Method for preparing givosiran

Abstract

Provided is a method for preparing givosiran. Givosiran is a double-stranded siRNA composed of a sense strand and an antisense strand by means of complementary pairing. The preparation method therefor comprises mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, wherein the sense strand substrate can form the sense strand, the antisense strand substrate can form the antisense strand, the sense strand substrate and the antisense strand substrate are linked via a hydrogen bond formed by means of base complementary pairing, and the terminal bases of the sense strand substrate and antisense strand substrate are not linked to each other, thereby forming a double-stranded nucleotide structure containing a nick. The RNA ligase is used to link the bases at both ends of the nick via a phosphodiester bond to form givosiran. The preparation method can solve the problem of the relatively low purity of givosiran in the preparation in the prior art.

Description

A method for preparing Givosiran

[0001] This application is based on and claims priority to Chinese application CN application number 202410921746.X, 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 Givosiran. Background Technology

[0003] Small interfering RNA (siRNA) is a double-stranded RNA of 19-25 nt in length. After entering the cell, siRNA dissociates into single strands. The sense strand specifically binds to the messenger RNA (mRNA) of the target gene through base matching, inducing a series of actions that ultimately degrade the target gene's mRNA, preventing mRNA translation and thus inhibiting target gene expression. In recent years, the development of siRNA drugs has received widespread attention. siRNA drugs act on mRNA, making their target sites significantly larger than those of traditional small molecule drugs that act on proteins. Furthermore, siRNA drugs can target new sites by changing their sequence, resulting in relatively short development times.

[0004] Givosiran is an siRNA-based double-stranded RNA drug developed by Alnylam for the treatment of acute hepatic porphyria (AHP) in adults. It was approved by the FDA in 2019 and marketed under the brand name GIVLAARI.

[0005] Currently, Givosiran is prepared using a chemical method, employing a solid-phase support such as controlled-porous glass (CPG) or polystyrene resin. It is synthesized cyclically via a phosphoramidite process, extending the oligonucleotide chain along the 3' to 5' direction. After the synthesis cycle is complete, the Givosiran chain is removed from the solid-phase support via ammonolysis, followed by purification to obtain the pure product. However, this solid-phase synthesis method is cyclical, and the yield decreases with increasing chain length. Furthermore, 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. These impurities are difficult to remove, leading to a complex and inefficient purification process. Therefore, Givosiran preparation is costly and difficult to scale up for production. Thus, a more efficient Givosiran synthesis method needs to be developed. Summary of the Invention

[0006] The main objective of this invention is to provide a method for preparing Givosiran, thereby solving the problem of low purity in the preparation of Givosiran in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing Givosiran is provided, wherein the Givosiran is a double-stranded siRNA composed of a sense strand and an antisense strand through complementary pairing; the method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase;

[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 Givosiran; the bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with a phosphate group at the 5' end and a hydroxyl group at the 3' end; the phosphate group at the 5' end and the hydroxyl group at the 3' end of the nick are linked by RNA ligase to form phosphodiester bonds, obtaining Givosiran; the RNA ligase is an RNA ligase from RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase from RNA ligase family 1 is selected from: having the following characteristics as shown in SEQ ID Proteins having the amino acid sequence shown in NO:3; RNA ligases of RNA ligase family 2 are selected from: proteins having the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2; or enzymes having more than 70% identity with any RNA ligase having the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:3 and having catalytic activity for the formation of phosphodiester bonds.

[0009] Furthermore, the nucleotide sequence of the sense strand is SEQ ID NO: 21, and the nucleotide sequence of the antisense strand is SEQ ID NO: 22.

[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 2-19 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 2-21 nt, more preferably 4-15 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; under the catalysis of RNA ligase, the first sense substrate fragment and the second sense substrate fragment are ligated to form a sense strand; the first antisense substrate fragment and the second antisense substrate fragment are ligated to form an antisense strand; the sense strand and the antisense strand form Givosiran through base complementarity pairing; preferably, the sense substrate fragment and the antisense substrate fragment are annealed and then mixed with RNA ligase to obtain Givosiran.

[0012] Further, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated under the catalysis of 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 under the catalysis of 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 L96 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.

[0013] Further, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 7, and the nucleotide sequence of the second sense substrate fragment is AmUmCmUmUmAm; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 10, and the nucleotide sequence of the second antisense substrate fragment is SEQ ID NO: 9.

[0014] Further, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 11, and the nucleotide sequence of the second sense substrate fragment is SEQ ID NO: 12; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 14, and the nucleotide sequence of the second antisense substrate fragment is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm.

