Preparation method for nedosiran

Abstract

Provided in the present invention is a preparation method for Nedosiran, Nedosiran being a double-stranded RNA consisting of a sense strand and an antisense strand by means of complementary pairing. The preparation method comprises: mixing sense strand substrate fragments, antisense strand substrate fragments and an RNA ligase, wherein the sense strand substrate fragments can form the sense strand, the antisense strand substrate fragments can form the antisense strand, the sense strand substrate fragments and the antisense strand substrate fragments are linked via hydrogen bonds formed by base complementation, bases on the heads and tails of the sense strand substrate fragments are not linked to each other, and bases on the heads and tails of the antisense strand substrate fragments are not linked to each other, thereby forming a double-stranded nucleotide structure containing nicks; and by using the RNA ligase, linking bases at both ends of the nicks via phosphodiester bonds, so as to form Nedosiran. The preparation method can solve the problem of low purity of prepared Nedosiran in the prior art.

Description

A method for preparing Nedosiran

[0001] This application is based on and claims priority to Chinese application CN application number 2024109217332 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 pharmaceutical biosynthesis, and more specifically, to a method for preparing Nedosiran. 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 translation and thus blocking the expression of the target gene—essentially, inducing gene silencing through RNA interference. The development of siRNA drugs has brought enormous potential to gene therapy and is now widely used in disease treatment. Because siRNA acts on mRNA, its target sites are significantly larger than those of traditional small molecule drugs that act on proteins, and it can target new sites by changing its sequence. This allows for intervention in gene expression.

[0004] Nedosiran is a gene-targeted therapy siRNA double-stranded RNA drug developed by Dicerna for the treatment of primary hyperoxaluria (PH). Nedosiran is indicated for three types of primary hyperuricemia (PH1, PH2, and PH3), and is currently the only investigational drug that can treat all three types of PH simultaneously.

[0005] The synthesis of Nedosiran primarily employs a chemical solid-phase synthesis method. This method utilizes a solid support and employs a cyclic synthesis via phosphoramidite trimerization. After the synthesis cycle is complete, the Nedosiran chain is cleaved from the solid support through ammonolysis, followed by purification to obtain the target product. However, the yield of this method 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 resulting in a complex and inefficient purification process. As Nedosiran is increasingly widely used in the treatment of primary hyperbilirubinemia, its synthesis scale is limited by the limitations of synthetic equipment, resulting in high costs and hindering large-scale production. Therefore, there is a need to develop a more efficient method for the synthesis of Nedosiran. Summary of the Invention

[0006] The main objective of this invention is to provide a method for preparing Nedosiran, in order to solve the problem of low purity of Nedosiran prepared in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing Nedosiran is provided, wherein the Nedosiran is a double-stranded siRNA composed of complementary sense and antisense strands; the preparation method includes:

[0008] A positive-strand substrate fragment, an antisense substrate fragment, and an RNA ligase are mixed, wherein the positive-strand substrate fragment can form the positive strand, and the antisense substrate fragment can form the antisense strand. The positive-strand and antisense substrate fragments are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive-strand and antisense substrate fragments are not linked to each other, forming a double-stranded nucleotide structure with a nick. The bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form Nedosiran. The bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group. The 5' end phosphate group and the 3' end hydroxyl group upstream and downstream of the nick are linked by RNA ligase to form phosphodiester bonds, obtaining Nedosiran. The RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme with more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 and having catalytic activity in forming phosphodiester bonds.

[0009] Furthermore, the nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO: 20.

[0010] Furthermore, the positive chain substrate fragment includes two or more segments, and the negative chain substrate fragment includes two or more segments; preferably, the length of the positive chain substrate fragment is 2-34 nt, more preferably 6-30 nt; preferably, the length of the negative chain substrate fragment is 2-20 nt, more preferably 6-16 nt.

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

[0012] Further, the nucleotide sequence of the first positive strand substrate fragment is AmsUmGfUmUfGmUmCfCf, and the nucleotide sequence of the second positive strand substrate fragment is SEQ ID NO: 6; the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is CmAfAmCfAmUmsGmsGm; preferably, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 9, the nucleotide sequence of the second positive strand substrate fragment is SEQ ID NO: 10, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 11.

