RNAi agent for inhibiting expression of complement factor B as well as preparation method and application of RNAi agent

Abstract

The present invention relates to RNAi agents for inhibiting the expression of complement factor B, methods of preparation and uses thereof, which can inhibit the expression of complement factor B for the treatment of complement factor B related diseases and disorders.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to an RNAi agent for inhibiting the expression of complement factor B, and a preparation method and use thereof. Background Art

[0002] The complement system is part of the innate immune system. Compared to the adaptive immune system, it is evolutionarily older and conserved in most taxa. Its functions include decorating potentially pathogenic microorganisms (a process called opsonization) and targeting them for destruction, which is achieved by macromolecular assemblies called membrane attachment complexes (MACs). Certain components of the complement system, once activated, promote chemoattraction and activation of leukocytes.

[0003] Currently known complement activation pathways mainly include three: the classical pathway, the lectin pathway, and the alternative pathway. Complement factor B (CFB) is a trypsin-like serine protease that circulates in human blood in the form of a potential zymogen. The alternative pathway is a magnesium-dependent cascade that is activated by the deposition and activation of C3 on certain susceptible surfaces (such as cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials).

[0004] Other diseases associated with the complement cascade include membranous nephropathy (MN), C3 glomerulonephritis (C3G), age-related macular degeneration (AMD), geographic atrophy (GA), atypical hemolytic uremic syndrome (aHUS), hemolytic uremic syndrome (HUS), complications of hemodialysis, hemolytic anemia or hemodialysis, neuromyelitis (NMO), hepatic inflammation, inflammatory bowel disease, dermatomyositis and amyotrophic lateral sclerosis, myasthenia gravis (MG), respiratory diseases and cardiovascular diseases.

[0005] Although international application numbers WO2023018523, WO2023097291, WO2023031359, WO2017135397 and WO2023076451 disclose RNA compounds that can inhibit the expression of complement factor B gene, no small molecule or nucleic acid drugs have been approved for the treatment of complement factor B-related diseases. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an RNAi agent for inhibiting the expression of complement factor B, and a preparation method and use thereof. The RNAi agent has a good inhibitory effect on the expression of complement factor B gene.

[0007] In a first aspect, the present invention provides an RNAi agent for inhibiting the expression of complement factor B, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in Table 1.

[0008] The sense strand and the antisense strand may be partially, substantially or completely complementary to each other, for example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0009] The lengths of the nucleotides of the sense strand and the antisense strand may be the same or different. For example, the sense strand includes 19 nucleotides and the antisense strand includes 21 nucleotides, or the sense strand includes 19 nucleotides and the antisense strand also includes 19 nucleotides, or the sense strand includes 21 nucleotides and the antisense strand includes 19 nucleotides, or the sense strand includes 21 nucleotides and the antisense strand includes 21 nucleotides.

[0010] As a preferred technical solution of the present invention, the sense strand and / or antisense strand contains at least one modified nucleotide.

[0011] Wherein, the modified nucleotide is selected from:

[0012] Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA); wherein the alkyl nucleotides are selected from methyl nucleotides and ethyl nucleotides; glycol nucleic acids include (S)-glycol nucleic acids ((S)-GNA) and (R)-glycol nucleic acids ((R)-GNA).

[0013] Wherein, the sense strand and antisense strand of the RNAi agent each include at least one modified nucleotide, in a specific embodiment, the sense strand includes at least one modified nucleotide, and the nucleotides of the antisense strand are not modified, or the nucleotides of the sense strand are not modified, and the antisense strand includes at least one modified nucleotide. In a specific embodiment, each nucleotide of the sense strand is modified, and each nucleotide of the antisense strand is also modified.

[0014] As a preferred technical solution of the present invention, the modified RNAi is selected from the sequences shown in Table 2.

[0015] As a preferred technical solution of the present invention, the RNAi agent further comprises a targeting ligand, and the targeting ligand is connected to the sense strand and / or the antisense strand.

