, broad therapeutic range, and long-lasting effects, endow them with unique clinical applications. Utilizing GalNAc ligand-mediated endocytosis via desialization protein receptor (ASGPR), highly efficient targeted delivery of nucleic acid drugs such as siRNA and ASO to the liver has been perfectly achieved. Currently, six GalNAc-nucleic acid drugs have been successfully marketed, and several others are in clinical trials. The disease areas treated with small nucleic acid drugs have also gradually expanded from early rare disease indications to basic chronic diseases. With a deeper understanding of disease mechanisms, the development of many diseases involves abnormalities in multiple genes or proteins, and single-target drugs often fail to achieve ideal therapeutic effects. In some diseases, there may be synergistic effects between two targets. Simultaneously regulating these two targets through dual-target drugs or combination therapy can produce synergistic and / or synergistic effects, thereby improving therapeutic efficacy, reducing drug dosage, decreasing side effects, or increasing administration convenience. Based on the characteristics of small nucleic acid drugs, the design and development of dual-target small nucleic acid drugs to achieve one or more of the above effects has enormous medical application potential. Summary of the Invention

[0003] One aspect of the present invention provides a dual-target RNAi agent comprising a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9. The first RNAi molecule comprises a first sense strand and a first antisense strand, and the second RNAi molecule comprises a second sense strand and a second antisense strand. The 3' end of the first antisense strand is connected to the 5' end of the second antisense strand via a linker -dTdTdT-. A first ligand is attached to the 3' end of the first sense strand, and a second ligand is attached to the 5' end of the second sense strand, wherein:

[0004] The first sense strand contains the nucleotide sequence shown in SEQ ID NO: 3, and the first antisense strand contains the nucleotide sequence shown in SEQ ID NO: 4; and

[0005] The second sense strand contains the nucleotide sequence shown in SEQ ID NO: 1, and the second antisense strand contains the nucleotide sequence shown in SEQ ID NO: 2;

[0006] The structure of the first ligand is as follows:

[0007]

[0008] The wavy line represents the position connecting to the 3' end of the chain of justice.

[0009] The structure of the second ligand is as follows:

[0010]

[0011] The wavy line represents the position where it connects to the 5' end of the chain of justice.

[0012] In a preferred embodiment, the dual-target RNAi agent comprises a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9. The first RNAi molecule consists of a first sense strand and a first antisense strand, and the second RNAi molecule consists of a second sense strand and a second antisense strand. The 3' end of the first antisense strand is connected to the 5' end of the second antisense strand via a linker -dTdTdT-. The 3' end of the first sense strand is connected to 3'-L96, and the 5' end of the second sense strand is connected to 5'-L96, wherein:

[0013] The first positive strand consists of the nucleotide sequence shown in SEQ ID NO: 5 and 3'-L96;

[0014] The second sense strand consists of the nucleotide sequence shown in SEQ ID NO: 6 and 5'-L96;

[0015] The chain formed by the 3' end of the first antisense strand being covalently linked to the 5' end of the second antisense strand via -dTdTdT- consists of the nucleotide sequence shown in SEQ ID NO: 7.

[0016] Another aspect of the invention provides a pharmaceutical composition comprising the RNAi agent provided by the invention and a pharmaceutically acceptable carrier.

[0017] Another aspect of the invention provides the use of the RNAi agent in the preparation of medicaments for the prevention or treatment of metabolic diseases, cardiovascular diseases, rare diseases, complement-related diseases, and liver diseases. Accordingly, the invention provides methods for the prevention or treatment of said diseases.

[0018] These and other aspects of the invention will become clearer from the following detailed description. Attached Figure Description

[0019] Figure 1 shows a schematic diagram of the structure of the RNAi agent according to the present invention.

[0020] Figure 2 shows the results of serum hPCSK9 protein content detected by ELISA after mice were given the RNAi agent according to the present invention.

[0021] Figure 3 shows the results of serum mANGPTL3 protein content detected by ELISA after mice were given the RNAi agent according to the present invention.

