Method for preparing sirna for inhibiting FXI gene expression

Abstract

Provided is a method for preparing siRNA for inhibiting FXI gene expression. The siRNA is Fitusiran, and the Fitusiran is a double-stranded RNA composed of a sense strand and an antisense strand that complement and are paired with each other. The preparation method comprises: mixing sense strand substrates, antisense strand substrates and an RNA ligase, wherein the sense strand substrates can constitute the sense strand, the antisense strand substrates can constitute the antisense strand, the sense strand substrates and the antisense strand substrates are connected via hydrogen bonds formed by means of complementary base pairing, and no linkage is formed between head and tail bases of the sense strand substrates and between head and tail bases of the antisense strand substrates, thereby forming a nicked double-stranded nucleotide structure; and ligating the bases at both ends of the nick via a phosphodiester bond using the RNA ligase to form the Fitusiran. The preparation method can solve the problem of Fitusiran prepared by means of the prior art having low purity.

Description

Preparation method of siRNA to inhibit FXI gene expression

[0001] This application is based on and claims priority to Chinese application CN application number 202410921752.5 filed on July 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of drug biosynthesis, and more specifically, to a method for preparing siRNA that inhibits the expression of the FXI gene. Background Technology

[0003] In current technologies, diseases caused by gene expression and mutations cannot be cured by traditional small molecule drugs; these diseases can only be treated through gene expression intervention. siRNA is a 19-25 nt double-stranded RNA oligonucleotide that specifically binds to the mRNA of the same target gene, inhibiting the translation process and thus blocking the expression of the target gene—that is, silencing the gene through RNA interference (RNAi). The development of siRNA drugs has brought enormous potential to gene therapy and is now widely used in the treatment of diseases. siRNA acts on mRNA, allowing for drug targets significantly larger than those of traditional small molecule drugs whose action sites are proteins, and it can also target new sites by changing its sequence. This allows for intervention in gene expression.

[0004] Fitusiran is a gene-targeted therapy siRNA-based double-stranded RNA drug, first announced in 2016 and developed by Alnylam in collaboration with Regeneron. It is currently in Phase 3 clinical trials. Fitusiran works by targeting small RNA fragments of specific genes to inhibit their transcription and expression, reducing antithrombin expression, inhibiting the synthesis of clotting factors, preventing bleeding events, and enhancing the body's blood clotting ability. It is used to treat universal hemophilia.

[0005] Fitusiran is primarily synthesized using a solid-phase support via a cyclic synthesis via phosphoramidite. After the synthesis cycle, the Fitusiran chain is cleaved from the solid-phase support by ammonolysis, followed by purification to obtain the target product. In this method, the yield decreases with increasing chain length. Impurities generated during synthesis, such as those with one more nucleotide than the target sequence (N+1 impurities) or one less nucleotide than the target sequence (N-1 impurities), also increase with increasing chain length, making them difficult to remove and purifying the product complex. As Fitusiran is increasingly widely used in the treatment of universal hemophilia, its synthesis scale is limited by the synthetic equipment, resulting in high costs and difficulty in scaling up production. Therefore, a more efficient method for Fitusiran synthesis is needed. Summary of the Invention

[0006] The main objective of this invention is to provide a method for preparing siRNA that inhibits the expression of the FXI gene, thereby solving the problem of low purity in the preparation of Fitusiran in the prior art.

[0007] To achieve the above objective, according to a first aspect of the present invention, a method for preparing siRNA that inhibits the expression of the FXI gene is provided. The siRNA is Fitusiran, a double-stranded RNA composed of complementary positive and negative strands. The preparation method includes: mixing a positive strand substrate, an antisense strand substrate, and an RNA ligase, wherein the positive strand substrate can form a positive strand, and the antisense strand substrate can form an antisense strand; the positive and antisense strand substrates are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrates are not interconnected, forming a nicked double-stranded nucleotide structure; and using the RNA ligase to link the bases at both ends of the nick with phosphodiester bonds to form Fitusiran.

