C3 complement targeting RNAi reagents and uses thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a C3 complement targeting RNAi reagent and application thereof. The RNAi reagent provided by the invention can effectively inhibit the expression of C3 complement in cells, so that the RNAi reagent can be used for treating diseases related to C3 complement.

Description

[0001] Cross-references to related applications

[0002] This invention claims priority to Chinese Patent Application No. 2024111277755, filed on August 16, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine, specifically relating to RNAi reagents targeting C3 complement and their uses. Background Technology

[0004] The complement system is a human immune defense system composed of more than 30 proteins (Janeway, CA Jr et al., 2001). There are three main complement activation pathways: the classical pathway, the alternative pathway, and the lectin pathway (Abbas AK et al., 2010). Complement activation leads to a series of chain enzymatic reactions, forming anaphylatoxins C3a and C5a, which further trigger downstream physiological responses.

[0005] The classical pathway is activated by the binding of C1 to classical pathway activators (primarily antigen-antibody complexes containing IgM, IgG1, IgG2, or IgG3). The reaction sequence of the classical complement activation pathway is: C1, 4, 2, 3, 5, 6, 7, 8, 9. This biochemical pathway creates a pore in the cell membrane, causing the target cell to swell and rupture due to the inability to maintain osmotic pressure.

[0006] The activation of the alternative pathway depends on the natural hydrolysis of C3 into C3a and C3b, and C3b then binds to factors B, D, and P to enter a reaction similar to the classical pathway.

[0007] The full name of the lectin pathway is the mannose-binding lectin pathway. Mannose-binding lectins can recognize mannose and fructose residues on the surface of pathogens, and then bind to the pathogen surface and carry out a reaction similar to the activation of the classical pathway.

[0008] Abnormal complement activation can lead to a series of pathological changes in many diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), generalized myasthenia gravis (GMG), rheumatoid arthritis (RA), and neuromyelitis optica (NMO).

[0009] Complement Component 3 (C3) plays a crucial role in the entire complement system. Both the classical and alternative activation pathways rely on C3 activation (Lambris 2001). C3 is also the most abundant complement in human blood, primarily produced in the liver (de Bruijn MH and Fey GH, 1985). Currently, only one monoclonal antibody, Pegacetacoplan, is available for C3 complement; it is expensive and requires frequent injections.

[0010] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded small interfering RNA (siRNA) during evolution. RNAi drugs also have the advantage of longer duration of action compared to antibody drugs. Therefore, the research and development of siRNAs targeting C3 complement is of great significance. Summary of the Invention

[0011] The first object of the present invention is to provide an RNAi reagent comprising: an antisense strand containing at least 15 consecutive nucleotides, wherein the at least 15 consecutive nucleotides differ by no more than 3 nucleotides from any at least 15 consecutive nucleotides in any of the sequences shown in SEQ ID NO: 54 to 106; and a sense strand containing a nucleotide sequence that is at least partially complementary to the antisense strand.

[0012] The present invention also provides an isolated cell containing the aforementioned RNAi reagent.

[0013] The present invention also provides a pharmaceutical composition comprising the aforementioned RNAi reagent.

[0014] A second objective of the present invention is to provide a method for inhibiting the expression of C3 complement in cells, the method comprising: contacting the cells with the RNAi reagent or the pharmaceutical composition thereof to inhibit the expression of C3 complement in the cells.

[0015] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the treatment and / or prevention of diseases related to C3 complement.

[0016] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the preparation of medicaments for the treatment and / or prevention of diseases related to C3 complement.

[0017] The RNAi reagent of the present invention can effectively inhibit the expression of C3 complement in cells, and therefore can be used to treat diseases related to C3 complement. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The efficacy of different siRNAs in human C3 transgenic mice in Example 3 of this invention.

[0020] Figure 2 This is the result of long-term inhibition of PAT-0038T in human C3 transgenic mice in Example 5 of the present invention. Detailed Implementation

[0021] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0022] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0024] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0025] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0026] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 80% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0027] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0028] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0029] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0030] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0031] In this invention, the term "RNAi reagent" (also known as "RNAi trigger") means a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting (e.g., under appropriate conditions, degrading or inhibiting) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. As used herein, RNAi reagents may function via RNA interference mechanisms (i.e., by interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC) or via any alternative mechanism or pathway. Although it is considered that RNAi reagents function primarily through RNA interference mechanisms as used herein, the disclosed RNAi reagents are not bound to or limited by any particular pathway or mechanism of action. The RNAi reagents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi reagent described herein is at least partially complementary to the target mRNA (i.e., C3 complement mRNA). The RNAi reagent may include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0032] In this invention, when it is mentioned that "the antisense strand (positive strand) contains at least 15 consecutive nucleotides, and the at least n consecutive nucleotides differ from any at least 15 consecutive nucleotides in a reference sequence (e.g., the sequence shown in SEQ ID No. X or the nucleotide sequence of positions 1-21 of the shown sequence) by no more than 3 nucleotides," the alignment involved includes alignment with at least 15 consecutive nucleotides starting at any position (e.g., positions 1, 2, 3, ..., 7, or 8) in the reference sequence. As an example, in some embodiments, the antisense strand (positive strand) contains 21 consecutive nucleotides, wherein the sequence of positions 1-15 differs from the sequence of positions 2-16 of the reference sequence in one, two, or three places, and the nucleotides 16-20 in the antisense strand (positive strand) are all identical or all different from the sequence of positions 17-21 of the reference sequence; such sequences are also within the scope of this invention. In some embodiments, the antisense strand (positive strand) contains 21 consecutive nucleotides, wherein the sequence of nucleotides 1 to 15 is identical to the sequence of nucleotides 1 to 15 of the reference sequence, and nucleotides 16 to 21 of the antisense strand (positive strand) are either identical or different from the sequence of nucleotides 16 to 21 of the reference sequence; such sequences are also within the scope of this invention.

[0033] In this invention, when referring to descriptions related to sequence alignment, the term "difference" or "separation" includes one or more of substitution, insertion, and deletion.

[0034] In this invention, when referring to the expression of a given gene, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” mean, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, when the cell, cell population, tissue, organ, or subject is treated with the RNAi reagent described herein, the expression of the gene is reduced compared to a second cell, cell population, tissue, organ, or subject who has not been treated in this way.

[0035] In this invention, "completely complementary" means that in a hybridization pair of nucleobase or nucleotide sequences, all (100%) bases in the adjacent sequence of the first oligonucleotide hybridize with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0036] In this invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0037] In this invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0038] In this invention, the term "at least partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary, or completely complementary.

[0039] In this invention, the term "treatment" refers to a method or procedure taken to provide relief or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, preventative treatment, and / or suppression or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject.

[0040] In this invention, the term "link" means the combination of two compounds or molecules through a covalent bond. Unless otherwise stated, as used herein, the term "link" may refer to a link between a first compound and a second compound, with or without any intermediate atoms or groups of atoms.

[0041] RNAi reagents

[0042] The present invention first provides an RNAi reagent comprising: an antisense strand containing at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides differ from any of the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in any of the sequences shown in SEQ ID NO: 54 to 106 by no more than 0, 1, 2, or 3 nucleotides; and a sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or completely complementary) to the antisense strand.