[0022] Figure 4 shows a schematic diagram of the structure of an RNAi agent according to a preferred embodiment of the present invention. Detailed Implementation

[0023] definition

[0024] As used herein, the term "RNAi agent" refers to an agent containing an RNA molecule that, upon introduction into a cell, can downregulate the expression of a target gene via an RNA interference mechanism. The terms "RNAi agent of the present invention," "RNAi agent described herein," or similar expressions include the modified RNAi agents of the present invention, regardless of sequence and target gene. RNA interference refers to the process by which nucleic acid molecules induce the cleavage and degradation of target RNA molecules (such as mRNA molecules) in a sequence-specific manner, for example, through the RNA-induced silencing complex (RISC) pathway. RNAi agents herein include siRNA, shRNA, and DNA / RNA hybrid molecules, sometimes collectively referred to herein as double-stranded RNA (dsRNA), which comprises two antiparallel consecutive nucleotide chains that are sufficiently complementary to each other to hybridize and form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonds (e.g., Watson-Crick, Wobble, Hoogsteen, or anti-Hoogsteen bonds) between complementary bases in the two polynucleotides. A "double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that hybridize to form a base pair, thereby creating a double strand between the two polynucleotide chains.

[0025] The term "antisense strand" refers to a strand of dsRNA containing a region substantially complementary to the target sequence. The term "sense strand" or "positive strand" refers to a strand of dsRNA containing a region substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is either perfectly complementary or imperfectly complementary. When a complementary region is not perfectly complementary to the target sequence, the mismatch may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in terminal regions, such as the 5, 4, 3, or 2 digits at the 5′ and / or 3′ ends of the dsRNA.

[0026] "siRNA" refers to a nucleic acid that forms a double-stranded RNA. When siRNA and a target gene are present in the same cell, the double-stranded RNA has the ability to reduce or inhibit the expression of the target gene. siRNA is typically about 15 to 30 base pairs in length, most commonly about 19 to 25 base pairs, such as 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs.

[0027] shRNA refers to short hairpin RNA, which consists of two short inverted repeat sequences and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. The stem-loop may have 10 or fewer nucleotides. The stem-loop may have 8 or fewer unpaired nucleotides. The stem-loop may have 4 to 10 unpaired nucleotides. The stem-loop may have 4 to 8 nucleotides.

[0028] The two substantially complementary strands of dsRNA do not need to be, but may be, covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the double helix. In addition to the double helix structure, dsRNA may also contain one or more nucleotide overhangs. An overhanging nucleotide is one or more unpaired nucleotides that extend beyond the double helix region at the end of the strand. Nucleotide overhangs typically occur when the 3′ end of one strand extends beyond the 5′ end of the other strand, or when the 5′ end of one strand extends beyond the 3′ end of the other strand. For example, at least one strand contains a 3′ overhang of at least one nucleotide, such as 1 to 4 nucleotide overhangs. As another example, at least one strand contains a 5′ overhang of at least one nucleotide, such as 1 to 4 nucleotide overhangs. In other embodiments, both the 3′ and 5′ ends of one strand of the dsRNA contain overhangs of at least one nucleotide.

[0029] As used herein, the term "flat-ended" or "brief-end" for dsRNA refers to a dsRNA that has no unpaired nucleotide or nucleotide analogue at a given end, i.e., no nucleotide overhang. One or both ends of a dsRNA can be flat. If both ends of a dsRNA are flat-ended, then the dsRNA is said to be flat-ended. To be clear, a "flat-ended" dsRNA is a dsRNA with both ends flat, i.e., neither end of the molecule has a nucleotide overhang. In most cases, such molecules are double-stranded along their entire length. As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a dsRNA. For example, a nucleotide overhang exists when the 3′ end of one strand of a dsRNA extends beyond the 5′ end of the other strand, or vice versa. Nucleotide overhangs may contain or consist of nucleotides / nucleoside analogues, including deoxynucleotides / nucleosides. Overhangs can be on the sense strand, antisense strand, or any combination thereof. In addition, the nucleotides at the overhang can be present at the 5′ end, 3′ end, or both ends of the antisense or sense strand of dsRNA.

[0030] dsRNA molecules may include chemical modifications of ribonucleotides, including modifications to the ribose, bases, or backbone components of ribonucleic acid, as described herein or known in the art. Any such modifications, as used in double-stranded ribonucleic acid molecules (such as siRNA, shRNA, etc.), are covered by the term "dsRNA" for the purposes of this disclosure. A "modified" nucleotide refers to a nucleotide that independently has a modified sugar moiety, modified internucleotide bonds, and / or modified nucleobases. Thus, the term "modified nucleotide" includes substitution, addition, or removal of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases.

[0031] The term "ligand" refers to a cell or tissue target that binds to a specified cell type (such as hepatocytes). For example, a ligand may be a derivative containing GalNAc. In a preferred embodiment, the ligand is one or more N-acetylgalactosamine derivatives attached via a divalent or trivalent branched ligand head.