[0013] Further, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate fragment is a phosphate group, and the 3' end is an R group; preferably, the 5' end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0014] Furthermore, both the sense and antisense substrate fragments comprise three segments: the sense substrate fragment includes a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; the antisense substrate fragment includes a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the preparation method includes: mixing the first, second, and third sense substrate fragments, the first and second antisense substrate fragments, the second and third antisense substrate fragments, and under the catalysis of RNA ligase, the first, second, and third sense substrate fragments are ligated to form a sense strand; catalyzing the ligation of the first, second, and third antisense substrate fragments to form an antisense strand; the sense and antisense strands form Nedosiran through base complementarity pairing. Preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain Nedosiran.

[0015] Further, the nucleotide sequence of the first positive strand substrate fragment is AmsUmGfUmUfGm; the nucleotide sequence of the second positive strand substrate fragment is UmCfCfUfUfUmUfUm; and the nucleotide sequence of the third positive strand substrate fragment is SEQ ID NO: 15. Preferably, the nucleotide sequence of the first antisense strand substrate fragment is CfAmUmsGmsGm; the nucleotide sequence of the second antisense strand substrate fragment is AfAmGmGmAfCmAfAm; and the nucleotide sequence of the third antisense strand substrate fragment is U*sCfsAfsGmAfUmAfAmAf.

[0016] Furthermore, the concentrations of the sense and antisense substrate fragments are each independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense and antisense substrate fragments 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.

[0017] By applying the technical solution of this invention and utilizing the above-described preparation method, under the catalysis of RNA ligase, the sense strand substrate fragments are ligated to form a Nedosiran sense strand, and the antisense strand substrate fragments are ligated to form a Nedosiran antisense strand, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with the chemical synthesis method for preparing Nedosiran, 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

[0018] 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:

[0019] Figure 1 shows a schematic diagram of the enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

[0020] Figure 2 shows a structural diagram of the R-based group according to an embodiment of the present invention.

[0021] Figure 3 shows the electrophoresis results of the RNA ligase Ligase25 and Ligase11 catalyzed products according to Example 1 of the present invention.

[0022] Figure 4 shows the HPLC detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention.

[0023] Figure 5 shows the LC-MS detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention. Detailed Implementation

[0024] 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.

[0025] Terminology Explanation:

[0026] N+1 impurities: Nucleic acid impurities that have an additional single nucleotide link compared to the target synthetic sequence.

[0027] N-1 impurities: Nucleic acid impurities that have a single nucleotide deletion compared to the target synthetic sequence.

[0028] As mentioned in the background section, existing technologies for preparing Nedosiran employ 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 Nedosiran using enzyme-catalyzed synthesis, and thus propose a series of protective solutions.

[0029] In a first typical embodiment of this application, a method for preparing Nedosiran is provided. Nedosiran is a double-stranded siRNA composed of complementary positive and negative strands. The preparation method includes: mixing a positive strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the positive strand substrate fragment can form a positive strand, and the antisense strand substrate fragment can form an antisense strand; the positive and antisense strand substrate fragments are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrate fragments are not linked to each other, forming a double-stranded nucleotide structure with a nick; using an RNA ligase, the bases at both ends of the nick are linked by phosphodiester bonds to form Nedosiran; the bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; using an RNA ligase, the phosphate group at the 5' end and the hydroxyl group at the 3' end of the nick are linked upstream and downstream to form phosphodiester bonds to obtain Nedosiran; the RNA ligase is a [SEQ ID] RNA ligases with the amino acid sequence shown in SEQ ID NO: 1; or enzymes that have more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 and have catalytic activity in forming phosphodiester bonds.

[0030] In the above preparation method, the positive-sense substrate fragment is two or more nucleotide sequences that can form the positive-sense chain. That is, multiple nucleotide sequences of the positive-sense substrate fragment can be spliced ​​together to form a sequence identical to the positive-sense chain. The difference between the positive-sense substrate fragment and the positive-sense chain is that there are nicks between the positive-sense substrate fragments, and they are not linked by phosphodiester bonds. Similarly, the antisense substrate fragment and the antisense chain have the above characteristics. Using RNA ligase, two or more positive-sense substrate fragments or antisense substrate fragments are linked by phosphodiester bonds to obtain the positive and antisense chains of Nedosiran.