[0043] In some embodiments, the positive strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differ from any of the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in any of the sequences shown in SEQ ID No. 1 to 53 by no more than 0, 1, 2, or 3 nucleotides.

[0044] In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0045] In some embodiments, the positive strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0046] In this invention, the justice chain and the antisense chain can have the same length or different lengths.

[0047] In practical implementation, those skilled in the art can combine the sequences provided in this invention by taking into account the complementarity of the sense and antisense strands, thereby obtaining combined RNAi reagents (such as siRNA).

[0048] In a preferred embodiment of the present invention, as shown in Table 1, the RNAi reagent is selected from siRNA-1 with a sense strand sequence of SEQ ID NO: 1 and an antisense strand sequence of SEQ ID NO: 54, siRNA-2 with a sense strand sequence of SEQ ID NO: 2 and an antisense strand sequence of SEQ ID NO: 55, siRNA-3 with a sense strand sequence of SEQ ID NO: 3 and an antisense strand sequence of SEQ ID NO: 56, siRNA-4 with a sense strand sequence of SEQ ID NO: 4 and an antisense strand sequence of SEQ ID NO: 57, siRNA-5 with a sense strand sequence of SEQ ID NO: 55 and an antisense strand sequence of SEQ ID NO: 58, siRNA-6 with a sense strand sequence of SEQ ID NO: 6 and an antisense strand sequence of SEQ ID NO: 59, siRNA-7 with a sense strand sequence of SEQ ID NO: 7 and an antisense strand sequence of SEQ ID NO: 60, siRNA-8 with a sense strand sequence of SEQ ID NO: 8 and an antisense strand sequence of SEQ ID NO: 61, and siRNA-8 with a sense strand sequence of SEQ ID NO: 54. siRNA-9 with SEQ ID NO: 9 and antisense sequence of SEQ ID NO: 62; siRNA-10 with SEQ ID NO: 10 and antisense sequence of SEQ ID NO: 63; siRNA-11 with SEQ ID NO: 11 and antisense sequence of SEQ ID NO: 64; siRNA-12 with SEQ ID NO: 12 and antisense sequence of SEQ ID NO: 65; siRNA-13 with SEQ ID NO: 13 and antisense sequence of SEQ ID NO: 66; siRNA-14 with SEQ ID NO: 14 and antisense sequence of SEQ ID NO: 67; siRNA-15 with SEQ ID NO: 15 and antisense sequence of SEQ ID NO: 68; siRNA-16 with SEQ ID NO: 16 and antisense sequence of SEQ ID NO: 69; siRNA-16 with SEQ ID NO: 17 and antisense sequence of SEQ ID NO: 68. siRNA-17 (NO: 70), siRNA-18 (sense strand sequence is SEQ ID NO: 18), siRNA-18 (sense strand sequence is SEQ ID NO: 71), siRNA-19 (sense strand sequence is SEQ ID NO: 19), siRNA-19 (sense strand sequence is SEQ ID NO: 72), siRNA-20 (sense strand sequence is SEQ ID NO: 20), siRNA-20 (sense strand sequence is SEQ ID NO: 73), siRNA-20 (sense strand sequence is SEQ ID NO: 21), and siRNA-19 (sense strand sequence is SEQ ID NO: 73).siRNA-21 (SEQ ID NO: 74), siRNA-22 (sense strand sequence is SEQ ID NO: 22), siRNA-22 (sense strand sequence is SEQ ID NO: 75), siRNA-23 (sense strand sequence is SEQ ID NO: 23), siRNA-23 (sense strand sequence is SEQ ID NO: 76), siRNA-24 (sense strand sequence is SEQ ID NO: 24), siRNA-24 (sense strand sequence is SEQ ID NO: 77), siRNA-25 (sense strand sequence is SEQ ID NO: 25), siRNA-25 (sense strand sequence is SEQ ID NO: 78), siRNA-26 (sense strand sequence is SEQ ID NO: 26), siRNA-26 (sense strand sequence is SEQ ID NO: 79), siRNA-27 (sense strand sequence is SEQ ID NO: 27), siRNA-27 (sense strand sequence is SEQ ID NO: 80), siRNA-28 (sense strand sequence is SEQ ID NO: 28), siRNA-28 (sense strand sequence is SEQ ID NO: 81), siRNA-28 (sense strand sequence is SEQ ID NO: 29), and siRNA-27 (sense strand sequence is SEQ ID NO: 80). siRNA-29 with SEQ ID NO: 82, siRNA-30 with the positive strand sequence of SEQ ID NO: 30 and the antisense strand sequence of SEQ ID NO: 83, siRNA-31 with the positive strand sequence of SEQ ID NO: 31 and the antisense strand sequence of SEQ ID NO: 84, siRNA-32 with the positive strand sequence of SEQ ID NO: 32 and the antisense strand sequence of SEQ ID NO: 85, siRNA-33 with the positive strand sequence of SEQ ID NO: 33 and the antisense strand sequence of SEQ ID NO: 86, siRNA-34 with the positive strand sequence of SEQ ID NO: 34 and the antisense strand sequence of SEQ ID NO: 87, siRNA-35 with the positive strand sequence of SEQ ID NO: 35 and the antisense strand sequence of SEQ ID NO: 88, siRNA-36 with the positive strand sequence of SEQ ID NO: 36 and the antisense strand sequence of SEQ ID NO: 89, and siRNA-36 with the positive strand sequence of SEQ ID NO: 37 and the antisense strand sequence of SEQ ID NO: 88. siRNA-37 (SEQ ID NO: 90), siRNA-38 (SEQ ID NO: 38) with the positive strand sequence of SEQ ID NO: 91, siRNA-39 (SEQ ID NO: 39) with the positive strand sequence of SEQ ID NO: 92, siRNA-40 (SEQ ID NO: 40) with the positive strand sequence of SEQ ID NO: 93, siRNA-41 (SEQ ID NO: 41) with the positive strand sequence of SEQ ID NO: 94, and siRNA-42 (SEQ ID NO: 42) with the positive strand sequence of SEQ ID NO: 90.siRNA-42 with SEQ ID NO: 95, siRNA-43 with the positive strand sequence of SEQ ID NO: 43 and the antisense strand sequence of SEQ ID NO: 96, siRNA-44 with the positive strand sequence of SEQ ID NO: 44 and the antisense strand sequence of SEQ ID NO: 97, siRNA-45 with the positive strand sequence of SEQ ID NO: 45 and the antisense strand sequence of SEQ ID NO: 98, siRNA-46 with the positive strand sequence of SEQ ID NO: 46 and the antisense strand sequence of SEQ ID NO: 99, siRNA-47 with the positive strand sequence of SEQ ID NO: 47 and the antisense strand sequence of SEQ ID NO: 100, siRNA-48 with the positive strand sequence of SEQ ID NO: 48 and the antisense strand sequence of SEQ ID NO: 101, siRNA-49 with the positive strand sequence of SEQ ID NO: 49 and the antisense strand sequence of SEQ ID NO: 102, and siRNA-49 with the positive strand sequence of SEQ ID NO: 50 and the antisense strand sequence of SEQ ID NO: 98. At least one of the following: siRNA-50 with the positive strand sequence SEQ ID NO: 103, siRNA-51 with the positive strand sequence SEQ ID NO: 51 and the negative strand sequence SEQ ID NO: 104, siRNA-52 with the positive strand sequence SEQ ID NO: 52 and the negative strand sequence SEQ ID NO: 105, and siRNA-53 with the positive strand sequence SEQ ID NO: 53 and the negative strand sequence SEQ ID NO: 106.