[0032] The term "therapeutic effective amount" refers to the amount of an RNAi agent or composition thereof of the present invention that effectively produces certain desired therapeutic effects in at least one subpopulation of cells in an animal, given a reasonable benefit / risk ratio suitable for any medical treatment.

[0033] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the range of correct medical judgment, suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0034] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or delivery vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulation material, that participates in carrying or delivering an RNAi agent from one organ or site of the body to another. Each carrier must be "acceptable" in the sense of compatibility with the other components of the composition and harmlessness to the patient.

[0035] The term "treatment" encompasses prevention, treatment, and cure. Patients receiving this treatment are typically any animal in need, including primates (especially humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0036] The term "angiopoietin-like 3" (ANGPTL3) is a member of the angiopoietin-like protein family involved in regulating lipid metabolism. The protein encoded by the ANGPTL3 gene is produced only in the liver and then secreted into the circulatory system. The ANGPTL3 protein structure contains an amino-terminal coiled-coil domain (CCD) that mediates homologous oligomer formation, a carboxyl-terminal fibrinogen-like domain (FLD) that mediates ligand activity, and a linker domain between the CCD and FLD. A highly hydrophobic region precedes the CCD and serves as a typical signal peptide sequence guiding protein secretion. Previous studies have shown that the biological effects of ANGPTL3 are manifested in two aspects: regulation of angiogenesis and lipid metabolism. Its induction of angiogenesis is relatively weak, while its regulation of lipid metabolism is more significant, regulating lipid metabolism by inhibiting lipoprotein lipase (LPL) activity. An exemplary mRNA sequence of human ANGPTL3 is available at NCBI Reference Sequence: NM_014495.4.

[0037] The term "proprotein convertase subtilisin / kexin type 9" (PCSK9) is a major regulator of low-density lipoprotein receptor (LDLR) levels on the hepatocyte surface, inhibiting the LDLR circulation pathway. LDLR function is crucial for cholesterol homeostasis as it is responsible for the cellular uptake and subsequent degradation of LDL. Circulating LDL binds to the N-terminal ligand-binding domain of LDLR via apolipoprotein B100, and the LDL / LDLR complex is internalized via receptor-mediated endocytosis. After migrating to the endosome, the low pH environment allows LDLR to release LDL, which is then recycled back to the cell surface. Free LDL is transported to lysosomes and degraded. PCSK9 interferes with the LDLR circulation capacity by binding to LDLR on the hepatocyte surface. An exemplary mRNA sequence of human PCSK9 is referenced in the NCBI Reference Sequence: XM_054335799.1.

[0038] The terms “dual-target RNAi agent,” “dual-site RNAi agent,” or “RNAi agent of the present invention,” or similar expressions, refer to a single RNAi molecule that can bind to and inhibit the expression of both ANGPTL3 and PCSK9, and do not include a mixture of two independent RNAi molecules that bind to only a single target gene.

[0039] Dual-target RNAi agents

[0040] A first aspect of the present invention provides a dual-target RNAi agent comprising a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9. The first RNAi molecule comprises a first sense strand and a first antisense strand, and the second RNAi molecule comprises a second sense strand and a second antisense strand. The 3' end of the first antisense strand is connected to the 5' end of the second antisense strand via a linker -dTdTdT-. A first ligand is attached to the 3' end of the first sense strand, and a second ligand is attached to the 5' end of the second sense strand, wherein:

[0041] The first sense strand contains the nucleotide sequence shown in SEQ ID NO: 3, and the first antisense strand contains the nucleotide sequence shown in SEQ ID NO: 4; and

[0042] The second sense strand contains the nucleotide sequence shown in SEQ ID NO: 1, and the second antisense strand contains the nucleotide sequence shown in SEQ ID NO: 2; wherein the first ligand is 3′-L96 and the second ligand is 5′-L96, and their structures are shown in this document.

[0043] The chemical structures of 5′-L96 and 3′-L96 are shown below, where the wavy line represents the position where they are connected to the 5′ or 3′ end of the positive chain.

[0044]

[0045]

[0046] In this invention, neither the first antisense strand of the first RNAi molecule nor the second antisense strand of the second RNAi molecule is connected to a ligand.

[0047] In this invention, the first sense strand and the second sense strand are not covalently connected. In this invention, the first sense strand forms part of the first RNAi agent by forming a double-stranded region with the first antisense strand through base pairing, and the second sense strand forms part of the second RNAi agent by forming a double-stranded region with the second antisense strand through base pairing.