[0008] The bases at both ends of the notch are the 5' and 3' ends of different substrates, respectively. The 5' end is a phosphate group, and the 3' end is a hydroxyl group. The phosphate group at the 5' end and the hydroxyl group at the 3' end of the notch are connected by an RNA ligase to form a phosphodiester bond, thus obtaining Fitusiran. The RNA ligase is selected from one or more of the RNA ligases containing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or an enzyme that has more than 70% identity with any RNA ligase having the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has catalytic activity in forming phosphodiester bonds.

[0009] Furthermore, the nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO: 24.

[0010] Further, the positive chain substrate includes two or more strands, and the negative chain substrate includes two or more strands; preferably, the length of the positive chain substrate is 2-19 nt, more preferably 4-17 nt, and even more preferably 8-13 nt; preferably, the length of the negative chain substrate is 2-21 nt, more preferably 4-19 nt, and even more preferably 8-15 nt.

[0011] Furthermore, both the sense and antisense strands of the substrate comprise two substrates: the sense strand substrate comprises a first sense strand substrate and a second sense strand substrate, and the antisense strand substrate comprises a first antisense strand substrate and a second antisense strand substrate. The preparation method includes: mixing the first sense strand substrate, the second sense strand substrate, the first antisense strand substrate, and the second antisense strand substrate; using RNA ligase to catalyze the ligation of the first sense strand substrate and the second sense strand substrate to form a sense strand; catalyzing the ligation of the first antisense strand substrate and the second antisense strand substrate to form an antisense strand; and the sense and antisense strands forming Fitusiran through base complementarity pairing. Preferably, the sense and antisense strand substrates are annealed and then mixed with RNA ligase to obtain Fitusiran.

[0012] Further, the 3' end of the first sense strand substrate and the 5' end of the second sense strand are ligated under the catalysis of RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated under the catalysis of RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate is a phosphate group, and the 3' end is an L96 group; preferably, the 5' end of the first antisense strand substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate is a phosphate group, and the 3' end is a hydroxyl group.

[0013] Further, the nucleotide sequence of the first sense substrate is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate is CmUfUmCfAmAf; preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate is SEQ ID NO: 11.

[0014] Further, the nucleotide sequence of the first sense substrate is SEQ ID NO: 13, and the nucleotide sequence of the second sense substrate is UmAfCmUfUmCfAmAf; preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate is SEQ ID NO: 15.

[0015] Furthermore, both the positive and negative substrates comprise three substrates: the positive substrate comprises a first positive substrate, a second positive substrate, and a third positive substrate; the negative substrate comprises a first negative substrate, a second negative substrate, and a third negative substrate. Preferably, the nucleotide sequence of the first positive substrate is GfsGmsUfUmAfAmCf; the nucleotide sequence of the second positive substrate is SEQ ID NO: 18; and the nucleotide sequence of the third positive substrate is UmCfAmAf. Preferably, the nucleotide sequence of the first negative substrate is UmsUfsGmAfAmGfUm; the nucleotide sequence of the second negative substrate is SEQ ID NO: 21; and the nucleotide sequence of the third negative substrate is AmCfCmsAmsGm. Furthermore, the concentrations of the sense and antisense substrates are each independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense substrate, antisense substrate, and RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the reaction temperature of the preparation method is 10-40℃, more preferably 15-30℃; preferably, the reaction time of the preparation method is 2-48 h, more preferably 12-24 h.

[0016] By applying the technical solution of this invention and utilizing the above-described preparation method, under the catalysis of RNA ligase, the sense strand substrates are linked to form a Fitusiran sense strand, and the antisense strand substrates are linked to form a Fitusiran antisense strand, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with chemical synthesis methods for preparing Fitusiran, the preparation method of this application yields a product with high purity, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 shows a schematic diagram of the structure of L96 according to an embodiment of the present invention.

[0019] Figure 2 shows a schematic diagram of the substrate reaction according to Embodiment 1 of the present invention.