[0049] In a preferred embodiment of the present invention, the RNAi reagent is siRNA-1 with a sense strand sequence of SEQ ID NO: 1 and an antisense strand sequence of SEQ ID NO: 54, siRNA-2 with a sense strand sequence of SEQ ID NO: 2 and an antisense strand sequence of SEQ ID NO: 55, siRNA-3 with a sense strand sequence of SEQ ID NO: 3 and an antisense strand sequence of SEQ ID NO: 56, siRNA-4 with a sense strand sequence of SEQ ID NO: 4 and an antisense strand sequence of SEQ ID NO: 57, siRNA-5 with a sense strand sequence of SEQ ID NO: 55 and an antisense strand sequence of SEQ ID NO: 58, siRNA-6 with a sense strand sequence of SEQ ID NO: 6 and an antisense strand sequence of SEQ ID NO: 59, siRNA-7 with a sense strand sequence of SEQ ID NO: 7 and an antisense strand sequence of SEQ ID NO: 60, siRNA-8 with a sense strand sequence of SEQ ID NO: 8 and an antisense strand sequence of SEQ ID NO: 61, and siRNA-8 with a sense strand sequence of SEQ ID NO: 9 and an antisense strand sequence of SEQ ID NO: 54. siRNA-9 (NO: 62), siRNA-10 (positive strand sequence is SEQ ID NO: 10) and siRNA-10 (antisense strand sequence is SEQ ID NO: 63), siRNA-11 (positive strand sequence is SEQ ID NO: 11) and siRNA-11 (antisense strand sequence is SEQ ID NO: 64), siRNA-11 (positive strand sequence is SEQ ID NO: 12) and siRNA-12 (antisense strand sequence is SEQ ID NO: 65), siRNA-12 (positive strand sequence is SEQ ID NO: 13) and siRNA-13 (antisense strand sequence is SEQ ID NO: 66), siRNA-13 (positive strand sequence is SEQ ID NO: 14) and siRNA-14 (antisense strand sequence is SEQ ID NO: 67), siRNA-14 (positive strand sequence is SEQ ID NO: 15) and siRNA-15 (antisense strand sequence is SEQ ID NO: 68), siRNA-15 (positive strand sequence is SEQ ID NO: 16) and siRNA-16 (antisense strand sequence is SEQ ID NO: 69), siRNA-16 (positive strand sequence is SEQ ID NO: 17) and siRNA-15 (antisense strand sequence is SEQ ID NO: 68). siRNA-17 (SEQ ID NO: 70), siRNA-18 (SEQ ID NO: 18) with the positive strand sequence of SEQ ID NO: 71, siRNA-19 (SEQ ID NO: 19) with the positive strand sequence of SEQ ID NO: 72, siRNA-19 (SEQ ID NO: 20) with the positive strand sequence of SEQ ID NO: 73, siRNA-20 (SEQ ID NO: 21) with the positive strand sequence of SEQ ID NO: 74, siRNA-21 (SEQ ID NO: 74) with the positive strand sequence of SEQ ID NO: 70.siRNA-22 with SEQ ID NO: 22 and antisense sequence of SEQ ID NO: 75, siRNA-23 with SEQ ID NO: 23 and antisense sequence of SEQ ID NO: 76, siRNA-24 with SEQ ID NO: 24 and antisense sequence of SEQ ID NO: 77, siRNA-25 with SEQ ID NO: 25 and antisense sequence of SEQ ID NO: 78, siRNA-26 with SEQ ID NO: 26 and antisense sequence of SEQ ID NO: 79, siRNA-27 with SEQ ID NO: 27 and antisense sequence of SEQ ID NO: 80, siRNA-28 with SEQ ID NO: 28 and antisense sequence of SEQ ID NO: 81, siRNA-29 with SEQ ID NO: 29 and antisense sequence of SEQ ID NO: 82, and siRNA-29 with SEQ ID NO: 30 and antisense sequence of SEQ ID NO: 75. siRNA-30 (SEQ ID NO: 83), siRNA-31 (sense strand sequence is SEQ ID NO: 31 and antisense strand sequence is SEQ ID NO: 84), siRNA-32 (sense strand sequence is SEQ ID NO: 32 and antisense strand sequence is SEQ ID NO: 85), siRNA-33 (sense strand sequence is SEQ ID NO: 33 and antisense strand sequence is SEQ ID NO: 86), siRNA-34 (sense strand sequence is SEQ ID NO: 34 and antisense strand sequence is SEQ ID NO: 87), siRNA-35 (sense strand sequence is SEQ ID NO: 35 and antisense strand sequence is SEQ ID NO: 88), siRNA-36 (sense strand sequence is SEQ ID NO: 36 and antisense strand sequence is SEQ ID NO: 89), siRNA-37 (sense strand sequence is SEQ ID NO: 37 and antisense strand sequence is SEQ ID NO: 90), and siRNA-37 (sense strand sequence is SEQ ID NO: 38 and antisense strand sequence is SEQ ID NO: 89). siRNA-38 (NO: 91), with the positive strand sequence of SEQ ID NO: 39 and the antisense strand sequence of SEQ ID NO: 92; siRNA-39, with the positive strand sequence of SEQ ID NO: 40 and the antisense strand sequence of SEQ ID NO: 93; siRNA-40, with the positive strand sequence of SEQ ID NO: 41 and the antisense strand sequence of SEQ ID NO: 94; siRNA-41, with the positive strand sequence of SEQ ID NO: 42 and the antisense strand sequence of SEQ ID NO: 95; siRNA-42, with the positive strand sequence of SEQ ID NO: 95.siRNA-43 with SEQ ID NO: 43 and antisense sequence of SEQ ID NO: 96; siRNA-44 with SEQ ID NO: 44 and antisense sequence of SEQ ID NO: 97; siRNA-45 with SEQ ID NO: 45 and antisense sequence of SEQ ID NO: 98; siRNA-46 with SEQ ID NO: 46 and antisense sequence of SEQ ID NO: 99; siRNA-47 with SEQ ID NO: 47 and antisense sequence of SEQ ID NO: 100; siRNA-48 with SEQ ID NO: 48 and antisense sequence of SEQ ID NO: 101; siRNA-49 with SEQ ID NO: 49 and antisense sequence of SEQ ID NO: 102; siRNA-50 with SEQ ID NO: 50 and antisense sequence of SEQ ID NO: 103; siRNA-44 with SEQ ID NO: 43 and antisense sequence of SEQ ID NO: 96; siRNA-45 with SEQ ID NO: 45 and antisense sequence of SEQ ID NO: 98; siRNA-46 with SEQ ID NO: 46 and antisense sequence of SEQ ID NO: 99; siRNA-47 with SEQ ID NO: 47 and antisense sequence of SEQ ID NO: 100; siRNA-48 with SEQ ID NO: 48 and antisense sequence of SEQ ID NO: 101; siRNA-49 with SEQ ID NO: 49 and antisense sequence of SEQ ID NO: 102; siRNA-50 with SEQ ID NO: 50 and antisense sequence of SEQ ID NO: 103; siRNA-45 with SEQ ID NO: 46 and antisense sequence of SEQ ID NO: 99; siRNA-45 with SEQ ID NO: 47 and antisense sequence of SEQ One of the following: siRNA-51 with SEQ ID NO: 51 and antisense sequence of SEQ ID NO: 104; siRNA-52 with SEQ ID NO: 52 and antisense sequence of SEQ ID NO: 105; and siRNA-53 with SEQ ID NO: 53 and antisense sequence of SEQ ID NO: 106.