[0031] In the above preparation method, Nedosiran can be prepared by mixing the positive and negative substrate fragments with RNA ligase. In this preparation method, the positive and negative substrate fragments can complementarily pair to form a double-stranded nucleotide structure with sticky ends. After forming the sticky ends, they continue to bind with other substrates to form a double-stranded nucleotide structure with notches. RNA ligase can recognize the notches in this double-stranded structure and connect the notches with phosphodiester bonds to prepare the target product Nedosiran. Preferably, the positive and negative substrate fragments are annealed and then mixed with RNA ligase to obtain Nedosiran.

[0032] In the above preparation method, the sense and antisense substrate fragments are first mixed and annealed. The sense and antisense substrate fragments can form a double-stranded RNA structure through base complementarity, and this double-stranded RNA structure contains nicks between the different substrate fragments. 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 thus obtaining the target product Nedosiran with a complete double-stranded structure.

[0033] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO: 20.

[0034] SEQ ID NO: 19:

[0035] AmsUmGfUmUfGmUmCfCfUfUfUmUfUmAfUmCfUmGmAmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmUmGmCm.

[0036] SEQ ID NO: 20:

[0037] U*sCfsAfsGmAfUmAfAmAfAfAmGmGmAfCmAfAmCfAmUmsGmsGm.

[0038] 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, s before the ribonucleotide in notations such as sUm and sAf indicates thio modification of the 5' phosphate of the ribonucleotide, and * indicates 2' R group modification. The structure of the R group is shown in Figure 2.

[0039] In a preferred embodiment, the positive chain substrate fragment includes two or more substrate fragments, and the negative chain substrate includes two or more substrate fragments; preferably, the length of the positive chain substrate fragment is 2-34 nt, more preferably 6-30 nt; preferably, the length of the negative chain substrate fragment is 2-20 nt, more preferably 6-16 nt.

[0040] Preferably, the positive-sense substrate fragment used to form the 3' end of the positive-sense strand has a length ≥18 nt, including but not limited to 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 nt. This length of 3' end positive-sense substrate fragment allows for complementary pairing with other substrate fragments, forming a notched secondary structure that facilitates the function of RNA ligase and the preparation of the target product, Nedosiran.

[0041] In a preferred embodiment, the RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having more than 70% identity with the RNA ligase shown in SEQ ID NO: 1, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (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 more, or even 99.9% or more) and having catalytic activity for the formation of phosphodiester bonds.

[0042] All of the above RNA ligases can recognize the notched double-stranded structure formed by complementary pairing of substrate fragments, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

[0043] SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

[0044] SEQ ID NO: 2: (Ligase 11, Thermococcus):

[0045] SEQ ID NO: 3: (Ligase 20, Archaea):

[0046] SEQ ID NO: 4: (Ligase 32, bacteria):

[0047] 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.

[0048] 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 are highly likely to have the same active site, active pocket, active mechanism, and protein structure as the proteins provided by the above sequences.

[0049] 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).

[0050] 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:

[0051] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0052] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0053] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0054] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0055] 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 score matrix" in the prior art.

[0056] In this application, only the RNA ligase shown in SEQ ID NO: 1, or an enzyme with more than 70% identity to the RNA ligase shown in SEQ ID NO: 1, can catalyze the formation of a phosphodiester bond between the phosphate group and the hydroxyl group of the substrate in this application to obtain the product Nedosiran. In the relevant experiments of this application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 that can synthesize Nedosiran by screening a large number of enzymes. However, the large proportion of negative results in the experiments showed that most RNA ligases are difficult to catalyze the synthesis of Nedosiran, including but not limited to the RNA ligases shown in SEQ ID NO: 2 to SEQ ID NO: 4. In this application specification, only SEQ ID NO: 2 to SEQ ID NO: 4 are used as examples to illustrate this type of RNA ligase that does not have the activity to catalyze the synthesis of Nedosiran.

[0057] In a preferred embodiment, both the sense and antisense substrate fragments comprise two fragments. The sense substrate fragment includes a first sense substrate fragment and a second sense substrate fragment, and the antisense substrate includes a first antisense substrate fragment and a second antisense substrate fragment. The preparation method includes: mixing the first sense substrate fragment, the second sense substrate fragment, the first antisense substrate fragment, and the second antisense substrate fragment; using RNA ligase to catalyze the ligation of the first sense substrate fragment and the second sense substrate fragment to form a sense strand; catalyzing the ligation of the first antisense substrate fragment and the second antisense substrate fragment to form an antisense strand; and the sense and antisense strands forming a Nedosiran through complementary base pairing.