[0020] Figure 3 shows the gel electrophoresis results according to Example 1 of the present invention.

[0021] Figure 4 shows the HPLC detection results according to Example 2 of the present invention. Detailed Implementation

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0023] Terminology Explanation:

[0024] N+1 impurities: Nucleic acid impurities that have an additional single nucleotide link compared to the target synthetic sequence.

[0025] N-1 impurities: Nucleic acid impurities that have a single nucleotide deletion compared to the target synthetic sequence.

[0026] As mentioned in the background section, existing techniques for preparing Fitusiran involve chemical synthesis, which is not only complex and costly but also generates numerous N+1 and N-1 impurities, affecting subsequent product purification. In this application, the inventors attempt to develop a method for preparing siRNA that inhibits FXI gene expression, utilizing an enzyme-catalyzed synthesis method for Fitusiran.

[0027] In a first typical embodiment of this application, a method for preparing siRNA that inhibits FXI gene expression is provided. The siRNA is Fitusiran, a double-stranded RNA composed of complementary sense and antisense strands. The preparation method includes:

[0028] Preparation methods include:

[0029] The sense and antisense substrates are mixed with RNA ligase, where the sense substrate forms the sense strand and the antisense substrate forms the antisense strand. The sense and antisense substrates are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the sense and antisense substrates are not linked to each other, forming a double-stranded nucleotide structure with a nick. The RNA ligase is then used to link the bases at both ends of the nick with phosphodiester bonds to form Fitusiran.

[0030] The bases at both ends of the notch are the 5' and 3' ends of different substrates, respectively. The 5' end is a phosphate group, and the 3' end is a hydroxyl group. The phosphate group at the 5' end and the hydroxyl group at the 3' end of the notch are connected by an RNA ligase to form a phosphodiester bond, thus obtaining Fitusiran. The RNA ligase is selected from one or more of the RNA ligases with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or an enzyme that has more than 70% identity with any RNA ligase with the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has the activity of catalyzing the formation of the above-mentioned phosphodiester bond.

[0031] The aforementioned positive and negative chain substrates can be synthesized by solid-phase chemical synthesis or by liquid-phase chemical synthesis.

[0032] In the above preparation method, the positive-sense substrate is two or more nucleotide sequences that can form the positive-sense chain. That is, multiple nucleotide sequences of the positive-sense substrate can be spliced ​​together to form a sequence identical to the positive-sense chain, differing from the positive-sense chain in that the positive-sense substrate has notches and is not linked by phosphodiester bonds. Similarly, the antisense substrate and antisense chain have the above characteristics. Using RNA ligase, two or more positive-sense substrates or antisense substrates are linked by phosphodiester bonds to obtain the positive and antisense chains of Fitusiran.

[0033] In this application, Fitusiran can be prepared by mixing the positive and negative substrates with RNA ligase. The positive and negative substrates are linked by hydrogen bonds formed through base complementarity, forming a double-stranded nucleotide structure with sticky ends. This sticky-end double-stranded nucleotide is further linked to other substrates through base complementarity pairing, wherein the head and tail bases of the positive and negative substrates are not linked to each other, forming a nicked double-stranded nucleotide structure. Finally, RNA ligase is used to link the nicks with phosphodiester bonds to obtain the target product Fitusiran. Preferably, the positive and negative substrates are annealed and then mixed with RNA ligase to obtain Fitusiran.

[0034] In the above preparation method, the sense and antisense substrates are first mixed and annealed. The sense and antisense substrates can form a double-stranded RNA structure through base complementarity, and this double-stranded RNA structure contains nicks between the different substrates. The annealed reaction system is then mixed with RNA ligase, which uses phosphodiester bonds to connect the phosphate and hydroxyl groups on both sides of the nick, repairing the nick and obtaining the target product Fitusiran with a complete double-stranded structure. In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO: 24.

[0035] SEQ ID NO: 23:

[0036] GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96.

[0037] SEQ ID NO: 24:

[0038] UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm.