[0016] As a preferred technical solution of the present invention, the targeting ligand includes an N-acetyl-galactosamine (GalNAc) portion.

[0017] As a preferred technical solution of the present invention, the targeting ligand is selected from:

[0018]

[0019]

[0020] As a preferred technical solution of the present invention, the targeting ligand is connected to the 3' or 5' end of the sense strand.

[0021] As a preferred technical solution of the present invention, the targeting ligand is connected to the 3' or 5' end of the antisense strand.

[0022] In some specific embodiments, the targeting ligand is connected to the 5' end of the sense strand. In some specific embodiments, the targeting ligand is connected to the 3' end of the sense strand. In some specific embodiments, the targeting ligand can also be internally connected to the nucleotides on the sense strand and / or antisense strand of the double-stranded siRNA analog. In some specific embodiments, the targeting ligand can also be connected to the double-stranded siRNA analog via a joint, for example, the targeting ligand can also be connected to the 3' or 5' end of the sense strand via a joint, or the targeting ligand can also be connected to the 3' or 5' end of the antisense strand via a joint, or the targeting ligand can also be internally connected to the nucleotides on the sense strand and / or antisense strand of the double-stranded siRNA analog via a joint.

[0023] In a second aspect, the present invention provides a pharmaceutical composition for inhibiting complement factor B gene expression, wherein the pharmaceutical composition comprises the RNAi agent as described above.

[0024] In a third aspect, the present invention also provides a use of the RNAi agent as described above in the preparation of a method for treating complement factor B-related diseases, disorders or symptoms.

[0025] As a preferred technical solution of the present invention, the disease is selected from:

[0026] Paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HEL LP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; oligoimmune vasculitis; epidermolysis bullosa; recurrent miscarriage; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, bullous dermatomyositis pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic-uremic syndrome, C3 neuropathy, antineutrophil cytoplasmic antibody-associated vasculitis (eg, granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis), Churg-Strauss syndrome, and microscopic polyangiitis) , humoral and vascular transplant rejection, graft dysfunction, myocardial infarction (e.g., tissue damage and ischemia in myocardial infarction), allogeneic transplantation, sepsis (e.g., poor prognosis in sepsis), coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disease, myocarditis, cerebrovascular disease, peripheral (e.g., musculoskeletal) vascular disease, renovascular disease, mesenteric / enteric vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and postoperative restenosis with stenting, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA).

[0027] The RNAi agent for inhibiting complement factor B gene expression provided by the present invention has good inhibitory activity on complement factor B and can be used to prevent and / or treat related diseases mediated by complement factor B gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the modified template used for the modified RNAi of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0030] As used herein, the term "comprising" is used to mean, and is used interchangeably with, the phrase "including, but not limited to," unless the context clearly dictates otherwise.

[0031] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0032] As used herein, the terms "sequence" and "nucleotide sequence" mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0033] In the present invention, the term "RNAi agent" refers to a complex of ribonucleic acid molecules, which has a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid chains, which have "sense" and "antisense" orientations relative to the target RNA.

[0034] In the present invention, "complementary" has a meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. The purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with uracil on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain.

[0035] The term "antisense strand" generally refers to a strand of a RNAi agent that includes a region that is substantially complementary to a target sequence. As used herein, the term "complementarity region" generally refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) defined herein. When the complementary region is not completely complementary to the target sequence, the mispairing may be in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0036] The term "sense strand" generally refers to a strand of an RNAi agent that includes a region that is substantially complementary to a region of the term antisense strand as defined herein. A "sense" strand is sometimes referred to as a "sense" strand, a "passenger" strand, or an "anti-guide" strand. With their sequence, the antisense strand targets the desired mRNA, while the sense strand targets different targets. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications or using 5' end caps on the sense strand.

[0037] In the present invention, modified nucleotides include but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyribose residues (invAb), and glycol nucleic acids (GNA).