[0058] In a preferred embodiment, the nucleotide sequence of the first positive strand substrate fragment is AmsUmGfUmUfGmUmCfCf, and the nucleotide sequence of the second positive strand substrate fragment is SEQ ID NO: 6; the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is CmAfAmCfAmUmsGmsGm; preferably, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 9, the nucleotide sequence of the second positive strand substrate fragment is SEQ ID NO: 10, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 11.

[0059] Nedosiran can be prepared using the above-described preparation method and the substrate fragments shown in SEQ ID NO: 5-SEQ ID NO: 8 or SEQ ID NO: 9-SEQ ID NO: 12. However, it should be noted that the choice of substrate is not limited to the substrate fragments shown in SEQ ID NO: 5-SEQ ID NO: 8 or SEQ ID NO: 9-SEQ ID NO: 12. Substrates capable of forming both the positive and negative strands can be used in the above preparation method. The above preparation method is applicable to the preparation of Nedosiran but is not limited to different substrate connection positions. The above preparation method shows good connection effects for both the positive and negative strand sequences of Nedosiran. The number of positive or negative strand substrates includes, but is not limited to, 2, 3, 4, or even more.

[0060] SEQ ID NO: 6: UmCfUmGmAmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmUmG mCm.

[0061] SEQ ID NO: 8: U*sCfsAfsGmAfUmAfAmAfAfAmGm.

[0062] SEQ ID NO: 9: AmsUmGUmUfGmUmCfCf.

[0063] SEQ ID NO: 10: UfUfUmUfUmAfUmCfUmGmAmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmUmGmCm.

[0064] SEQ ID NO: 11: CmAfAmCfAmUmsGmsGm.

[0065] SEQ ID NO: 12: U*sCfsAfsGmAfUmAfAmAfAfAmGmGmAf.

[0066] In a preferred embodiment, the 3' end of the first positive strand substrate fragment and the 5' end of the second positive strand substrate fragment are ligated under the catalysis of RNA ligase to form a positive strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment 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 positive strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second positive strand substrate fragment is a phosphate group, and the 3' end is an R group; the 5' end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0067] In a preferred embodiment, both the sense and antisense substrate fragments comprise three segments: a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the preparation method includes: mixing the first, second, and third sense substrate fragments, the first and second antisense substrate fragments, and under the catalysis of RNA ligase, ligating the first, second, and third sense substrate fragments to form a sense strand; catalyzing the ligation of the first, second, and third antisense substrate fragments to form an antisense strand; and forming Nedosiran through base complementarity pairing between the sense and antisense strands. Preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain Nedosiran.

[0068] In a preferred embodiment, the nucleotide sequence of the first positive strand substrate fragment is AmsUmGfUmUfGm; the nucleotide sequence of the second positive strand substrate fragment is UmCfCfUfUfUmUfUm; and the nucleotide sequence of the third positive strand substrate fragment is SEQ ID NO: 15. Preferably, the nucleotide sequence of the first antisense strand substrate fragment is CfAmUmsGmsGm; the nucleotide sequence of the second antisense strand substrate fragment is AfAmGmGmAfCmAfAm; and the nucleotide sequence of the third antisense strand substrate fragment is U*sCfsAfsGmAfUmAfAmAf.

[0069] SEQ ID NO: 15: AfUmCfUmGmAmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmU mGmCm.

[0070] In a preferred embodiment, the concentrations of the sense and antisense substrate fragments are each independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense substrate fragment, antisense substrate fragment, and RNA ligase further 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.

[0071] 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.

[0072] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0073] Example 1

[0074] Substrate fragments 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 sequences of substrate fragments 1-4 are shown in Table 1, with length in nt. The substrate fragment mixture was annealed to obtain an RNA fragment mixture. The reaction system was set to 10 μL, including 100 μM RNA fragment mixture, 50 mM Tris-HCl, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM), and RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 to a final concentration of 0.2 mg / mL, respectively. The reaction system was incubated at 16 °C for 16 h. The resulting reaction system was inactivated by centrifugation at 80 °C for 5 min to remove the ligases and centrifuged at 12000 rpm to remove the precipitate. A schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

[0075] The products catalyzed by RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 were analyzed by SDS-PAGE. Figure 3 shows the electrophoresis results of the products catalyzed by Ligase 25 and Ligase 11. In Figure 3, lane M represents the RNA molecule marker, lane 1 represents the reaction system of Ligase 25, and lane 2 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0076] Four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0077] Table 1

[0078] In this designation, a single 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide, 'f' indicates a 2' fluorine modification of the ribonucleotide, and 'm*' indicates a modifying group on the 2' R-group side chain of the ribose in the ribonucleotide. The 's' preceding A, C, G, or U (e.g., 'sUm', 'sAf', etc.) indicates a thiolation modification of the 5' phosphate group of the ribonucleotide. The following structural formula illustrates the connection of the 2' R-group using 'Gm*' as an example.