[0039] In this application, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in notations such as sGm, sUm, etc., indicates thiomodification of the 5' phosphate of the ribonucleotide.

[0040] All of the above RNA ligases can recognize the notched double-stranded structure formed by complementary substrate pairing, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

[0041] SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

[0042] SEQ ID NO: 2 (Ligase 26, Escherichia phage AR1):

[0043] SEQ ID NO: 3 (Ligase 31, Vibrio phage VH12019):

[0044] SEQ ID NO: 4 (Ligase 41, Vibrio phage VH7D):

[0045] SEQ ID NO: 5 (Ligase 42, Escherichia phage JN02):

[0046] In this application, "identity" refers to the "identity" between amino acid sequences or nucleotide sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0047] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) of identicality and function have an active site, active pocket, active mechanism, protein structure, etc., which are highly likely to be the same as the protein provided by sequence a).

[0048] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0049] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substitution are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0050] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0051] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0052] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0053] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0054] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62" scoring matrix in the prior art.

[0055] Fitusiran has a complex secondary structure and exhibits varying specificity in binding to different enzymes. In this application, Fitusiran can only be obtained by catalyzing the formation of a phosphodiester bond between the phosphate and hydroxyl groups of the substrate using the RNA ligases shown in SEQ ID NO: 1–SEQ ID NO: 5, or an enzyme with more than 70% identity to any of the RNA ligases shown in SEQ ID NO: 1–SEQ ID NO: 5. In the relevant experiments of this application, the inventors screened a large number of enzymes and obtained the aforementioned RNA ligases shown in SEQ ID NO: 1–SEQ ID NO: 5 that are capable of synthesizing Fitusiran. However, the large proportion of negative results in the experiments showed that most RNA ligases are unable to catalyze the synthesis of Fitusiran, including but not limited to the RNA ligases shown in SEQ ID NO: 6–SEQ ID NO: 8. This application specification only uses SEQ ID NO: 6–SEQ ID NO: 8 as examples to illustrate this type of RNA ligase that lacks activity in catalyzing Fitusiran synthesis.

[0056] SEQ ID NO: 6 (Ligase 11, Thermococcus):

[0057] SEQ ID NO: 7 (Ligase 20, Archaea):

[0058] SEQ ID NO: 8 (Ligase 32, bacteria):

[0059] In a preferred embodiment, the positive chain substrate includes two or more substrates, and the negative chain substrate includes two or more substrates; preferably, the length of the positive chain substrate is 2-19 nt, more preferably 4-17 nt, and even more preferably 8-13 nt; preferably, the length of the negative chain substrate is 2-21 nt, more preferably 4-19 nt, and even more preferably 8-15 nt.

[0060] In a preferred embodiment, both the sense and antisense substrates comprise two substrates. The sense substrate comprises a first sense substrate and a second sense substrate, and the antisense substrate comprises a first antisense substrate and a second antisense substrate. The preparation method includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate; using RNA ligase to catalyze the ligation of the first and second sense substrates to form a sense strand; catalyzing the ligation of the first and second antisense substrates to form an antisense strand; and the sense and antisense strands forming the aforementioned Fitusiran through complementary base pairing.

[0061] In a preferred embodiment, the 3' end of the first sense substrate and the 5' end of the second sense substrate are ligated under the catalysis of RNA ligase to form a sense strand; the 3' end of the first antisense substrate and the 5' end of the second antisense substrate are ligated under the catalysis of RNA ligase to form an antisense strand; preferably, in the above preparation method, the 5' end of the first sense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense substrate is a phosphate group, and the 3' end is an L96 group; the 5' end of the first antisense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense substrate is a phosphate group, and the 3' end is a hydroxyl group.

[0062] In a preferred embodiment, the nucleotide sequence of the first sense substrate is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate is CmUfUmCfAmAf; preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate is SEQ ID NO: 11.

[0063] In a preferred embodiment, the nucleotide sequence of the first sense substrate is SEQ ID NO: 13, and the nucleotide sequence of the second sense substrate is UmAfCmUfUmCfAmAf; preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate is SEQ ID NO: 15.