[0048] Some embodiments of the present invention provide RNAi agents having the schematic structure shown in FIG1, wherein siRNA-1 represents a first RNAi molecule, siRNA-2 represents a second RNAi molecule, AS1 represents the antisense strand of the first RNAi molecule, SS1 represents the sense strand of the first RNAi molecule, AS2 represents the antisense strand of the second RNAi molecule, SS2 represents the sense strand of the second RNAi molecule, L96 represents 3′-L96, L96-2 represents 5′-L96, and AS1 and AS2 are connected by dTdTdT.

[0049] In this invention, in the first RNAi molecule, the first sense strand and the first antisense strand form a first double-stranded region; in the second RNAi molecule, the second sense strand and the second antisense strand form a second double-stranded region. The first double-stranded region is 19 base pairs long, and the second double-stranded region is 21 base pairs long. The first RNAi molecule has a -UU- overhang at the 3′ end of the antisense strand, and the 3′ end of the sense strand (or the 5′ end of the antisense strand) is blunt. The second RNAi molecule has an -AA- overhang at the 3′ end of the antisense strand, and the 3′ end of the sense strand (or the 5′ end of the antisense strand) is blunt.

[0050] In a particularly preferred embodiment, the dual-target RNAi agent provided by the present invention is A1.4-10-P1-DT-d3 (structural schematic diagram shown in Figure 4), which comprises a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9. The first RNAi molecule consists of a first sense strand and a first antisense strand, and the second RNAi molecule consists of a second sense strand and a second antisense strand. The 3' end of the first antisense strand is connected to the 5' end of the second antisense strand via a linker -dTdTdT-. The 3' end of the first sense strand is connected to 3'-L96, and the 5' end of the second sense strand is connected to 5'-L96, wherein:

[0051] The first positive strand consists of the nucleotide sequence shown in SEQ ID NO: 5 and 3'-L96;

[0052] The second sense strand consists of the nucleotide sequence shown in SEQ ID NO: 6 and 5'-L96;

[0053] The chain formed by the 3' end of the first antisense strand being covalently linked to the 5' end of the second antisense strand via -dTdTdT- consists of the nucleotide sequence shown in SEQ ID NO: 7.

[0054] The dual-target RNAi agent provided by this invention inhibits the expression levels of PCSK9 and ANGPTL3 in cells. Experiments have shown that it can effectively inhibit the expression of target proteins PCSK9 and ANGPTL3 for a long time, and its in vivo activity and long-term effect are superior to those of the single-drug combination group.

[0055] Pharmaceutical Composition

[0056] Another aspect of the invention provides pharmaceutical compositions or formulations comprising any of the RNAi agents described herein and pharmaceutically acceptable carriers, excipients, diluents, and / or delivery polymers. The pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application, taking into account clinical use. Typically, this will require the preparation of compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals. The pharmaceutical compositions and formulations may be used to treat subjects at risk of developing a disease, condition, or ailment that benefits from reduced levels of the target mRNA or suppression of target gene expression.

[0057] The ingredients and methods used to formulate a pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the type and severity of the disease or condition to be treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended route of delivery. For example, in some embodiments, the pharmaceutical composition is formulated for delivery via local administration (e.g., direct injection, implantation, or local administration), systemic administration, or via subcutaneous, intravenous, intraperitoneal, or extraintestinal routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or local (including buccal and sublingual) administration. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may include a lipid-based delivery carrier. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery.

[0058] In some embodiments, the pharmaceutical composition comprises a therapeutically or preventively effective amount of the RNAi agent described herein. "Effective amount" means an amount sufficient to produce a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce the expression of a specific gene in a specific organ, tissue, cell population, or cell type (e.g., liver or hepatocytes) of a patient. The effective amount of the RNAi agent of the present invention can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, and can be administered daily, weekly, monthly, or at longer intervals. Accurate determination of the specific effective dosage and frequency of administration may be based on several factors, including the patient's body type, age, and general condition, the type of disease to be treated (e.g., hyperlipidemia, abnormal lipid and / or cholesterol metabolism, myocardial infarction, coronary artery disease, peripheral artery disease, stroke, metabolic-associated steatohepatitis, metabolic-associated fatty liver disease), the specific RNAi agent used, and the route of administration. In some embodiments, the effective amount of the active ingredient ranges from about 0.010 to 100 mg / kg body weight per dose. The dosage may also depend on variables such as the patient's overall health status, the relative biological efficacy of the delivered compound, the drug formulation, the presence and type of excipients in the formulation, and the route of administration. Furthermore, it should be understood that the initial dose may be increased beyond the above upper limits to rapidly achieve the desired blood or tissue levels, or the initial dose may be less than the optimal dose.