[0050] In some preferred embodiments, the antisense strand contains a nucleotide sequence differing from any of the sequences shown in SEQ ID NO: 54, 55, 57, 59, 62, 63, 67–69, 76, 80–82, 88–95, 98, 99, 101, 105 by 0, 1, or 2 nucleotides, and the sense strand contains a nucleotide sequence at least partially complementary to the antisense strand (e.g., partially complementary, substantially complementary, or completely complementary). In some preferred embodiments, the sense strand contains a nucleotide sequence differing from any of the sequences shown in SEQ ID NO: 1, 2, 4, 6, 9, 10, 14–16, 23, 27–29, 35–42, 45, 46, 48, 52 by 0, 1, or 2 nucleotides. When such sequences are present, the inhibitory effect of the RNAi reagent is superior.

[0051] In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 9 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 62 by 0, 1, or 2 nucleotides.

[0052] In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 10 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 63 by 0, 1, or 2 nucleotides.

[0053] In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 38 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 91 by 0, 1, or 2 nucleotides.

[0054] The inhibitory effect of the RNAi reagent is further enhanced when the above sequences are present. The inhibitory effect is particularly optimal when the sense strand sequence is as shown in SEQ ID NO: 38 and the antisense strand sequence is as shown in SEQ ID NO: 91.

[0055] All nucleotide groups in the above RNAi reagents may be unmodified or contain at least one modified nucleotide group, and the modification may be on nucleotides at any position.

[0056] In some implementations, the justice chain and the antisense chain may be partially complementary, substantially complementary, or completely complementary to each other.

[0057] In some embodiments, when the sequence identity of the sense or antisense strand in the RNAi reagent is less than 100% or differs by more than one nucleotide from the corresponding sequence mentioned in this invention, it still has an inhibitory effect on C3 complement that is similar to (e.g., still has an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent to (e.g., still has an efficacy equivalent to 95-105% of the corresponding sequence). For example, the two bases at the 3' end of the antisense strand (such as any of the sequences shown in SEQ ID NO: 54-106) can be replaced with UU, AA, CU, UC, AG, CC, GG, or UG, or any combination of two nucleic acids. Such nucleic acid sequences are also within the scope of protection of this invention.

[0058] In some preferred embodiments, the RNAi reagent has an inhibitory efficiency of not less than 50% against C3 complement (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0059] The present invention also provides target gene sites for nucleic acids as described above. In some embodiments, the target gene sites are marked as any item in column 1 of Table 1.

[0060] Table 1

[0061]

[0062]

[0063] Note: Column 1 indicates the position of the first base of the target gene in the human C3 complement mRNA sequence, and so on; the numbers in columns 3 and 5 represent sequence numbers, for example, "1" represents SEQ ID NO: 1.

[0064] The reference sequence for the target gene is the coding sequence of human C3 complement mRNA, NM_000064.4.

[0065] In some embodiments, the positive strand of the RNAi reagent has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the isochronous sequences at positions 200-400, 400-600, 600-900, 1200-1400, 1300-1500, 2500-2600, 2800-3000, 3700-3900, or 4000-4700 of NM_000064.4.

[0066] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 200-400, 400-600, 600-900, 1200-1400, 1300-1500, 2500-2600, 2800-3000, 3700-3900, or 4000-4700 in NM_000064.4.

[0067] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 200-300, 400-500, 650-750, 700-850, 1250-1350, 1350-1450, 2500-2600, 2800-2950, ​​3750-3850, 4100-4300, 4400-4550, or 4550-4650 in NM_000064.4.

[0068] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 760-810 in NM_000064.4.

[0069] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 2840-2900 in NM_000064.4.

[0070] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 3770-3820 in NM_000064.4.

[0071] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 4110-4160 in NM_000064.4.

[0072] In some embodiments, the RNAi reagent targets any position within the region of nucleotides 4590-4640 in NM_000064.4.

[0073] The advantages of the technical solutions regarding bare sequences (i.e., unmodified sequences) mentioned in this invention do not depend on the choice of modification method or target vector. The following details the applicable modification schemes and further preferred modification schemes:

[0074] In some embodiments, the RNAi reagent contains nucleotide groups as basic structural units, the nucleotide groups containing phosphate groups, ribose groups and bases, and preferably, the RNAi reagent contains at least one modified nucleotide group. RNAi reagents containing modified groups have an inhibition efficiency of not less than 50% against C3 complement (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0075] In some embodiments, the modified nucleotide group is a nucleotide group whose phosphate group and / or ribose group are modified. The modified site may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in the sense strand and / or antisense strand.

[0076] In some embodiments, modification of the phosphate group refers to modifying the oxygen in the phosphate group, including phosphate thioate modification and boranophosphate modification. The oxygen in the phosphate group is replaced by sulfur, borane, amino, alkyl, or alkoxy groups, as shown in the following formulas. These modifications can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing.

[0077]

[0078]

[0079] In the above structural formulas, BASE represents a base A, U, C, G, or T. X can be oxygen (O) or sulfur (S). R can be the same or different in the above structures, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc. R' and R” can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.

[0080] In some embodiments, modification of the ribose group refers to the modification of the 2′-hydroxyl group (2′-OH) in the ribose group. Introducing substituents such as methoxy or fluorine at the 2′-hydroxyl position of the ribose group makes the nucleic acid less susceptible to cleavage by ribonucleases, thereby increasing the stability of the nucleic acid and giving it stronger resistance to nuclease hydrolysis. Modification of the 2′-hydroxyl group in the pentose of nucleotides includes 2′-fluoromodification (e.g., 2′-arabino-fluoromodification), 2′-methoxymodification (2′-OME), 2′-methoxyethylmodification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), 2′,4′-constrained ethylmodification, 2′-aminomodification (2′-Amino modification), 2′-deoxymodification (2′-Deoxy modification), BNA, acyclic nucleic acid modification, misaligned nucleic acid modification, L-type nucleic acid modification, etc. BNA (inner-ring bridging nucleotide) refers to a constrained or inaccessible nucleotide. BNAs can contain bridging structures with "fixed" C 3'-endoglycosylation rings, including five-, six-, or seven-membered rings. This bridge is typically incorporated into the 2', 4' positions of the ribosome to provide 2',4'-BNA nucleotides, such as locked-ethyl (LNA), ring-locked-ethyl (ENA), and ethyl-locked nucleic acid (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) and glycerol nucleic acid (GNA). Misalignment refers to the replacement of the 3',5'-phosphate bond with a 2',5'-phosphate bond. L-type nucleic acid modification refers to the replacement of a naturally occurring D-type nucleic acid with its mirror-image L-type nucleic acid.