[0038] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides,

[0039] 2'-methoxy modified nucleotides, e.g. 2'-methoxyethyl nucleotides, e.g. 2'-Fluoronucleotides, e.g. 5'-C-methylphosphononucleotides, e.g. Vinylphosphonate deoxyribonucleotides (VP), e.g. Phosphorothioate nucleotides (S), e.g. 2'-deoxyribonucleotides, such as: Inverted abasic deoxyribose residues (invAb), for example: Glycol nucleic acids (GNAs), including (S)-glycol nucleic acids ((S)-GNAs), for example: and (R)-glycol nucleic acids ((R)-GNA), for example:

[0040] Among them, Base represents a base, R represents an alkyl group, Me represents a methyl group, and Et represents an ethyl group.

[0041] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0042] Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125 and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0043] The locked nucleic acid structure is as follows:

[0044]

[0045] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0046] The term "morpholino" means a sugar surrogate having the formula:

[0047]

[0048] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar substitutes are referred to herein as "modified morpholino groups".

[0049] In the present invention, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides. Lowercase letter m indicates that a nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that a nucleotide adjacent to the left of letter f is a fluorinated-modified nucleotide; LNA indicates that a nucleotide adjacent to the right of letter m is a locked nucleic acid (LNA) modified; GNA indicates that a nucleotide adjacent to the left of letter f is a GNA modified; lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; VP indicates that a nucleotide on the right of letter VP is a vinyl phosphate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymine nucleotide; dU represents a deoxyuridine nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0050] It should be emphasized that the "modification" of nucleotides described in the present disclosure includes but is not limited to the above examples, and the nucleotides may also be replaced with other nucleotides, for example, (S)-glycerol nucleic acid, etc.

[0051] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0052] Targeting ligands can also be cell or tissue targeting agents that bind to a specified cell type, such as renal cells, such as lectins, glycoproteins, lipids or proteins, such as antibodies. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0053] The targeting ligand can also be a protein, for example, a glycoprotein, or a peptide, for example, a molecule with specific affinity for the auxiliary ligand, or an antibody, for example, an antibody that binds to a specified cell type, for example, a hepatocyte. The ligand can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand can be, for example, an activator of lipopolysaccharide, p38MAP kinase, or an activator of NF-KKB.

[0054] The targeting ligand can be a substance, e.g., a drug, that can increase the uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cell microtubules, microfilaments and / or intermediate filaments). The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, red sea sponge A, phalloidin, swinholide A, indanocine, or myoservin.

[0055] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known to the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dose form is generally the amount of the compound that produces the therapeutic effect. Generally speaking, in units of one percent, the amount is about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0056] The term "prevention and / or treatment" includes not only prevention and / or treatment of diseases, but also generally includes preventing the onset of diseases, slowing or reversing the progression of diseases, preventing or slowing the onset of one or more symptoms associated with diseases, reducing and / or alleviating one or more symptoms associated with diseases, reducing the severity and / or duration of diseases and / or any symptoms associated therewith, and / or preventing further increase in the severity of diseases and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by diseases, and any pharmacological effects that are generally beneficial to patients being treated. The nucleic acid or pharmaceutical composition of the present application does not need to achieve complete cure or eradication of any symptoms or manifestations of diseases to form a viable therapeutic agent. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, prophylactically administered treatments constitute viable preventive agents that do not need to be completely effective in preventing the onset of symptoms. It is sufficient to simply reduce the impact of the disease in the subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reduce the likelihood of disease occurrence or exacerbation.

[0057] The terms "disease" or "disorder" are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the state of the body or certain organs that prevents or disrupts the performance of functions, and / or causes symptoms such as discomfort, dysfunction, pain or even death in the person suffering from the disease or contacting it. Disease or disorder may also be referred to as distemper, ailment, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.

[0058] The term "inhibit" may be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms and includes any level of inhibition.

[0059] The phrase "inhibiting the expression of complement factor B (CFB)" in the present invention includes inhibiting the expression of any complement factor B (CFB) gene (e.g., mouse complement factor B (CFB) gene, rat complement factor B (CFB) gene, monkey complement factor B (CFB) gene, or human complement factor B (CFB) gene) as well as variants or mutants of the complement factor B (CFB) gene encoding complement factor B (CFB) protein.