[0059] The pharmaceutical compositions of the present invention can be administered via any common route, as long as the target tissue is accessible through that route. These routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, oral, intradermal, transdermal, and sublingual routes, or by direct injection into liver tissue or delivery via the portal vein. In some embodiments, the pharmaceutical compositions are administered parenterally. For example, in some embodiments, the pharmaceutical compositions are administered intravenously. In other embodiments, the pharmaceutical compositions are administered subcutaneously.

[0060] Liposome formulations are particularly well-suited for topical administration, offering several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target site, and the ability to deliver RNAi agents into the skin. In some embodiments, liposomes are used to deliver RNAi agents to epidermal cells and also enhance the penetration of RNAi agents into dermal tissues such as the skin.

[0061] Suitable pharmaceutical compositions for injection include, for example, sterile aqueous solutions or dispersions and sterile powders, for the immediate preparation of sterile injectable solutions or dispersions. Suitable carriers include physiological saline, antibacterial water, phosphate-buffered saline (PBS), or... ELTM (BASF, Parsippany, NJ). Generally, these formulations are sterile and to some extent fluid, making them easy to inject. The formulation should remain stable under production and storage conditions and should be protected against contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed case, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferred, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Extended absorption of the injectable composition can be achieved by using agents with delayed absorption in the composition, such as aluminum monostearate and gelatin.

[0062] Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound along with any other ingredients (such as those listed above) to a solvent, followed by filtration and sterilization. Typically, dispersions are prepared by adding various sterilized active ingredients to an alkaline dispersion medium and other desired ingredients, for example, as described above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired ingredients from their previously sterile filtered solutions.

[0063] The compositions of the present invention can generally be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed from free amino groups) derived from inorganic acids (such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, hydrobromic acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, maleic acid, salicylic acid, citric acid, malonic acid, succinic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, magnesium, or iron oxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the RNAi agent of the present invention is formulated as a sodium salt.

[0064] For example, for parenteral administration in aqueous solution form, the solution is typically appropriately buffered, and the liquid diluent is first made isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used for administration, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion fluid, or injected at the recommended infusion site. For human administration, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local Food and Drug Administration. In some embodiments, the pharmaceutical composition of the present invention comprises, or is composed of, the sterile saline solution described herein and an RNAi agent. In other embodiments, the pharmaceutical composition of the present invention comprises, or is composed of, the RNAi agent described herein and sterile water (e.g., water for injection, WFI). In other embodiments, the pharmaceutical composition of the present invention comprises, or is composed of, the RNAi agent described herein and phosphate-buffered saline (PBS).

[0065] In some embodiments, the pharmaceutical compositions of the present invention are packaged or stored together with a delivery device within the delivery device. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, autoinjectors, infusion pumps, in vivo syringes, and injection pens. Devices for nebulized or powdered formulations include, but are not limited to, inhalers, blowpipes, aspirators, etc. Therefore, the present invention includes delivery devices containing the pharmaceutical compositions of the present invention for treating or preventing one or more diseases or conditions described herein.

[0066] Treatment methods and uses

[0067] This invention provides a method for reducing or inhibiting the expression of a specific gene in cells (e.g., hepatocytes) by exposing cells to any of the RNAi agents described herein. The cells may be in vitro or in vivo. Gene expression can be assessed by measuring the amount or level of transcribed or translated proteins of the corresponding gene. The reduction in the expression of the corresponding gene in cells or animals treated with the RNAi agent of this invention can be determined relative to the expression of the corresponding gene in cells and animals not treated with the RNAi agent or treated with a control RNAi agent. For example, in some embodiments, the reduction in the expression of the corresponding gene is assessed by (a) measuring the amount or level of the corresponding gene mRNA in hepatocytes treated with the RNAi agent of this invention, (b) measuring the amount or level of the corresponding gene mRNA in hepatocytes treated with a control RNAi agent (e.g., an RNAi agent targeting RNA molecules not expressed in hepatocytes or an RNAi agent having a nonsense or scrambled sequence) or without an RNAi agent, and (c) comparing the corresponding gene mRNA level measured in the treated cells in (a) with the corresponding gene mRNA level in the control cells in (b). Before comparison, the mRNA levels of the corresponding genes in the treated and control cells can be normalized to the RNA levels of the control genes (e.g., 18S ribosomal RNA or housekeeping genes). The mRNA levels of the corresponding genes can be measured using various methods, including Northern blotting, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and droplet digital PCR.