[0079] The ribonucleotides at positions 1, 2, 4, 6, 7, 12, and 14 of substrate 1 have a 2' methoxy modification, the ribonucleotides at positions 3, 5, 8, 9, 10, 11, 13, and 15 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotide at position 2 has a thiomodification.

[0080] The ribonucleotides at positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of substrate 2 have a 2' methoxy group modification, the ribonucleotide at position 2 has a 2' fluorine group modification, and the ribonucleotides at positions 12, 13, 14, and 15 have a 2' R group side chain modification.

[0081] The ribonucleotides at positions 1, 3, 5, 7, 8, 9, and 10 of substrate 3 have a 2' methoxy group, and the ribonucleotides at positions 2, 4, and 6 have a 2' fluorine group. The 5' phosphate group at positions 9 and 10 has a thio group.

[0082] The ribonucleotides at positions 4, 6, 8, 11, and 12 of substrate 4 have a 2' methoxy group modification, and the ribonucleotides at positions 2, 3, 5, 7, 9, and 10 have a 2' fluorine group modification. The 5' phosphate group on the ribonucleotides at positions 2, 3, and 4 has a thio group modification. The ribonucleotide at position 1 has a 2' R group side chain modification.

[0083] The prepared Nedosiran has the following positive chain: AmsUmGfUmUfGmUmCfCfUfUfUmUfUmAfUmCfUmGmAmGmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmUmGmCm (SEQ ID NO: 19) and the negative chain: U*sCfsAfGmsAfUmAfAmAfAfAmGmGmAfCmAfAmCfAmUmsGmsGm (SEQ ID NO: 20).

[0084] Table 2

[0085] In the expression of product yield, "++" indicates a yield of 25-50% (excluding the 50% endpoint), "+++" indicates a yield of 50-75%, and "++++" indicates a yield >75%.

[0086] 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).

[0087] Example 2

[0088] Substrate fragments 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 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, 100 eq MgCl2, and 10 eq DTT (1 eq = 800 μM). Ligase 25, the RNA ligase with high catalytic activity shown in Example 1, was added to each ligase 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.

[0089] The product obtained by Ligase 25 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.

[0090] Table 3

[0091] In the representation of product yield, "+++" indicates a yield of 90-95% (excluding the 95% endpoint), and "++++" indicates a yield >95%.

[0092] The molecular weight of the positive chain product was determined by LC-MS to be 11871.88, and the molecular weight of the antisense chain product was 7396.14. The theoretical values ​​for the positive chain product were 11871.85±8 and the theoretical values ​​for the antisense chain product were 7396.12±8, indicating that Ligase 25 was linked to generate Nedosiran. The detection results are shown in Figure 5.

[0093] Example 3

[0094] 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, 1.25 eq DTT (1 eq = 800 μM), and RNA ligase Ligase 25 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 76.60% and the purity was 96.00%.

[0095] Example 4

[0096] Substrate 5–8 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 sequences of substrate 5–8 are shown in Table 4. 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 inactivated by ligase at 80℃ for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product obtained by Ligase 25 catalysis was analyzed by HPLC. The enzyme activity was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the yield was "+++" and the proportion of the target peak in the sample was 90.1%.

[0097] Table 4

[0098] In this designation, a single 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide; 'f' indicates a 2' fluorine modification of the ribonucleotide; and 's' preceding A, C, G, or U (e.g., 'sUm', 'sAf', etc.) indicates a thiolation modification of the 5' phosphate group of the ribonucleotide. 'm*' indicates a modification group on the 2' R-side chain of the ribose in the ribonucleotide.

[0099] The ribonucleotides at positions 1, 2, 4, 6, and 7 of substrate 5 have a 2' methoxy group, and the ribonucleotides at positions 3, 5, 8, and 9 have a 2' fluorine group. The 5' phosphate group at position 2 has a thio group.