[0015] Furthermore, both the positive and negative substrate fragments comprise three segments: the positive substrate fragment includes a first positive substrate fragment, a second positive substrate fragment, and a third positive substrate fragment; the negative substrate fragment includes a first negative substrate fragment, a second negative substrate fragment, and a third negative substrate fragment. Preferably, the nucleotide sequence of the first positive substrate fragment is CmsAmsGmAmAm; the nucleotide sequence of the second positive substrate fragment is AmGfAmGfUmGfUm; and the nucleotide sequence of the third positive substrate fragment is CfUmCfAmUmCmUmUmAm. Preferably, the nucleotide sequence of the first negative substrate fragment is UfGmsGmsUm; the nucleotide sequence of the second negative substrate fragment is CmUfCmUfUmUf; and the nucleotide sequence of the third negative substrate fragment is SEQ ID NO: 20.

[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 the Givosiran sense strand, and the antisense strand substrate fragments are ligated to form the Givosiran antisense strand, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with the chemical synthesis method for preparing Givosiran, 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 structure of the L96 group according to an embodiment of the present invention.

[0020] Figure 2 shows a schematic diagram of the enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

[0021] Figure 3 shows the electrophoresis results of the RNA ligase Ligase25 and Ligase26 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 Givosiran 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 Givosiran using enzymatic catalytic synthesis, thus proposing a series of protective solutions.

[0029] In a first typical embodiment of this application, a method for preparing Givosiran is provided. The Givosiran is a double-stranded RNA composed of complementary positive and negative strands. The method includes: mixing a positive strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase; mixing the positive strand substrate fragment, the antisense strand substrate fragment, and the RNA ligase; wherein the positive strand substrate fragment can form the positive strand, and the antisense strand substrate fragment can form the antisense strand; the positive strand substrate fragment and the antisense strand substrate fragment are base-paired... The hydrogen bonds formed by the notch are not interconnected between the head and tail bases of the sense and antisense substrate fragments, resulting in a double-stranded nucleotide structure with a nick. Using RNA ligase, the bases at both ends of the nick are linked by phosphodiester bonds to form Givosiran. The bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, with the 5' end being a phosphate group and the 3' end a hydroxyl group. Using RNA ligase, the phosphate group at the 5' end and the hydroxyl group at the 3' end of the nick are connected upstream and downstream to form phosphodiester bonds, yielding Givosiran.

[0030] The RNA ligase is an RNA ligase from RNA ligase family 1 and / or RNA ligase family 2; the RNA ligase from RNA ligase family 1 is selected from proteins having the amino acid sequence shown in SEQ ID NO: 3; the RNA ligase from RNA ligase family 2 is selected from proteins having the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or has more than 70% identity with any RNA ligase having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 3, 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 is an enzyme that catalyzes the formation of phosphodiester bonds. 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.

[0031] 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 Givosiran.

[0032] In the above preparation method, Givosiran is prepared by mixing the sense and antisense substrate fragments with RNA ligase. In this method, the sense and antisense substrate fragments can complement each other to form a double-stranded nucleotide with sticky ends. This double-stranded nucleotide with sticky ends can then bind to other substrates to form a nicked double-stranded nucleotide structure. The RNA ligase can recognize the specific recognition site of this nicked double-stranded nucleotide and ligate the nick, thereby preparing the target product Givosiran.

[0033] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 21, and the nucleotide sequence of the antisense strand is SEQ ID NO: 22.

[0034] SEQ ID NO: 21: CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm.

[0035] SEQ ID NO: 22: UmsAfsAfGfAmUfGmAfGmAfCmAf CmUfCmUfUmUfCmUfGmsGms Um.

[0036] 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 sAm, sGm, etc., indicates thiomodification of the 5' phosphate of the ribonucleotide.

[0037] SEQ ID NO: 1 (Ligase-25, Vibrio phage NT-1):

[0038] SEQ ID NO: 2 (Ligase 26, Escherichia phage AR1):

[0039] SEQ ID NO: 3 (Ligase 42, Escherichia phage JN02):

[0040] SEQ ID NO: 4 (Ligase 11, Thermococcus):

[0041] SEQ ID NO: 5 (Ligase 20, Archaea):

[0042] SEQ ID NO: 6 (Ligase 32, bacteria):

[0043] 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.

[0044] 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.

[0045] 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).

[0046] 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:

[0047] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0048] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0049] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0050] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0051] 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.