[0068] In some embodiments, the method for assessing the expression levels of the target genes (ANGPTL3 and PCSK9) is performed in vitro in cells that naturally express the target genes (e.g., hepatocytes) or in cells engineered to express the target genes. In some embodiments, the method is performed in hepatocytes in vitro. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human or non-human primate hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the hepatocytes are HuH-7 cells. In another embodiment, the hepatocytes are human primary hepatocytes.

[0069] In other embodiments, the method for assessing target gene expression levels is performed in vivo. The RNAi agent and any control RNAi agent may be administered to animals (e.g., transgenic animals expressing the target gene, corresponding disease model animals, or non-human primates), and the protein expression level corresponding to the target gene may be measured in plasma or serum harvested from the animals after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with target gene expression, such as levels of low-density lipoprotein, high-density lipoprotein, cholesterol, or triglycerides, may be assessed in the treated animals. In some embodiments, the present invention provides the use of the said RNAi agent or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of one or more diseases. In some embodiments, the present invention provides the use of the said RNAi agent or pharmaceutical composition for the prevention or treatment of one or more diseases. In some embodiments, the present invention provides a method for the prevention or treatment of one or more diseases or a method for lowering triglyceride (TG) and / or low-density lipoprotein cholesterol (LDL-C) levels, the method comprising administering a preventively effective amount or a therapeutically effective amount of the present invention's RNAi agent or pharmaceutical composition to a subject in need. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0070] In the various embodiments described above in this section, the disease may be, for example, a metabolic disease, a cardiovascular disease, a rare disease, a complement-related disease, or a liver disease.

[0071] In a preferred embodiment, the metabolic disease is, for example, metabolic-associated steatohepatitis (MASH), metabolic-associated fatty liver disease (MAFLD), hyperlipidemia, hypertriglyceridemia (HTG), familial chylomicronemia syndrome (FCS), hypercholesterolemia, familial hypercholesterolemia (FH), statin-resistant hypercholesterolemia, mixed dyslipidemia, pancreatitis caused by hypertriglyceridemia, type II diabetes mellitus (T2DM), insulin resistance (IR), and obesity.

[0072] In a preferred embodiment, the cardiovascular disease is, for example, atherosclerosis (ASCVD), coronary artery disease, myocardial infarction, stroke, or cardiovascular risk associated with elevated lipoprotein(a).

[0073] In a preferred embodiment, the rare disease is, for example, transthyretin amyloidosis (ATTR), hereditary angioedema (HAE), α1-antitrypsin deficiency (AATD), familial hypercholesterolemia (HOFH / HEFH), or familial chylomicron syndrome (FCS).

[0074] In a preferred embodiment, the complement-related diseases include, for example, paroxysmal nocturnal hemoglobinuria (PNH), IgA nephropathy (IgAN), C3 glomerulonephropathy (C3G), immune complex membrane proliferative glomerulonephritis (IC-MPGN), generalized myasthenia gravis (gMG), antimyelin-associated glycoprotein antibody-associated peripheral neuropathy (MAG-PN), age-related macular degeneration (AMD), and macular geographic atrophy (GA).

[0075] In a preferred embodiment, the liver disease is, for example, liver fibrosis, cirrhosis, and liver tumors, wherein the liver tumor is, for example, hepatocellular carcinoma (HCC) or metastatic liver cancer.

[0076] sequence list

[0077] Table 1. RNAi molecules targeting PCSK9

[0078] Table 2. RNAi molecules targeting ANGPTL3

[0079] Among them, mA, mC, mG, and mU are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

[0080] fA, fC, fG, and fU are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively.

[0081] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively.

[0082] * indicates a thiophosphate linker;

[0083] SS stands for the justice chain, and AS stands for the antithesis chain.

[0084] Table 3. Exemplary RNAi agents of the present invention

[0085] Among them, mA, mC, mG, and mU are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

[0086] fA, fC, fG, and fU are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively.

[0087] dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively.

[0088] The underlined nucleotides represent connectors between two strands;

[0089] * indicates a thiophosphate linker;

[0090] L96 in sequences 5 and 8 represents 3′-L96, and L96-2 in sequence 6 represents 5′-L96.