[0100] The ribonucleotides at positions 2, 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 of substrate 6 have a 2' methoxy group modification, the ribonucleotides at positions 1, 4, 6, and 8 have a 2' fluorine group modification, and the ribonucleotides at positions 18, 19, 20, and 21 have a 2' R group side chain modification.

[0101] The ribonucleotides at positions 1, 3, 5, 6, 7, and 8 of substrate 7 have a 2' methoxy group, and the ribonucleotides at positions 2 and 4 have a 2' fluorine group. The 5' phosphate group at positions 7 and 8 has a thio group.

[0102] The ribonucleotides at positions 4, 6, 8, 11, 12, and 13 of substrate 8 have a 2' methoxy group modification, and the ribonucleotides at positions 2, 3, 5, 7, 9, 10, and 14 have a 2' fluorine group modification. The 5' phosphate group on the ribonucleotides at positions 2, 3, and 4 has a thio group modification. The ribonucleotide at position 1 has a 2' R group side chain modification.

[0103] The prepared Nedosiran has the following positive chain: AmsUmGfUmUfGmUmCfCfUfUfUmUfUmAfUmCfUmGmAmGmGmCmAmGmCmCmGm*Am*Am*Am*GmGmCmUmGmCm (SEQ ID NO: 19) and the negative chain: U*sCfsAfsGmAfUmAfAmAfAfAmGmGmAfCmAfAmCfAmUmsGmsGm (SEQ ID NO: 20).

[0104] Example 5

[0105] Substrate 9–14 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 sequences of substrate 9–14 are shown in Table 5. After annealing, the substrate mixture was used to obtain a mixture of RNA fragments of substrate 5–8. The reaction system was set to 50 μL. The reaction system consisted of 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), and RNA ligase Ligase 25 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 inactivated by ligase at 80℃ for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product obtained by Ligase 25 catalysis was detected by HPLC. The enzyme activity was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the yield was "+++" and the proportion of the target peak in the sample was 93.5%.

[0106] Table 5

[0107] In this designation, a single 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide; 'f' indicates a 2' fluorine modification of the ribonucleotide; and 's' preceding A, C, G, or U (e.g., 'sUm', 'sAf', etc.) indicates a thiolation modification of the 5' phosphate group of the ribonucleotide. 'm*' indicates a modification group on the 2' R-side chain of the ribose in the ribonucleotide.

[0108] The ribonucleotides at positions 1, 2, 4, and 6 of substrate 9 have 2' methoxy modifications, the ribonucleotides at positions 3 and 5 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotide at position 2 has a thio modification.

[0109] The ribonucleotides at positions 1, 6, and 8 of substrate 10 have a 2' methoxy modification, and the ribonucleotides at positions 2, 3, 4, 5, and 7 have a 2' fluorine modification.

[0110] The ribonucleotides at positions 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 of substrate 11 have a 2' methoxy group modification, the ribonucleotides at positions 1 and 3 have a 2' fluorine group modification, and the ribonucleotides at positions 13, 14, 15, and 16 have a 2' R group side chain modification.

[0111] The ribonucleotides at positions 2, 3, 4, and 5 of substrate 12 have a 2' methoxy group, and the ribonucleotide at position 1 has a 2' fluorine group. The 5' phosphate groups at positions 4 and 5 have a thio group.

[0112] The ribonucleotides at positions 2, 3, 4, 6, and 8 of substrate 13 have 2' methoxy modifications, and the ribonucleotides at positions 1, 5, and 7 have 2' fluorine modifications.

[0113] The ribonucleotides at positions 4, 6, and 8 of substrate 14 have 2' methoxy modifications, the ribonucleotides at positions 2, 3, 5, 7, and 9 have 2' fluorine modifications, the 5' phosphate group on the ribonucleotides at positions 2, 3, and 4 has a thio modification, and the ribonucleotide at position 1 has a 2' R group side chain modification.

[0114] The prepared Nedosiran has the following positive chain: AmsUmGfUm UfGmUmCf CfUfUfUm UfUmAfUm CfUmGmAm GmCmAmGmCmCmGm*Am*Am*Am*GmGm CmUmGmCm (SEQ ID NO: 19) and the negative chain: U*sCfsAfsGmAfUmAfAmAfAfAmGmGmAfCmAfAmCfAmUmsGmsGm (SEQ ID NO: 20).