[0064] Fitusiran can be prepared using the above-described preparation method and substrates containing the above-described nucleotide sequences. However, it should be noted that the choice of substrate is not limited to the substrates described above; any substrate capable of forming both the sense and antisense strands can be used in the above preparation method. The above preparation method is applicable to the preparation of Fitusiran but is not limited to different substrate connection positions. The above preparation method shows good ligation effects for both the sense and antisense strand sequences of Fitusiran. The number of sense or antisense substrates includes, but is not limited to, 2, 3, 4, or even more.

[0065] SEQ ID NO: 9: GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAf.

[0066] SEQ ID NO: 11: GmUfGmUfUmAfAmCfCmsAmsGm.

[0067] SEQ ID NO: 12: UmsUfsGmAfAmGfUmAfAmAfUmGm.

[0068] SEQ ID NO: 13: GfsGmsUfUmAfAmCfAmCfCfAfUmUf.

[0069] SEQ ID NO: 14: UfGmUfUmAfAmCfCmsAmsGm.

[0070] SEQ ID NO: 15: UmsUfsGmAfAmGfUmAfAmAfUmGmGm.

[0071] In a preferred embodiment, both the positive and negative substrates comprise three substrates: a first positive substrate, a second positive substrate, and a third positive substrate; and a first antisense substrate, a second antisense substrate, and a third antisense substrate. Preferably, the nucleotide sequence of the first positive substrate is GfsGmsUfUmAfAmCf; the nucleotide sequence of the second positive substrate is SEQ ID NO: 18; and the nucleotide sequence of the third positive substrate is UmCfAmAf. Preferably, the nucleotide sequence of the first antisense substrate is UmsUfsGmAfAmGfUm; the nucleotide sequence of the second antisense substrate is SEQ ID NO: 21; and the nucleotide sequence of the third antisense substrate is AmCfCmsAmsGm.

[0072] SEQ ID NO: 18: AmCfCfAfUmUfUmAfCmUf.

[0073] SEQ ID NO: 21: AfAmAfUmGmGmUfGmUfUmAf.

[0074] In a preferred embodiment, the concentrations of the sense substrate and the antisense substrate are each independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense substrate, the antisense substrate, and the RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the reaction temperature of the preparation method is 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48 h, more preferably 12-24 h.

[0075] The concentrations of the aforementioned sense and antisense substrate fragments are each selected from, but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mM; the reaction temperatures of the aforementioned preparation methods are, but not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 °C; and the reaction times of the aforementioned preparation methods are, but not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, or 48 h.

[0076] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0077] Example 1

[0078] Substrates 1–4 (length: nt) were added to a clean reagent bottle in an equimolar ratio and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrates 1–4 are shown in Table 1.

[0079] Table 1

[0080] In this sequence, m after A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, f indicates a 2' fluorine modification of the ribonucleotide, s before the ribonucleotide in sequences such as "sGm" and "sAm" indicates a thiomodification of the 5' phosphate of the ribonucleotide, and L96 indicates that the 3' end of the positive strand is modified with an L96 group. The structure is shown in Figure 1, where the wavy line represents the base connected to the L96 group.

[0081] After annealing the substrate mixture, a mixture of RNA fragments from substrates 1-4 was obtained. The reaction system was set to 10 μL, containing 100 μM RNA fragment mixture, 50 mM Tris-HCl, 10 eq ATP, 100 eq MgCl2, and 10 eq DTT (1 eq = 100 μM). RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase 11, Ligase 20, and Ligase 32 were added to a final concentration of 0.2 mg / mL, as shown in Figure 2. The reaction system was incubated at 16 °C for 16 h. The resulting reaction system was then subjected to 80 °C for 5 min to inactivate the ligases, and the precipitate was removed by centrifugation at 12000 rpm.