[0091]

[0092]

[0093] Example 1. Synthesis of dual-target Bis-siRNA compounds

[0094] The synthesis method of dual-target Bis-siRNA is no different from the conventional solid-phase synthesis method of phosphorus amide (both were synthesized by Suzhou Beixin Biotechnology Co., Ltd.), including four steps: deprotection, coupling, capping, and oxidation or sulfidation. After solid-phase synthesis, ammonia deprotection was completed by treatment with ammonia solution at 55°C for 16 hours, followed by HPLC purification and lyophilization to obtain single-chain compounds. The obtained sense and antisense strands were annealed according to the molar ratio to obtain complementary paired dual-target Bis-siRNA compounds, and the concentrations were adjusted to the required levels. Using a solid-phase support (Universal CPG or CPG support with GalNAc ligand) as the starting cycle, nucleoside monomers were sequentially linked from 3′ to 5′. Nucleoside phosphorus amide monomers, 2′-fluoromodified monomers, 2′-methoxymodified monomers, and other raw materials were purchased from Shanghai Zhaowei Technology Development Co., Ltd. The obtained Bis-siRNA has sense and antisense strands as shown in Table 3, A1.4-10-P1-DT-d3.

[0095] Example 2. Activity screening of PCSK9 and ANGPTL3 single-target modified compounds in Huh7 cells

[0096] Huh7 cells were cultured in DMEM (CORNING-10-0130CV) medium, and cell density was measured using a Countstar Rigel S2 cell counter. The cell suspension was diluted to 2×10⁵ cells / mL in DMEM medium containing 10% FBS. At the same time, siRNA was diluted to 12-fold working concentration. Lipofectamine RNAiMax (Invitrogen-13778150) and Opti-MEM medium (Gibco-31985070) were mixed in a 1:1 ratio with the siRNA dilution to prepare a transfection complex. The mixture was incubated at room temperature. 20 μL of transfection complex and 100 μL of cell suspension were added to each well, and the 96-well plate was incubated at 37°C and 5% CO₂ for 48 hours.

[0097] RNA was extracted from cells in 96-well plates using the EZ-Press 96RNA Purification Kit. After discarding the culture medium, the plates were washed once with PBS. 150 μL of lysis buffer was added to each well, and the plates were incubated on a horizontal shaker for 5 min. An equal volume of anhydrous ethanol was added to each well, and the mixture was transferred to the RNA extraction plate. The plates were centrifuged for 4 min. The waste liquid was discarded. 600 μL of Buffer RW1 was added to each well, and the plates were centrifuged for 4 min. The waste liquid was discarded. 600 μL of Buffer RW2 was added to each well, and the plates were centrifuged for 12 min. The centrifuged RNA extraction plates were transferred to new elution plates and incubated at room temperature for 3 min. 30 μL of Nuclease-free ddH2O was added to each well, and the plates were incubated at room temperature for 1 min, then centrifuged for 4 min. Another 30 μL of Nuclease-free ddH2O was added to each well, and the plates were incubated at room temperature for 1 min, then centrifuged for 4 min. III. Reverse transcription was performed using the RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323-01) kit. After reverse transcription, 10 μl of Buffer EB (from the EZ-Press 96 RNA Purification Kit) was added to each well. The prepared qPCR system was added to the 96-well plate, sealed with a sealing membrane, and PCR was performed on a QuantStudio 7Flex system (Applied Biosystem). Data were analyzed using the ΔΔCt method. The results are shown in Tables 4 and 5.

[0098] Table 4. Inhibition of PCSK9 by PCSK9-targeting compounds in Huh7 cells

[0099] Table 5. Inhibition of ANGPTL3 by ANGPTL3 single-target modified compounds in Huh7 cells

[0100] Example 3. In vivo activity assessment of PCSK9 / ANGPTL3 dual-target compound in transgenic mice

[0101] Humanized 3′-UTR PCSK9 mice were obtained from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0102] Four days prior to drug administration, serum was collected from the jaw of mice after a 6-hour fast. The levels of hPCSK9 and mANGPTL3 proteins in the mouse serum were measured using the Abcam hPCSK9 ELISA kit (ab209884) and the R&D mANGPTL3 ELISA kit (MANL30), respectively. Mice were randomly divided into groups of five based on hPCSK9 protein levels. siRNA was dissolved in physiological saline, and mice were subcutaneously injected with a single dose of 6 mg / kg (3 mg / kg + 3 mg / kg for the combination group). After drug administration, serum was collected weekly from the jaw, and the levels of hPCSK9 and mANGPTL3 proteins were measured by ELISA.