[0115] Example 6

[0116] The average yield of the full-length Nedosiran product synthesized by solid-phase synthesis was 39.1%, with N+1 and N-1 impurities accounting for a total of 1.52%.

[0117] The yield of Nedosiran product prepared using the enzyme-linked method of this invention was 84.05%, while the yield of substrate prepared using solid-phase synthesis was 32.9%. The overall yield of the combined process was 31.6%. Although the overall yield was slightly lower than the average yield of the product obtained by solid-phase synthesis, the total proportion of N+1 and N-1 impurities was only 0.4%, which is much lower than the proportion of such impurities in the process of synthesizing Nedosiran by solid-phase synthesis.

[0118] 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, single-stranded RNA fragments designed based on the Nedosiran sequence are catalyzed by RNA ligase to form Nedosiran, 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, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production.

[0119] 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 Nedosiran, characterized in that, The Nedosiran is a double-stranded siRNA composed of complementary sense and antisense strands; The preparation method includes: A positive sense substrate fragment, an antisense substrate fragment, and an RNA ligase are mixed, wherein the positive sense substrate fragment is capable of forming the positive sense strand, and the antisense substrate fragment is capable of forming the antisense strand; The positive and negative substrate fragments are connected by hydrogen bonds formed by complementary bases. The head and tail bases of the positive and negative substrate fragments 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 the Nedosiran; The bases at both ends of the notch are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; The 5' phosphate group and the 3' hydroxyl group upstream and downstream of the notch are linked by the RNA ligase to form the phosphodiester bond, thereby obtaining the Nedosiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; Or an RNA ligase with more than 70% identity to the amino acid sequence shown in SEQ ID NO: 1, and an enzyme with catalytic activity in forming phosphodiester bonds.

3. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO:

20.

4. The preparation method according to any one of claims 1-3, characterized in that, The justice chain substrate fragment includes two or more fragments, and the antisense chain substrate fragment includes two or more fragments; The length of the substrate segment of the justice chain is 2-34 nt; The length of the antisense substrate fragment is 2-20 nt.

5. The preparation method according to claim 1, characterized in that, Both the justice chain substrate segment and the antisense chain substrate segment include two segments. The justice chain substrate segment includes a first justice chain substrate segment and a second justice chain substrate segment. The antisense chain substrate segment includes a first antisense chain substrate segment and a second antisense chain substrate segment. The preparation method includes: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, and the second antisense strand substrate fragment; under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are ligated to form the sense strand; the first antisense strand substrate fragment and the second antisense strand substrate fragment are ligated to form the antisense strand; and the sense strand and the antisense strand form the Nedosiran through complementary base pairing.

6. The preparation method according to claim 5, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is AmsUmGfUmUfGmUmCfCf, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO: 6; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense substrate fragment is CmAfAmCfAmUmsGmsGm.

7. The preparation method according to claim 5, characterized in that, The nucleotide sequence of the first positive-strand substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second positive-strand substrate fragment is SEQ ID NO:

10. The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate fragment is SEQ ID NO:

11.

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

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

10. The preparation method according to claim 1, characterized in that, Both the justice chain substrate segment and the antisense chain substrate segment include three segments. The substrate segment of the justice chain includes a first justice chain substrate segment, a second justice chain substrate segment, and a third justice chain substrate segment; The antisense substrate fragment includes a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment.

11. The preparation method according to claim 10, characterized in that, The preparation method includes: mixing the first positive strand substrate fragment, the second positive strand substrate fragment, the third positive strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment; under the catalysis of the RNA ligase, the first positive strand substrate fragment, the second positive strand substrate fragment, and the third positive strand substrate fragment are ligated to form the positive strand; the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment are ligated to form the antisense strand; and the positive strand and the antisense strand form the Nedosiran through base complementary pairing.

12. The preparation method according to claim 10, characterized in that, The first justice chain substrate fragment is AmsUmGfUmUfGm; The nucleotide sequence of the second positive-strand substrate fragment is UmCfCfUfUfUmUfUm; The nucleotide sequence of the third positive-strand substrate fragment is SEQ ID NO: 15; The nucleotide sequence of the first antisense substrate fragment is CfAmUmsGmsGm; The nucleotide sequence of the second antisense substrate fragment is AfAmGmGmAfCmAfAm; The nucleotide sequence of the third antisense substrate fragment is U*sCfsAfsGmAfUmAfAmAf.

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

14. 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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