[0052] In this application, only RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 3, or enzymes with more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 3, can catalyze the formation of phosphodiester bonds between the phosphate group and the hydroxyl group of the substrate in this application to obtain the product Givosiran. In the relevant experiments of this application, the inventors obtained the aforementioned RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 3 that are capable of synthesizing Givosiran by screening from 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 Givosiran, including but not limited to the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 6. In this application specification, only SEQ ID NO: 4 to SEQ ID NO: 6 are used as examples to illustrate this type of RNA ligase that does not have the activity of catalyzing the synthesis of Givosiran.

[0053] In a preferred embodiment, 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 2-19 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 2-21 nt, more preferably 4-15 nt.

[0054] 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 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 ligation of the first and second sense substrate fragments to form a sense strand under the catalysis of RNA ligase; catalyzing the ligation of the first and second antisense substrate fragments to form an antisense strand; and forming Givosiran 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 Givosiran.

[0055] 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 Givosiran with a complete double-stranded structure.

[0056] In a preferred embodiment, 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 L96 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.

[0057] In a preferred embodiment, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 7, and the nucleotide sequence of the second sense substrate fragment is AmUmCmUmUmAm; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 10, and the nucleotide sequence of the second antisense substrate fragment is SEQ ID NO: 9.

[0058] In a preferred embodiment, the nucleotide sequence of the first positive-sense substrate fragment is SEQ ID NO: 11, and the nucleotide sequence of the second positive-sense substrate fragment is SEQ ID NO: 12; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 14, and the nucleotide sequence of the second antisense substrate fragment is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm.

[0059] Givosiran can be prepared using the above-described preparation method and substrate fragments. However, it should be noted that the selection of substrate fragments is not limited to those described above; any substrate fragment 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 Givosiran but is not limited to different substrate fragment connection positions. The above preparation method shows good ligation effects for both the sense and antisense strand sequences of Givosiran. The number of sense or antisense substrate fragments includes, but is not limited to, 2, 3, 4, or even more.

[0060] SEQ ID NO: 7: CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCf.

[0061] SEQ ID NO: 9: CmUfCmUfUmUfCmUfGmsGmsUm.

[0062] SEQ ID NO: 10: UmsAfsAfGfAmUfGmAfGmAfCmAf.

[0063] SEQ ID NO: 11: CmsAmsGmAmAmAmGfAmGfUm.

[0064] SEQ ID NO: 12: GfUmCfUmCfAmUmCmUmUmAm.

[0065] SEQ ID NO: 14: UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm.

[0066] In a preferred embodiment, both the positive and negative substrate fragments comprise three segments: a first positive substrate fragment, a second positive substrate fragment, and a third positive substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the nucleotide sequence of the first positive substrate fragment is CmsAmsGmAmAm; the nucleotide sequence of the second positive substrate fragment is AmGfAmGfUmGfUm; and the nucleotide sequence of the third positive substrate fragment is CfUmCfAmUmCmUmUmAm. Preferably, the nucleotide sequence of the first antisense substrate fragment is UfGmsGmsUm; the nucleotide sequence of the second antisense substrate fragment is CmUfCmUfUmUf; and the nucleotide sequence of the third antisense substrate fragment is SEQ ID NO: 20.

[0067] SEQ ID NO: 20: UmsAfsAfGfAmUfGmAfGmAfCmAf.

[0068] 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 and antisense substrate fragments 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.

[0069] 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.

[0070] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0071] Example 1:

[0072] Based on the Givosiran sequence, the positive and negative strands (SEQ ID NO: 21 and SEQ ID NO: 22) were used to design four single-stranded RNA fragments (length: nt) as shown in Table 1:

[0073] Table 1

[0074] 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 "sAm" and "sGm" 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.

[0075] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, and 14 of substrate 1 have a 2' methoxy modification, the nucleotides at positions 7, 9, 11, 13, and 15 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has a thiomodification.

[0076] The ribonucleotides at positions 1, 2, 3, 4, 5, and 6 of substrate 2 have a 2' methoxy group modification.

[0077] The ribonucleotides at positions 1, 3, 5, 7, 9, 10, and 11 of substrate 3 have a 2' methoxy modification, the nucleotides at positions 2, 4, 6, and 8 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 10 and 11 has a thiomodification.

[0078] The ribonucleotides at positions 1, 5, 7, 9, and 11 of substrate 4 have a 2' methoxy modification, the nucleotides at positions 2, 3, 4, 6, 8, 10, and 12 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has a thiomodification.

[0079] The above four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0080] Four single-stranded RNA fragments were mixed in equimolar proportions to obtain a substrate mixture with a final concentration of 2.5 mM (2.5 mM for each substrate). The mixture was then annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzymatic ligation reaction in a 10 μL volume. The reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, and dithiothreitol (DTT). RNA ligases Ligase 25, Ligase 26, Ligase 11, Ligase 20, and Ligase 32 were added, respectively. 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. A schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 2.