[0082] The products obtained by different RNA ligases were analyzed by Urea-PAGE. The electrophoresis results of Ligase 25, Ligase 31, and Ligase 11 are shown in Figure 3. In Figure 3, lane M represents the RNA molecule marker, lane 1 represents the reaction system of Ligase 25, lane 2 represents the reaction system of Ligase 31, and lane 3 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis results of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0083] The ribonucleotides at positions 2, 4, 6, 8, 12, and 14 of substrate 1 have a 2' methoxy group, and the ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11, 13, and 15 have a 2' fluorine group. The 5' phosphate group on the ribonucleotides at positions 2 and 3 has a thio group.

[0084] The ribonucleotides at positions 1, 3, and 5 of substrate 2 have 2' methoxy modifications, and the ribonucleotides at positions 2, 4, and 6 have 2' fluorine modifications.

[0085] The ribonucleotides at positions 1, 3, 5, 7, 9, 10, and 11 of substrate 3 have 2' methoxy modifications, the ribonucleotides at positions 2, 4, 6, and 8 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 10 and 11 has thio modifications.

[0086] The ribonucleotides at positions 1, 3, 5, 7, 9, 11, and 12 of substrate 4 have 2' methoxy modifications, the ribonucleotides at positions 2, 4, 6, 8, and 10 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has thio modifications.

[0087] The prepared Fitusiran has a sense strand of GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23) and an antisense strand of UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0088] Table 2

[0089] "None" indicates that the target product band was not detected, "++" indicates a yield of 25-50% (excluding the 50% endpoint), "+++" indicates a yield of 50-90%, and "++++" indicates a yield >90%.

[0090] The grayscale data of the product and substrate were obtained by grayscale analysis of the Urea-PAGE gel electrophoresis results image. In this example, the yield calculation formula is: Yield = Product grayscale data / (Product grayscale data + Substrate grayscale data).

[0091] Example 2

[0092] Substrate 1–4 were added to a clean reagent bottle in equimolar proportions and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. After annealing, the substrate mixture yielded a mixed solution of RNA fragments of substrates 1–4. The reaction system was set to 50 μL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM). Ligase 25 and Ligase 31, RNA ligases with high catalytic activity as described in Example 1, were added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. After the reaction, the protein was inactivated by heating at 80 °C for 5 min, and the precipitate was removed by centrifugation.

[0093] The product obtained by Ligase25 catalysis was detected by HPLC and LC-MS, as shown in Figure 4. The yield was calculated based on the statistical results of the target peak area in the HPLC results, as shown in Table 3.

[0094] Table 3

[0095] "++" indicates a yield of 25-50% (excluding the 50% endpoint), "+++" indicates a yield of 50-90%, and "++++" indicates a yield >90%.

[0096] LC-MS analysis showed that the molecular weight of the positive-sense product was 8568.88, and the molecular weight of the antisense product was 7671.11. The theoretical values ​​for the positive-sense product were 8568.86±8 and the theoretical values ​​for the antisense product were 7671.11±8, indicating that Ligase 25 and Ligase 31 could connect to the substrate and generate Fitusiran.

[0097] Example 3

[0098] Substrate 1–4 were added to a clean reagent bottle in equimolar proportions and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 1–4. The reaction system was set to 10 mL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was heated at 50℃ for 10-20 min to inactivate the protein, and the precipitate was removed by centrifugation at 12000 rpm. The supernatant was purified using a Nano-Q column, and the obtained product was eluted with NaCl gradient, desalted by membrane coating (molecular weight cutoff of 1 kDa), and then lyophilized. The yield was calculated to be 76.16%, and the purity was 97.53% as determined by HPLC.

[0099] Example 4

[0100] Substrate 5–8 (length: nt) were added to a clean reagent bottle in an equimolar ratio and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrate 5–8 are shown in Table 4. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 5–8. The reaction system was set to 50 μL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was subjected to 80 °C for 5 min to inactivate the ligase, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was analyzed by HPLC, and the results showed that the target peak accounted for 83.4% of the sample.