[0081]

[0082]

[0083] In this context, BASE represents the bases A, U, C, G, or T. R can be the same or different in the above structures, for example: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

[0084] In some embodiments, the nucleotide group containing a uracil or cytosine base in the sense strand of the RNAi reagent is a nucleotide group whose riboside group has been modified; that is, the 2'-OH of the riboside group in the uracil or cytosine base-containing nucleotide group in the sense strand of the RNAi reagent is replaced by a methoxy or fluorine group. More preferably, the 3' ends of both the sense and antisense strands of the RNAi reagent may be linked with dTdT; or, the 3' end of the antisense strand of the RNAi reagent may be linked with AA or UU or any combination of two nucleic acids (which may be, but is not limited to, CC, GG, or UG), giving the sequence a specific inducing factor for mRNA degradation. The RNAi reagent with the above modifications exhibits superior in vivo inhibitory effects, and the above modifications can further reduce the immunogenicity of the RNAi reagent of the present invention in vivo.

[0085] The RNAi reagent of the present invention may further include a modification involving the attachment of a monophosphate nucleoside to the 5' end of the antisense strand. The 5'-monophosphate at the terminal of the siRNA strand is important for RISC recognition. Phosphorylation of the 5'-hydroxyl group plays a role in the efficient loading of siRNA onto Ago2 within the cell. The 5'-terminal monophosphate in the siRNA strand interacts with Argonaute-2 (Ago2) via an H-bond, thereby ensuring accurate target localization and precise cleavage of the mRNA. Several commonly used 5'-monophosphate nucleoside derivatives have been shown to exhibit stability in biological metabolic media and play a role in promoting the loading of the siRNA strand onto Ago2 within the cell (NucleicAcids Research, 2015, 43, 2993–3011). In the RNAi reagent of the present invention, trans-vinyl phosphate (VP) is preferred, but other monophosphate nucleoside derivatives may also be included.

[0086]

[0087]

[0088] In the above structures, BASE represents the base A, U, C, G, or T. R can be the same or different in the above structures, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

[0089] In this invention, The meaning is consistent, referring to a chemical element X being attached to any one or more groups.

[0090] In some embodiments, at least one nucleotide in the RNAi reagent is a modified nucleotide or includes a modified interphase bond.

[0091] In some embodiments, the modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-open-ring nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, debased nucleotides, ribitol, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides, peptide nucleic acids (PNA), glycerol nucleic acids (GNA), triazine-cyclic DNA (tcDNA), nucleotides containing non-natural bases, nucleotides containing vinylphosphonates, nucleotides containing cyclopropylphosphonates, and 3'-O-methyl nucleotides. In some embodiments, the modified nucleotide is further preferably selected from one or both of 2'-O-methyl nucleotides and 2'-fluoronucleotides.

[0092] In some embodiments, the modified inter-linked bonds are preferably one or more selected from phosphate thioester nucleotide inter-linked bonds and methylphosphonate nucleotide inter-linked bonds. In some embodiments, the modified inter-linked bonds are further preferably one or more selected from phosphate thioester monoester nucleotide inter-linked bonds and phosphate thioester diester nucleotide inter-linked bonds.

[0093] In some embodiments, the last two, three, or four nucleotides at the 5' and / or 3' ends of the antisense strand contain a phosphate thioester nucleotide bond, and the antisense strand contains four, five, six, seven, or eight 2'-fluoronucleotides, with the remaining nucleotides being 2'-O-methylnucleotides.

[0094] In some alternative embodiments, the last three nucleotides at the 5' and 3' ends of the antisense strand contain a phosphate thionucleotide bond, and at least one (preferably two, three, four, five, or six) of the nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 counting from the 5' end of the antisense strand is a 2'-fluoronucleotide, and the remaining nucleotides are 2'-O-methylnucleotides.

[0095] In some embodiments, the antisense strand comprises 5'-nNfnNfnNfnnnnnnnNfnNfnnn nnnn-3', 5'-nNfnnnNfnnnnnnnNfnNfnnnnnnn-3', 5'-nNfnNfnnnnnnnnnN fnNfnnnnnnn-3', 5'-nNfnNfnNfnNfnnnnnNfnNfnnnnnnn-3', 5'-nNfnNfn NfnnnNfnnnNfnNfnnnnnnn-3', 5'-nNfnNfnNfnnnnnNfnNfnNfnnnnnnn-3', or 5'-nNfnNfnNfnnnnnnnNfnNfnNfnnnnn-3'; where Nf represents 2'-fluoro nucleotide and n represents 2'-O-methyl nucleotide. Compared with known modification methods, the above-described antisense strand modification scheme is further beneficial to enhancing the inhibitory effect of the RNAi reagent on C3 complement.

[0096] In some preferred embodiments, the antisense chain comprises 5'-nNfnNfnNfnnnnnnnnNfnNfnnnnnnnn-3'. Compared to other modification methods, the above-described antisense chain modification scheme exhibits significantly better inhibition of C3 complement.

[0097] In some embodiments, the last two, three, or four nucleotides at the 5' end and / or 3' end of the positive strand contain a phosphate thioester nucleotide bond, and the positive strand contains 2, 3, 4, 5, or 6 2'-fluoronucleotides, with the remaining nucleotides being 2'-O-methylnucleotides.

[0098] In some embodiments, the positive strand has at least one (preferably two, three, or four) nucleotides at positions 7, 9, 10, and 11 counting from the 5' end that are 2'-fluoronucleotides, and the remaining nucleotides are 2'-O-methylnucleotides.

[0099] In some embodiments, the positive strand comprises 5'-nnnnnnNfnNfNfNfnnnnnnnnnnn n-3', where Nf represents a 2'-fluoronucleotide and n represents a 2'-O-methylnucleotide. Compared with known modification methods, the above-described positive strand modification scheme further enhances the inhibitory effect of the nucleic acid on C3 complement.

[0100] In some embodiments, the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' end and / or 3' end of the antisense strand contain thiophosphate nucleotide bonds, and the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' end and / or 3' end of the sense strand contain thiophosphate nucleotide bonds.

[0101] In some specific embodiments, the last three nucleotides at the 5' and 3' ends of the antisense strand contain a phosphate thioester nucleotide bond, and the last three nucleotides at the 5' end of the sense strand contain a phosphate thioester nucleotide bond.

[0102] In some embodiments, the antisense strand contains a nucleotide sequence that differs from any of the antisense strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0103] In some embodiments, the positive strand contains a nucleotide sequence that differs from any of the positive strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0104] In some embodiments, the RNAi reagent contains a double strand as shown in either Table 2 or Table 3.