[0081] The products obtained by RNA ligases Ligase 25, Ligase 26, Ligase 11, Ligase 20, and Ligase 32 were analyzed by SDS-PAGE. The electrophoresis results of the products catalyzed by Ligase 25 and Ligase 26 are shown in Figure 3. In Figure 3, lane M represents the RNA molecule marker, lane 1 represents the Ligase 25 reaction system, and lane 2 represents the Ligase 26 reaction system. 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.

[0082] Activity screening of six RNA ligases revealed that Ligase25 exhibited the best ligation performance, converting most of the substrate into Givosiran.

[0083] The sense chain of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCfAmUmCmUmUmAm (SEQ ID NO: 21), and the antisense chain is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22). The reaction results are shown in Table 2.

[0084] Table 2

[0085] Remark:

[0086] 1) Reaction conditions: 100 μM substrate fragment, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM), 0.2 mg / mL enzyme, 1891 V, 50 mM Tris-HCl, pH 7.5, 16℃, 16 h;

[0087] 2)++ indicates 25-50% (excluding the 50% endpoint value), and +++ indicates 50-75%.

[0088] 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).

[0089] Example 2:

[0090] Ligase 25, Ligase 26, and Ligase 42, which can catalyze the synthesis of Givosiran in Example 1, were used to perform enzymatic ligation reactions using annealed substrate fragments. The reaction conditions were as follows: the reaction system was set to 50 μL, and reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT), and RNA ligase were added sequentially to the reactor. The reaction was carried out at 16°C for 16 h.

[0091] After the reaction was completed, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The samples were analyzed by HPLC and LC-MS. The HPLC results of the Ligase 25 catalyzed product are shown in Figure 4. The yield was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.

[0092] Table 3

[0093] Remark:

[0094] 1) Reaction conditions: substrate fragment 800 μM, ATP 4 eq, MgCl2 100 eq, DTT 10 eq (1 eq = 800 μM), 0.2 mg / mL enzyme, 236 V, 50 mM Tris-HCl, pH 7.5, 16℃, 16 h;

[0095] 2) ND indicates no product generation detected, ++ indicates 50-70% (excluding the 70% endpoint value), and +++ indicates 70-80%.

[0096] The molecular weight of the positive-sense product was determined by LC-MS to be 8732.04, and the molecular weight of the antisense product was 7560.03. The theoretical values ​​for the positive-sense product were 8732.04±8 and the theoretical values ​​for the antisense product were 7560.03±8, indicating that Ligase 25, Ligase 26, and Ligase 42 were linked to form Givosiran. The LC-MS detection results for Ligase 25 are shown in Figure 5.

[0097] Example 3

[0098] The annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 10 mL, and reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT), and RNA ligase were added sequentially to the reactor. The reaction was carried out at 16 °C for 16 h. After the overnight reaction, the protein was inactivated by heating at 50 °C for 15 min. The supernatant was collected by centrifugation, purified using a Nano-Q column, eluted with a NaCl gradient, desalted by membrane coating (molecular weight cutoff 1 kDa), and lyophilized in a freeze dryer to obtain a dry powder. The yield was calculated to be 66.18%, and the purity (HPLC detection) was 91.88%.

[0099] Example 4

[0100] The sequence design based on Givosiran is shown in Table 4 as four single-stranded RNA fragments (length: nt).

[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 "sAm" and "sGm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

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

[0104] Substrate 6 has 2' methoxy groups at positions 2, 4, 6, 7, 8, 9, 10, and 11, and 2' fluorine groups at positions 1, 3, and 5. It also has an L96 group at the 5' end.

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

[0106] The ribonucleotides at positions 1, 5, 7, 9, 11, 13, and 15 of substrate 8 have a 2' methoxy modification, the nucleotides at positions 2, 3, 4, 6, 8, 10, 12, and 14 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has a thiomodification.

[0107] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 10 mL, and the following were added sequentially to the reactor: 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 25, 50 mM Tris-HCl, pH 7.5. The reaction was carried out at 16 °C for 16 h. After the reaction, the protein was inactivated by heating at 80 °C for 5 min, and the supernatant was collected by centrifugation. The results were analyzed by HPLC, and the yield was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample. The results showed a yield of ±++ (product peak percentage 75.2%).

[0108] The positive chain of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmAmUmCmUmUmAm (SEQ ID NO: 21), and the negative chain is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).