[0101] Table 4

[0102] In these sequences, m after A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, f indicates a 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in sequences such as "sGm" and "sAm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0103] The ribonucleotides at positions 2, 4, 6, 8, and 12 of substrate 5 have a 2' methoxy group, and the ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11, and 13 have a 2' fluorine group. The 5' phosphate group on the ribonucleotides at positions 2 and 3 has a thio group.

[0104] The ribonucleotides at positions 1, 3, 5, and 7 of substrate 6 have 2' methoxy modifications, and the ribonucleotides at positions 2, 4, 6, and 8 have 2' fluorine modifications.

[0105] The ribonucleotides at positions 2, 4, 6, 8, 9, and 10 of substrate 7 have 2' methoxy modifications, the ribonucleotides at positions 1, 3, 5, and 7 have 2' fluorine modifications, and the ribonucleotides at positions 9 and 10 have thio modifications on the 5' phosphate group.

[0106] The ribonucleotides at positions 1, 3, 5, 7, 9, 11, 12, and 13 of substrate 8 have 2' methoxy modifications, the ribonucleotides at positions 2, 4, 6, 8, and 10 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has thio modifications.

[0107] The prepared Fitusiran has a sense strand of GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23) and an antisense strand of UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0108] Example 5

[0109] Substrate 9–14 (length: nt) was added to a clean reagent bottle in an equimolar ratio and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrate 9–14 are shown in Table 5. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 9–14. The reaction system was set to 50 μL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was subjected to 80 °C for 5 min to inactivate the ligase, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was analyzed by HPLC, and the results showed that the target peak accounted for 83.4% of the sample.

[0110] Table 5

[0111] In these sequences, m after A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, f indicates a 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in sequences such as "sGm" and "sAm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0112] Substrate 9 has 2' methoxy groups at positions 2, 4, and 6, and 2' fluorine groups at positions 1, 4, 5, and 7. The 5' phosphate group at positions 2 and 3 has a thio group.

[0113] The ribonucleotides at positions 1, 5, 7, and 9 of substrate 10 have 2' methoxy modifications, and the ribonucleotides at positions 2, 3, 4, 6, 8, and 10 have 2' fluorine modifications.

[0114] The ribonucleotides at positions 1 and 3 of substrate 11 have 2' methoxy modifications, and the ribonucleotides at positions 2 and 4 have 2' fluorine modifications.

[0115] The ribonucleotides at positions 1, 3, 4 and 5 of substrate 12 have 2' methoxy modifications, the ribonucleotide at position 2 has a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 4 and 5 has thio modifications.

[0116] The ribonucleotides at positions 2, 4, 5, 6, 8, and 10 of substrate 13 have 2' methoxy modifications, and the ribonucleotides at positions 1, 3, 7, 9, and 11 have 2' fluorine modifications.

[0117] The ribonucleotides at positions 1, 3, 5, and 7 of substrate 14 have 2' methoxy modifications, the ribonucleotides at positions 2 and 3 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has thio modifications.

[0118] The prepared Fitusiran has a sense strand of GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23) and an antisense strand of UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0119] Comparative Example 1

[0120] The yield of Fitusiran product prepared using the enzyme ligation method of this invention was 30.9%, which is comparable to the average yield (31.2%) of full-length Fitusiran product synthesized by solid-phase synthesis. However, the total proportion of N+1 and N-1 impurities in the Fitusiran product synthesized using the method described in this application was 0.4%, lower than the total proportion of N+1 and N-1 impurities in the full-length Fitusiran product synthesized by solid-phase synthesis (1.42%).