[0105] In some preferred embodiments, the RNAi reagent contains a compound selected from PAT-0001, PAT-0002, PAT-0004, PAT-0006, PAT-0009, PAT-0009.1, PAT-0009.2, PAT-0010, PAT-0010.1, PAT-0010.2, PAT-0014, PAT-0015, PAT-0016, PAT-0023, PAT-0027, PAT-0028, PA. The double-stranded RNAi reagents containing any of the above double-stranded RNAi compounds exhibit excellent inhibitory effects on C3 complement in cell experiments. These compounds are T-0029, PAT-0035, PAT-0036, PAT-0037, PAT-0038, PAT-0038.1, PAT-0038.2, PAT-0038.3, PAT-0038.4, PAT-0038.5, PAT-0039, PAT-0040, PAT-0041, PAT-0042, PAT-0045, PAT-0046, PAT-0048, and PAT-0052.

[0106] In some further preferred embodiments, the RNAi reagent contains a double strand selected from any one of PAT-0009, PAT-0010, and PAT-0038. RNAi reagents containing the aforementioned double strands achieve excellent inhibitory effects on C3 complement both in vivo and in vitro.

[0107] In some further preferred embodiments, the RNAi reagent contains the double strand described in PAT-0038. RNAi reagents containing the aforementioned double strand achieve optimal inhibitory effects on C3 complement both in vivo and in vitro.

[0108] Table 2

[0109]

[0110]

[0111] Table 3

[0112]

[0113]

[0114] In the various modified sequences of this invention, the nucleotide represented by the lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to it on the left is a 2'-fluoro nucleotide; s indicates that the two adjacent nucleotides on the left and right are connected by a phosphate diester nucleotide bond.

[0115] The RNAi reagent according to the present invention can be obtained by conventional methods in the art, such as solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis is available through commercially available custom services and is therefore commercially available. The modified nucleotide groups can be introduced using nucleotide monomers with corresponding modifications.

[0116] Based on the RNAi reagent (siRNA) synthesized above, the present invention can further construct shRNA expression plasmids with the same or similar functions as the above RNAi reagents. The method for constructing the expression plasmid is known to those skilled in the art and will not be described in detail here.

[0117] In some embodiments, the RNAi reagent further comprises at least one ligand that is attached to one or more of the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and the 3' end of the sense strand.

[0118] Based on common knowledge in the art, the sense and antisense strands of this invention exhibit superior inhibitory effects when applied to different targeted drug delivery systems. In other words, the efficacy advantages of the naked and modified sequences in this invention do not depend on the choice of target vector. To further improve the bioavailability and therapeutic effect of siRNA, this invention also optimizes the targeted drug delivery system, resulting in the following technical solution.

[0119] In some embodiments, the ligand is attached to the 5' end or 3' end of the positive strand of the nucleic acid. In some embodiments, the ligand is attached to the 5' end of the positive strand of the nucleic acid. In some embodiments, the ligand is attached to the 3' end of the positive strand of the nucleic acid.

[0120] In some embodiments, the ligand is attached to the 5' end or 3' end of the antisense strand of the nucleic acid. In some embodiments, the ligand is attached to the 5' end of the antisense strand of the nucleic acid. In some embodiments, the ligand is attached to the 3' end of the antisense strand of the nucleic acid.

[0121] In some implementations, the ligand is attached to the 3' end of the positive strand of the nucleic acid.

[0122] In some embodiments, the ligand is a GalNAc derivative; more preferably, the ligand is one or more GalNAc derivatives linked by a single-chain, double-chain, or triple-chain branched ligand.

[0123] In some embodiments, the RNAi reagent comprises a compound with the structure shown in Formula I:

[0124]

[0125] In Formula I, Nu represents a nucleic acid composed of the sense strand and the antisense strand. This targeted drug delivery system utilizes the structural characteristics on its left side to improve the cell penetration ability of nucleic acid drugs (Nu), enhance their stability within cells, and has a simple preparation process and strong practicality.

[0126] cell

[0127] The present invention also provides an isolated cell containing the aforementioned RNAi reagent.

[0128] In some embodiments, the cells can be used for purposes such as gene function research, disease model research, or drug screening.

[0129] In some embodiments, the cells do not develop into an animal individual. In some specific embodiments, the cells may be microbial cells or animal cells, but the animal cells are not animal embryonic stem cells or cells at various stages of formation and development (e.g., germ cells, fertilized egg cells, etc.).

[0130] Pharmaceutical Composition

[0131] The present invention also provides a pharmaceutical composition comprising the aforementioned RNAi reagent.

[0132] The pharmaceutical composition can be prepared using conventional methods from the RNAi reagent and the pharmaceutically acceptable carrier. For example, the pharmaceutical composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular, or intravenous injection.

[0133] According to the pharmaceutical composition of the present invention, there are no particular requirements for the amount of RNAi reagent and pharmaceutically acceptable carrier. Generally, the content of pharmaceutically acceptable carrier can be 1-100,000 parts by weight relative to 1 part by weight of the RNAi reagent (e.g., 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1,000 parts by weight, 5,000 parts by weight, 10,000 parts by weight, 50,000 parts by weight, 100,000 parts by weight, or any value between any two of the above).

[0134] According to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be any of the carriers conventionally used in the art, for example, it may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight (e.g., 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, or any value between any two of the above). The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliohms / kg. The content of the osmotic pressure regulator can be determined by those skilled in the art based on the desired osmotic pressure.

[0135] According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any type of liposome capable of encapsulating nucleic acids, and its diameter can be 25-1000 nm, including but not limited to cholesterol and its analogues or derivatives.

[0136] The dosage of the pharmaceutical composition described in this invention can be a conventional dosage in the art, which can be determined based on various parameters, particularly the age, weight, and sex of the subject. For example, for female mice aged 3-4 months and weighing 25-30g, the dosage of the pharmaceutical composition can be 0.01-100 mg / kg body weight, preferably 1-10 mg / kg body weight, based on the amount of the RNAi reagent in the pharmaceutical composition.

[0137] Methods and Applications

[0138] The present invention also provides a method for inhibiting the expression of C3 complement in cells, the method comprising: contacting the cells with the RNAi reagent or the pharmaceutical composition to inhibit the expression of C3 complement in the cells.

[0139] In some implementations, the cells are in a subject, for example, a human subject, such as a subject with C3 complement-related disease, or a subject who needs to prevent the risk of C3 complement-related disease.

[0140] In some embodiments, the cells are located in vitro. The method is based on research purposes or is used to construct animal models.

[0141] In some embodiments, contacting the cells with the nucleic acid inhibits C3 complement expression by at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., compared to C3 complement expression levels prior to the cells' first contact with the nucleic acid; e.g., before administering a first dose of the nucleic acid to the subject). In some embodiments, inhibiting C3 complement expression reduces C3 complement protein levels in the subject's serum sample by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to C3 complement expression levels prior to the cells' first contact with the nucleic acid.

[0142] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the treatment and / or prevention of diseases related to C3 complement.

[0143] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the preparation of medicaments for the treatment and / or prevention of diseases related to C3 complement.

[0144] In some implementations, the disease is: (i) a disease associated with enhanced or elevated C3 complement; or (ii) a disease that would benefit from reduced C3 complement expression.