[0060] "Inhibiting the expression of complement factor B (CFB)" includes any level of inhibition of the complement factor B (CFB) gene, such as at least partially suppressing the expression of the complement factor B (CFB) gene, such as inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0061] The term "complement factor B-related disease" is a disease or disorder caused by complement activation or associated with complement activation. Such diseases are typically associated with inflammation and / or immune system activation, such as membrane attack complex-mediated lysis, allergic reactions and / or hemolysis. Non-limiting examples of complement component-related diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or invasive hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD) D)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent abortion; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome, C3 neuropathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, graft dysfunction, myocardial infarction, allogeneic transplantation, sepsis, Coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renal vascular disorders, mesenteric / enteric vascular disorders disorders, vasculitis, Henoch-Schönlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki's disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA) (see, e.g., Holers (2008) Immunological Reviews 223:300-316; Holers and Thurman (2004) Molecular Immunology 41:147-152; U.S. Patent Publication No. 20070172483).

[0062] Complement factor B related diseases are paroxysmal nocturnal hemoglobinuria (PNH). The PNH can be classic PNH or PNH in another bone marrow failure syndrome and / or myelodysplastic syndrome (MDS), such as a cell reduction environment. In another embodiment, complement factor B related diseases are atypical hemolytic uremic syndrome (aHUS). In yet another embodiment, complement factor B related diseases are rheumatoid arthritis.

[0063] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared from the compounds having specific substituents discovered in the present invention and pharmaceutically acceptable acids or bases.

[0064] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical drugs. For other information about the carrier, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0065] The term "pharmaceutically acceptable excipient" is a substance intentionally included in a drug delivery system other than an active pharmaceutical ingredient (API, therapeutic product, such as complement factor B (CFB). An excipient does not or is not intended to exert a therapeutic effect at the intended dose. An excipient may serve the following functions: a) aid in the handling of the drug delivery system during preparation, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other attribute of the overall safety, efficacy or delivery of the API during storage or use.

[0066] Among them, excipients include (but are not limited to): absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavoring agents, glidants, wetting agents, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents and fragrances.

[0067] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0068] Example 1 Synthesis of targeting ligands

[0069]

[0070] Wherein, the ligand L96 can be connected to the RNAi agent via a phosphate group, a thiophosphate group or another connecting group. The specific synthesis route is referred to the document with International Patent Publication No. WO2009073809.

[0071]

[0072] Among them, the synthesis route of NAG37 can refer to the document with patent number CN201780042047.6, among which, Indicates the siRNA binding site.

[0073]

[0074] Among them, the synthesis route of M10 (conjugates 9 and 10) can refer to the document with patent number CN202210705962.1, and R2 represents siRNA.

[0075]

[0076] Among them, the synthesis route of L10 can refer to the synthesis route of the patent with patent publication number WO2019105414A1, wherein Nu represents siRNA.

[0077]

[0078] Among them, the synthesis route of GLS-15 can refer to the synthesis route of the patent with patent publication number CN202280016262.X, wherein, Indicates the siRNA binding site.

[0079]

[0080] Among them, the synthesis route of GL6 can refer to the synthesis method of compound 6 in patent document WO2023241591. Indicates the siRNA binding site.

[0081] Example 2 Synthesis of RNAi Agents

[0082] The RNAi agent was prepared using OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthesis route can be found in patent document CN201480067917.1, and the RNAi agent sequence is shown in Table 1.

[0083] Table 1 is a sequence table of RNAi agents

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Example 3 Synthesis of modified RNAi agents

[0099] The modified RNAi agent was prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark), wherein the naked sequences S1-S1016 in Table 1 were modified using a modified template, and the modified RNAi agents were numbered S1M-S1016M, corresponding to the sequence numbers in Table 1, wherein the modified template used is shown in Figure 1 The sequences of some modified RNAi agents are shown in Table 2. The specific synthesis routes can be found in patent document CN201480067917.1.