[0121] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the preparation method of this application, a single-stranded RNA fragment designed based on the Fitusiran sequence is catalyzed by RNA ligase to form Fitusiran, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with chemical synthesis methods, the preparation method of this application yields products with high purity, produces fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing siRNA that inhibits FXI gene expression, characterized in that, The siRNA is Fitusiran, which is a double-stranded RNA composed of complementary sense and antisense strands; The preparation method includes: mixing a sense strand substrate, an antisense strand substrate, and an RNA ligase, wherein the sense strand substrate is capable of forming the sense strand, and the antisense strand substrate is capable of forming the antisense strand; The positive and negative substrates are connected by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and negative substrates are not connected to each other, forming a double-stranded nucleotide structure with notches. The bases at both ends of the notch are linked by phosphodiester bonds using the RNA ligase to form Fitusiran; The bases at both ends of the notch are the 5' and 3' ends of different substrates, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; The phosphate group at the 5' end and the hydroxyl group at the 3' end of the notch are linked together using the RNA ligase to form the phosphodiester bond, thereby obtaining Fitusiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is selected from one or more RNA ligases containing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; Or an RNA ligase that has more than 70% identity with any of the RNA ligases having the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5, and has the activity of catalyzing the formation of the phosphodiester bond.

3. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO:

24.

4. The preparation method according to any one of claims 1-2, characterized in that, The base of the justice chain includes two or more elements, and the base of the antisense chain includes two or more elements; The length of the base of the justice chain is 2-19nt; The length of the antisense substrate is 2-21 nt.

5. The preparation method according to claim 4, characterized in that, Both the justice chain substrate and the antisense chain substrate include two substrates. The justice chain substrate includes a first justice chain substrate and a second justice chain substrate. The antisense chain substrate includes a first antisense chain substrate and a second antisense chain substrate. The preparation method includes: mixing the first sense strand substrate, the second sense strand substrate, the first antisense strand substrate, and the second antisense strand substrate; using the RNA ligase to catalyze the ligation of the first sense strand substrate and the second sense strand substrate to form the sense strand; catalyzing the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand; and the sense strand and the antisense strand forming the Fitusiran through complementary base pairing.

6. The preparation method according to claim 5, characterized in that, The 3' end of the first sense strand substrate and the 5' end of the second sense strand are ligated together under the catalysis of the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated together under the catalysis of the RNA ligase to form the antisense strand.

7. The preparation method according to claim 6, characterized in that, The first positive-chain substrate has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second positive-chain substrate has a phosphate group at its 5' end and an L96 group at its 3' end. The first antisense substrate has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second antisense substrate has a phosphate group at its 5' end and a hydroxyl group at its 3' end.

8. The preparation method according to claim 6, characterized in that, The nucleotide sequence of the first sense substrate is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate is CmUfUmCfAmAf; The nucleotide sequence of the first antisense substrate is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate is SEQ ID NO:

11.

9. The preparation method according to claim 6, characterized in that, The nucleotide sequence of the first sense substrate is SEQ ID NO: 13, and the nucleotide sequence of the second sense substrate is UmAfCmUfUmCfAmAf; The nucleotide sequence of the first antisense substrate is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate is SEQ ID NO:

15.

10. The preparation method according to claim 4, characterized in that, Both the justice chain substrate and the antisense chain substrate include three substrates. The base of the justice chain includes a first justice chain base, a second justice chain base, and a third justice chain base; The antisense substrate includes a first antisense substrate, a second antisense substrate, and a third antisense substrate.

11. The preparation method according to claim 10, characterized in that, The nucleotide sequence of the first positive-strand substrate is GfsGmsUfUmAfAmCf; The nucleotide sequence of the second positive-strand substrate is SEQ ID NO: 18; The nucleotide sequence of the third positive-strand substrate is UmCfAmAf; The nucleotide sequence of the first antisense substrate is UmsUfsGmAfAmGfUm; The nucleotide sequence of the second antisense substrate is SEQ ID NO: 21; The nucleotide sequence of the third antisense substrate is AmCfCmsAmsGm.

12. The preparation method according to any one of claims 1-3, characterized in that, The concentrations of the sense and antisense substrates are each independently selected from 0.1-4.5 mM; The reaction system formed by mixing the sense substrate, the antisense substrate, and the RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT.

13. The preparation method according to claim 1, characterized in that, The reaction temperature of the preparation method is 10-40℃; The reaction time for the preparation method is 2-48 hours.

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