[0145] In some embodiments, the disease is selected from one or more of the following: paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, hereditary angioedema (HAE), multiple sclerosis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome, membranoproliferative glomerulonephritis (MPGN), IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulonephropathy (C3G), acute tubular necrosis (ATN), focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN), neuromyelitis optica (NMO), age-related macular degeneration (AMD), Guillain-Barré syndrome, multifocal motor neuropathy, and generalized myasthenia gravis (GMG).

[0146] In this invention, the subject can be a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In some embodiments, the subject is preferably a primate, and more preferably a human.

[0147] In some implementations, the medication can be administered via multiple routes, depending on whether local or systemic treatment is required. Dosage can be referenced as described above and will not be repeated here.

[0148] In some embodiments, administration can be local (e.g., percutaneous patch), pulmonary, such as by inhalation or blowing of powder or spray, including via nebulizer; intratracheal, nasal, epidermal, and percutaneous, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, such as via a transplant device; or intracranial, such as via intraparenchymal, intrathecal or intracardiac administration.

[0149] In some embodiments, the RNAi reagent or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

[0150] Example

[0151] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0152] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0153] Example 1

[0154] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) to a 96-well cell culture dish containing 10 4Hep3B (Procell, Cat#CL-0102) cells were cultured overnight at 37°C in a cell culture vessel with 5% CO2. RNAiMAX (1.5 μL / well) and small interfering nucleic acids (siRNA) from Table 2 were added to Opti-MEM medium to bring the final concentration per well to 1 nM or 10 nM. Cells were then cultured at 37°C with 5% CO2 for 48 hours. To extract RNA, the cell culture supernatant was aspirated, washed with PBS, and then 50 μL of prepared lysis buffer (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)) was added and mixed. After standing for 10 min, 2.5 μL of stop solution was added to terminate the reaction for 2 min. RT-PCR was performed according to the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number: 4368814), with each reaction containing 10 μL of lysed DNA. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for human C3 was Hs00163811_m1, and the probe for the internal control gene (human HPRT1) was Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 95℃ for 20 seconds per cycle, followed by 40 cycles of 95℃ for 1 second and 60℃ for 20 seconds. The real-time fluorescence PCR instrument was a QuantStudio. TM 6 Pro real-time quantitative PCR system (Thermo Fisher). C3 gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression used as an internal control. C3 gene expression level was expressed as a percentage relative to the control group with only RNAiMAX. Results are shown in Table 4 below.

[0155] Table 4. Knockdown effect of siRNA on C3 mRNA expression in Hep3B cells.

[0156]

[0157]

[0158] Example 2

[0159] The siRNA obtained in Example 1 was further diluted and validated in Hep3B cells as described above. The inhibitory effect of siRNA on C3 gene expression was expressed as a percentage relative to the control group containing only RNAiMAX. The results are shown in Table 5 below.

[0160] Table 5

[0161]

[0162]

[0163] Example 3

[0164] As shown in Table 6, the 3' end of the positive strand of the siRNA selected in Example 2 was coupled with Tri-GalNAc (the structure of which is shown in Formula I), and subcutaneously injected into human C3 transgenic mice (Shanghai Southern Model Biotechnology Co., Ltd.) at a dose of 3 mg / kg. Blood samples were collected on day 14, and the plasma level of human C3 protein was measured using an ELISA kit (Abcam, ab108823). PBS was used as a control group in this experiment. The results are as follows: Figure 1 As shown.

[0165] Table 6

[0166] siRNA ID Sense strand (5'-3') Antisense strand (5'-3') PAT-0009T csasgagaAfaUfUfCfuacuacaucu-TriGalNAc asGfsaUfgUfaguagaaUfuUfcucugsusu PAT-0010T gsasgaaaUfuCfUfAfcuacaucuau-TriGalNAc asUfsaGfaUfguaguagAfaUfuucucsusg PAT-0027T csgsgaagGfaAfUfCfagaaugaaca-TriGalNAc usGfsuUfcAfuucugauUfcCfuuccgsgsc PAT-0028T asasggaaUfcAfGfAfaugaacaaaa-TriGalNAc usUfsuUfgUfucauucuGfaUfuccuuscsc PAT-0036T csasgcuaAfaAfGfAfcuuugacuuu-TriGalNAc asAfsaGfuCfaaagucuUfuUfagcugscsa PAT-0038T gsasucaaCfuCfAfCfcuguaauaaa-TriGalNAc usUfsuAfuUfacaggugAfgUfugaucsusu PAT-0052T gscsugagGfaGfAfAfuugcuucaua-TriGalNAc usAfsuGfaAfgcaauucUfcCfuccucsasg

[0167] Example 4

[0168] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) to a 96-well cell culture dish containing 10 4Hep3B (Procell, Cat#CL-0102) cells were cultured overnight at 37°C in a cell culture vessel with 5% CO2. RNAiMAX (1.5 μL / well) and small interfering nucleic acids (siRNA) from Table 7 were added to Opti-MEM medium to achieve final concentrations of 0.000038 nM, 0.000305 nM, 0.002441 nM, 0.019531 nM, 0.156250 nM, 1.25 nM, or 10 nM per well. Cells were then cultured for another 48 hours at 37°C with 5% CO2. To extract RNA, the cell culture supernatant was aspirated, washed with PBS, and then 50 μL of prepared lysis buffer (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)) was added and mixed. After standing for 10 min, 2.5 μL of top solution was added to terminate the reaction for 2 min. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (ThermoFisher, catalog number: 4368814), with each reaction containing 10 μL of lysed liquid. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for human C3 was Hs00163811_m1, and the probe for the internal control gene (human HPRT1) was Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were 95℃ for 20 seconds per cycle, followed by 40 cycles of 95℃ for 1 second and 60℃ for 20 seconds. The real-time fluorescence PCR instrument was QuantStudio. TM 6 Pro real-time quantitative PCR system (Thermo Fisher). C3 gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression as an internal control. C3 gene expression level was expressed as a percentage relative to the control group with only RNAiMAX, and the IC50 value was calculated using Prism GraphPad. The results are shown in Table 8.

[0169] Table 7

[0170] siRNA ID Sense strand (5'-3') Antisense strand (5'-3') PAT-0009 csasgagaAfaUfUfCfuacuacaucu asGfsaUfgUfaguagaaUfuUfcucugsusu PAT-0009.1 csasgagaAfaUfUfCfuacuacaucu asGfsaugUfaguagaaUfuUfcucugsusu PAT-0009.1 csasgagaAfaUfUfCfuacuacaucu asGfsaUfguaguagaaUfuUfcucugsusu PAT-0010 gsasgaaaUfuCfUfAfcuacaucuau asUfsaGfaUfguaguagAfaUfuucucsusg PAT-0010.1 gsasgaaaUfuCfUfAfcuacaucuau asUfsagaUfguaguagAfaUfuucucsusg PAT-0010.2 gsasgaaaUfuCfUfAfcuacaucuau asUfsaGfauguaguagAfaUfuucucsusg PAT-0038 gsasucaaCfuCfAfCfcuguaauaaa usUfsuAfuUfacaggugAfgUfugaucsusu PAT-0038.1 gsasucaaCfuCfAfCfcuguaauaaa usUfsuAfuUfaCfaggugAfgUfugaucsusu PAT-0038.2 gsasucaaCfuCfAfCfcuguaauaaa usUfsuAfuUfacaGfgugAfgUfugaucsusu PAT-0038.3 gsasucaaCfuCfAfCfcuguaauaaa usUfsuAfuUfacaggUfgAfgUfugaucsusu PAT-0038.4 gsasucaaCfuCfAfCfcuguaauaaa usUfsuAfuUfacaggugAfgUfuGfaucsusu PAT-0038.5 gsasucaaCfuCfAfCfcuguaauaaa usUfsuauUfacaggugAfgUfugaucsusu

[0171] Table 8

[0172]

[0173]

[0174] Example 5

[0175] PAT-0038 also showed long-lasting inhibitory effects in human C3 transgenic mice. For example... Picture 2 As shown, a single subcutaneous injection of 3 mg / kg PAT-0038T can continuously inhibit C3 expression by approximately 90% for at least one month.