[0100] Table 2 shows the modified RNAi

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Tm = 2'-O-methylthymidine-3'-phosphate; Ams = 2'-O-methyladenosine-3'-phosphorothioate; Ums = 2'-O-methyluridine-3'-phosphorothioate; Cms = 2'-O-methylcytidine-3'-phosphate guanosine-3'-phosphorothioate; Gms = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-phosphorothioate; Ufs = 2'-fluorouridine-3'-phosphorothioate; Cfs = 2'-fluorocytidine-3'-phosphorothioate; Gfs = 2'-fluoroguanosine-3'-phosphorothioate; m = 2'-O-methyl; f = 2'-fluoro; s = phosphorothioate linkage.

[0109] Example 4 Synthesis of RNAi Agent Compounds

[0110] OligoMaker ApS192 RNA synthesizer (made in Denmark) was used to prepare the modified RNAi agent, and then the targeting ligand L96 of Example 1 was connected to the 3' end of the sense strand of the modified RNAi agent in Table 2. The specific synthesis route can be referred to patent document CN201480067917.1, the content of which can be incorporated into this application by reference.

[0111] Example 5 In vitro testing of RNAi agents in HuH7 cells

[0112] The exon region of human complement factor B (CFB) gene (including 5'UTR and 3'UTR sequences) was cloned into the reporter-based screening plasmid psiCHECK2 (Promega-C8021) to produce Renilla luciferase / CFB fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) medium containing 10% fetal bovine serum (Gibco-10099141C), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), and 1% penicillin-streptomycin (Gibco-15070063). CFB-psiCHECK2 plasmid, RNAi agent and Lipo2000 (Invitrogen-11668019) transfection reagent diluted with Opti-MEM (Gibico-11058021) were added to the HuH7 cell suspension and then incubated at 1×10 5 Cells were plated at a density of 100 μg / ml in 96-well plates, resulting in a final concentration of 3 nM or 0.1 nM RNAi agents. After 24 hours of culture, relative levels of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid were measured using a dual-luciferase reporter assay (Promega-E2920).

[0113] The inhibition rate of the CFB gene mediated by the RNAi agent was calculated according to the following formula.

[0114] The CFB gene inhibition rate of the sample (%) = (1-relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100. The test results are shown in Tables 3 and 4. The RNAi agent sequence of the present invention has a good inhibitory activity on the CFB gene.

[0115] Table 3 shows the inhibitory activity of the modified RNAi agents against the CFB gene

[0116]

[0117]

[0118]

[0119] Table 4 shows the inhibitory activity of RNAi agents against CFB gene

[0120]

[0121] Among them, A≥50%.

[0122] Example 6 In vitro testing of RNAi agents in HuH7 cells

[0123] HuH7 cells were cultured in DMEM (Pricella PM150210) containing 10% fetal bovine serum (Corning 35-081-CV) and 1% penicillin-streptomycin (HyClone SV30010). HuH7 cells in the logarithmic growth phase were taken and cultured at 1.0×10 4 / well density was spread on a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The culture medium was discarded, and 40 μL Opti-MEM (Gibco-31985-062) was added, followed by 10 μL Lipo2000 (Invitrogen-52887) and RNAi agent mixture, so that the final concentration of the RNAi agent mixture was 10 nM, 1 nM, and 0.02 nM. After 6 hours of transfection, the old culture medium was discarded and replaced with fresh complete culture medium. After 48 hours of continuous culture, cell RNA was extracted (BayBio-TIRM-48-KD) and reverse transcribed (Trans-AU341-02-V2). The TaqMan method (EZB-EZB-Probe-U2) was used to detect the expression abundance of CFBmRNA (NM_001710.6).