[0103] Protein residual amount % = protein concentration on day N / protein concentration on day -4 * 100%, and the results are shown in Figures 2 and 3. The results show that the dual-target compound A1.4-10-P1-DT-d3 of the present invention can inhibit the expression of target proteins PCSK9 and ANGPTL3 for a long time and with high efficiency, and its in vivo activity and long-term effect are superior to those of the single-drug combination group.

Claims

1. A dual-target RNAi agent comprising a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9, wherein the first RNAi molecule comprises a first sense strand and a first antisense strand, the second RNAi molecule comprises a second sense strand and a second antisense strand, the 3' end of the first antisense strand is linked to the 5' end of the second antisense strand via a linker -dTdTdT-, a first ligand is attached to the 3' end of the first sense strand, and a second ligand is attached to the 5' end of the second sense strand, wherein: The first sense strand contains the nucleotide sequence shown in SEQ ID NO: 3, and the first antisense strand contains the nucleotide sequence shown in SEQ ID NO: 4; and The second sense strand contains the nucleotide sequence shown in SEQ ID NO: 1, and the second antisense strand contains the nucleotide sequence shown in SEQ ID NO: 2; The structure of the first ligand is as follows: The wavy line represents the position connecting to the 3' end of the chain of justice. The structure of the second ligand is as follows: The wavy line represents the position where it connects to the 5' end of the chain of justice.

2. The dual-target RNAi agent according to claim 1, wherein the dual-target RNAi agent comprises a first RNAi molecule targeting ANGPTL3 and a second RNAi molecule targeting PCSK9, the first RNAi molecule comprising a first sense strand and a first antisense strand, the second RNAi molecule comprising a second sense strand and a second antisense strand, the 3' end of the first antisense strand being connected to the 5' end of the second antisense strand via a linker -dTdTdT-, the 3' end of the first sense strand being connected to 3′-L96, and the 5' end of the second sense strand being connected to 5′-L96, wherein: The first positive strand consists of the nucleotide sequence shown in SEQ ID NO: 5 and 3′-L96; The second sense strand consists of the nucleotide sequence shown in SEQ ID NO: 6 and 5′-L96; The chain formed by the 3' end of the first antisense strand being covalently linked to the 5' end of the second antisense strand via -dTdTdT- consists of the nucleotide sequence shown in SEQ ID NO:

7.

3. A pharmaceutical composition comprising the dual-target RNAi agent as described in claim 1 or 2; and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is an intravenous or subcutaneous injection.

5. Use of the dual-target siRNA of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of one or more diseases selected from metabolic diseases, cardiovascular diseases, rare diseases, complement-related diseases, and liver diseases.

6. The use according to claim 5, wherein it has one or more of the following features: (a) The metabolic diseases mentioned are selected from metabolic-associated steatohepatitis (MASH), metabolic-associated fatty liver disease (MAFLD), hyperlipidemia, hypertriglyceridemia (HTG), familial chylomicronemia syndrome (FCS), hypercholesterolemia, familial hypercholesterolemia (FH), statin-resistant hypercholesterolemia, mixed dyslipidemia, pancreatitis caused by hypertriglyceridemia, type II diabetes mellitus (T2DM), insulin resistance (IR), and obesity; (b) The cardiovascular and cerebrovascular diseases mentioned are selected from atherosclerosis (ASCVD), coronary artery disease, myocardial infarction, stroke and cardiovascular risks associated with elevated lipoprotein (a); (c) The rare diseases mentioned are selected from transthyretin amyloidosis (ATTR), hereditary angioedema (HAE), α1-antitrypsin deficiency (AATD), familial hypercholesterolemia (HOFH / HEFH) and familial chylomicron syndrome (FCS); (d) The complement-related diseases mentioned are selected from paroxysmal nocturnal hemoglobinuria (PNH), IgA nephropathy (IgAN), C3 glomerulonephropathy (C3G), immune complex membrane proliferative glomerulonephritis (IC-MPGN), generalized myasthenia gravis (gMG), antimyelin-associated glycoprotein antibody-associated peripheral neuropathy (MAG-PN), age-related macular degeneration (AMD), and macular geographic atrophy (GA); and (e) The liver diseases mentioned are selected from liver fibrosis, cirrhosis and liver tumors.

Images