[0176] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An RNAi reagent comprising: An antisense strand containing at least 15 consecutive nucleotides, wherein the at least 15 consecutive nucleotides differ by no more than 3 nucleotides from any at least 15 consecutive nucleotides in any of the sequences shown in SEQ ID NO: 54–106; and The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.

2. The RNAi reagent according to claim 1, wherein, The positive strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides from any of the at least 15 consecutive nucleotides in any of the sequences shown in SEQ ID No. 1 to 53.

3. The RNAi reagent according to claim 1, wherein, The antisense strand contains a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 54, 55, 57, 59, 62, 63, 67–69, 76, 80–82, 88–95, 98, 99, 101, 105 by 0, 1, or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand contains a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 1, 2, 4, 6, 9, 10, 14–16, 23, 27–29, 35–42, 45, 46, 48, 52 by 0, 1, or 2 nucleotides.

4. The RNAi reagent according to claim 1, wherein, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 9 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 62 by 0, 1, or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 10 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 63 by 0, 1, or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 38 by 0, 1, or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 91 by 0, 1, or 2 nucleotides.

5. The RNAi reagent according to any one of claims 1 to 4, wherein, At least one nucleotide in the RNAi reagent is a modified nucleotide or includes a modified interphase bond; The modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-open-ring nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, debased nucleotides, ribitol, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides, peptide nucleic acids, glycerol nucleic acids, triazine-cyclic DNA, nucleotides containing non-natural bases, nucleotides containing vinyl phosphonates, nucleotides containing cyclopropyl phosphonates, and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoronucleotides. The modified inter-linked bonds are preferably one or more of thiophosphate nucleotide inter-linked bonds and methylphosphonate nucleotide inter-linked bonds; the modified inter-linked bonds are further preferably one or more of thiophosphate monoester nucleotide inter-linked bonds and thiophosphate diester nucleotide inter-linked bonds.

6. The RNAi reagent according to claim 5, wherein, The antisense strand includes 5'-nNfnNfnNfnnnnnnnNfnNfnnnnnnn-3', 5'-nNfnnnNfnnnnnnnnNfnNfnnnnnnnn-3', 5'-nNfnNfnnnnnnnnnNfnNfnnnnnnn-3', 5'-nNfnNfnNfnNfnnnnnnNfnN fnnnnnnn-3', 5'-nNfnNfnNfnnnNfnnnNfnNfnnnnnnn-3', 5'-nNfnNfnNfn nnnnNfnNfnNfnnnnnnn-3', or 5'-nNfnNfnNfnnnnnnnNfnNfnNfnnnnn-3'; where Nf represents 2'-fluoronucleotide and n represents 2'-O-methyl nucleotide.

7. The RNAi reagent according to claim 5 or 6, wherein, The positive chain contains 5'-nnnnnnNfnNfNfNfnnnnnnnnnn-3', where Nf represents a 2'-fluoronucleotide and n represents a 2'-O-methylnucleotide.

8. The RNAi reagent according to any one of claims 5 to 7, wherein, The last 2 to 4 nucleotides at the 5' end and / or 3' end of the antisense strand contain phosphate thioester nucleotide bonds.

9. The RNAi reagent according to any one of claims 5 to 8, wherein, The last 2 to 4 nucleotides at the 5' end and / or 3' end of the positive strand contain phosphate thioester nucleotide bonds.

10. The RNAi reagent according to claim 1, wherein, The antisense strand contains a nucleotide sequence that differs from any of the antisense strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides; or The positive strand contains a nucleotide sequence that differs from any of the positive strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides; or The RNAi reagent contains any of the double strands shown in Table 2 or Table 3.

11. The RNAi reagent according to claim 10, wherein, The RNAi reagent contains a compound selected from PAT-0001, PAT-0002, PAT-0004, PAT-0006, PAT-0009, PAT-0009.1, PAT-0009.2, PAT-0010, PAT-0010.1, PAT-0010.2, PAT-0014, PAT-0015, PAT-0016, PAT-0023, PAT-0027, PAT-0028, and PAT-0029. The double-stranded polymer described in any of the following: PAT-0035, PAT-0036, PAT-0037, PAT-0038, PAT-0038.1, PAT-0038.2, PAT-0038.3, PAT-0038.4, PAT-0038.5, PAT-0039, PAT-0040, PAT-0041, PAT-0042, PAT-0045, PAT-0046, PAT-0048, and PAT-0052.

12. The RNAi reagent according to any one of claims 1 to 11, wherein, The RNAi reagent further comprises at least one ligand, which is attached to one or more of the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and the 3' end of the sense strand; the ligand is a GalNAc derivative; more preferably, the ligand is one or more GalNAc derivatives attached by a single-stranded, double-stranded, or triple-stranded ligand header.

13. The RNAi reagent according to claim 12, wherein, The RNAi reagent contains a compound with the structure shown below: In the formula, Nu represents a nucleic acid composed of the sense strand and the antisense strand.

14. An isolated cell containing the RNAi reagent according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the RNAi reagent according to any one of claims 1 to 13.

16. A method for inhibiting the expression of C3 complement in cells, the method comprising: The cells are contacted with the RNAi reagent of any one of claims 1 to 13 or the pharmaceutical composition of claim 15 to inhibit the expression of C3 complement in the cells.

17. Use of the RNAi reagent according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 15 in any of the following aspects: 1) Treatment and / or prevention of diseases related to C3 complement; 2) Prepare drugs for the treatment and / or prevention of diseases related to C3 complement.

18. The use according to claim 17, wherein, The disease in question is: (i) diseases associated with C3 complement enhancement or elevation; or (ii) Diseases that will benefit from reduced C3 complement expression.

19. The use according to claim 17, wherein, The disease is selected from one or more of the following: paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, neutrophil cytoplasmic antibody-associated vasculitis, hereditary angioedema, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, membranoproliferative glomerulonephritis, IgA nephropathy, primary membranous nephropathy, C3 glomerulonephropathy, acute tubular necrosis, focal segmental glomerulosclerosis, diabetic nephropathy, neuromyelitis optica, age-related macular degeneration, Guillain-Barré syndrome, multifocal motor neuropathy, and generalized myasthenia gravis.

20. The use according to any one of claims 17 to 19, wherein, The RNAi reagent or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

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