[0124] The expression level of target gene mRNA in each sample was calculated by ΔΔCT relative quantitative method. -ΔΔCT The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value, then subtract the ΔCT value of the control group containing only the transfection reagent (RNAiMAXControl) from the ΔCT of the drug group (sample) to obtain ΔΔCT, and finally multiply ΔΔCT by 2 -ΔΔCT Finally, the relative expression level of CFB mRNA (value of sample) was obtained by conversion.

[0125] The inhibition rate of CFB gene mediated by siRNA analogs was calculated according to the following formula.

[0126] CFB inhibition rate % = (1-value of sample / Average value of RNAi MAX Control) * 100. The RNAi agent of the present invention has a good inhibitory activity on the CFB gene.

[0127] Example 7 In vivo evaluation method

[0128] 7-8 week old male C57BL / 6 mice were given AAV8 adeno-associated virus (2×10 11vg / only / 0.2mL, iv, single) modeling. One week after modeling, under the action of AAV8 virus, CFB gene was universally expressed in mice. At this time, the mice were treated subcutaneously with a dose of 3mg / kg RNAi agent compound (the RNAi agent compound was the 3' end of the positive chain of the modified RNAi agent in Table 2 conjugated with L96), and liver samples were collected 2 weeks later for total RNA extraction (Transgen, EC521-96). The extracted total RNA was reverse transcribed (Takara, RR036B), and qRT-PCR analysis of CFBmRNA (Takara, RR820B) was performed, and the expression of the target gene in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method; this method measures the Ct difference (ΔCt) between the target gene and the internal reference gene (GAPDH).

[0129] The formula is as follows:

[0130] ΔCT = average Ct of target gene - average Ct of GAPDH; ΔΔCT = ΔCT (sample) - ΔCT (vehicle control); relative amount of target gene mRNA = 2 - ΔΔCt; % inhibition = (relative amount of vehicle control - relative amount of sample) / relative amount of vehicle control × 100%. The results show that the RNAi agent compound of the present invention has good inhibitory activity.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An RNAi agent for inhibiting complement factor B gene expression, characterized in that: The RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in Table 1.

2. The RNAi agent according to claim 1, characterized in that The sense strand and / or antisense strand comprises at least one modified nucleotide.

3. The RNAi agent according to claim 1, characterized in that The modified nucleotide is selected from: Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (vP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA).

4. The RNAi agent according to claim 2 or 3, characterized in that The modified RNAi is selected from the sequences shown in Table 2.

5. The RNAi agent according to claim 1, characterized in that The RNAi agent further includes a targeting ligand linked to the sense strand and / or the antisense strand.

6. The RNAi agent according to claim 5, characterized in that The targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

7. The RNAi agent according to claim 5, characterized in that The targeting ligand is selected from:

8. The RNAi agent according to claim 5, characterized in that The targeting ligand is linked to the 3' or 5' end of the sense strand or the antisense strand.

9. A pharmaceutical composition for inhibiting complement factor B gene expression, characterized in that: The pharmaceutical composition comprises the RNA agent according to any one of claims 1-8.

10. Use of the RNAi agent according to any one of claims 1 to 8 in the preparation of a method for treating a complement factor B-related disease condition or symptom, wherein the disease is selected from: Paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMo); multifocal motor neuropathy (MMN); multiple sclerosis (Ms); macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HEL LP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; oligoimmune vasculitis; epidermolysis bullosa; recurrent miscarriage; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, bullous dermatomyositis pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic-uremic syndrome, C3 neuropathy, antineutrophil cytoplasmic antibody-associated vasculitis (eg, granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis), Churg-Strauss syndrome, and microscopic polyangiitis) , humoral and vascular transplant rejection, graft dysfunction, myocardial infarction (e.g., tissue damage and ischemia in myocardial infarction), allogeneic transplantation, sepsis (e.g., poor prognosis in sepsis), coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AlHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disease, myocarditis, cerebrovascular disease, peripheral (e.g., musculoskeletal) vascular disease, renovascular disease, mesenteric / enteric vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and postoperative restenosis with stenting, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA).

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