[0109] Example 5

[0110] Based on the Givosiran sequence, six single-stranded RNA fragments (length: nt) were designed as shown in Table 5.

[0111] Table 5

[0112] 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 "sAm" and "sGm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0113] The ribonucleotides at positions 1, 2, 3, 4 and 5 of substrate 9 have a 2' methoxy modification, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has a thio modification.

[0114] The ribonucleotides at positions 1, 3, 5, and 7 of substrate 10 have 2' methoxy modifications, and the nucleotides at positions 2, 4, and 6 have 2' fluorine modifications.

[0115] The ribonucleotides at positions 2, 4, 5, 6, 7, 8, and 9 of substrate 11 have 2' methoxy modifications, and the nucleotides at positions 1 and 3 have 2' fluorine modifications.

[0116] The ribonucleotides at positions 2, 3, and 4 of substrate 12 have a 2' methoxy modification, the nucleotide at position 1 has a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 3 and 4 has a thio modification.

[0117] The ribonucleotides at positions 1, 3, 5, and 7 of substrate 13 have 2' methoxy modifications, and the nucleotides at positions 2, 4, and 6 have 2' fluorine modifications.

[0118] The ribonucleotides at positions 1, 5, 7, 9, and 11 of substrate 14 have a 2' methoxy modification, the nucleotides at positions 2, 3, 4, 6, 8, 10, and 12 have a 2' fluorine modification, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has a thiomodification.

[0119] The positive chain of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmAmUmCmUmUmAm (SEQ ID NO: 21), and the negative chain is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).

[0120] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 10 mL, and the following were added sequentially to the reactor: 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 25, 50 mM Tris-HCl, pH 7.5. The reaction was carried out at 16 °C for 16 h. After the reaction, the protein was inactivated by heating at 80 °C for 5 min, and the supernatant was collected by centrifugation. The protein was analyzed by HPLC, and the enzyme activity was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample. The results showed a yield of ±++ (product peak percentage 70.3%).

[0121] Comparative Example 1

[0122] The average yield of the full-length Givosiran product synthesized by solid-phase synthesis was 32.9%, with N+1 and N-1 impurities accounting for a total of 1.52%.

[0123] The yield of Givosiran product prepared using the enzyme-linked method of this invention was 87.53%, while the average yield of the substrates used in solid-phase synthesis was 39.1%. Multiplying these yields, the overall yield of the process was 34.2%, which is higher than the average yield of the product obtained by solid-phase synthesis. Furthermore, the total proportion of N+1 and N-1 impurities was 0.41%, which is lower than the proportion of such impurities in the solid-phase synthesis of Givosiran.

[0124] 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 Givosiran sequence are catalyzed by RNA ligase to form Givosiran, 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.

[0125] 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 Givosiran, characterized in that, Givosiran is a double-stranded siRNA, composed of a sense strand and an antisense strand through complementary base pairing. 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 Givosiran; 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 using the RNA ligase to form the phosphodiester bond, thereby obtaining Givosiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is one or more RNA ligases from RNA ligase family 1 and / or RNA ligase family 2. The RNA ligases of the RNA ligase family 1 are selected from proteins having the amino acid sequence shown in SEQ ID NO: 3; The RNA ligases of the RNA ligase family 2 are selected from proteins having the amino acid sequences shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or The RNA ligase is 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: 3, 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: 21, and the nucleotide sequence of the antisense strand is SEQ ID NO:

22.

4. The preparation method according to any one of claims 1-2, 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 2-19 nt; The antisense substrate fragment has a length of 2-21 nt.

5. The preparation method according to claim 3, 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; and, under the catalysis of the RNA ligase, ligating the first sense strand substrate fragment and the second sense strand substrate fragment to form the sense strand. Under the catalytic action of the RNA ligase, the first antisense substrate fragment and the second antisense substrate fragment are catalyzed to join to form the antisense strand; The sense strand and the antisense strand form the Givosiran through complementary base pairing.

6. The preparation method according to claim 4, 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.

7. The preparation method according to claim 4, 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 L96 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.

8. The preparation method according to claim 4, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO: 7, and the nucleotide sequence of the second sense strand substrate fragment is AmUmCmUmUmAm; The nucleotide sequence of the first antisense substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10, and the nucleotide sequence of the second antisense substrate fragment is the nucleotide sequence shown in SEQ ID NO:

9.

9. The preparation method according to claim 4, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO: 11, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO: 12; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 14, and the nucleotide sequence of the second antisense substrate fragment is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm.

10. The preparation method according to claim 3, 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 9, characterized in that, The nucleotide sequence of the first positive-strand substrate fragment is CmsAmsGmAmAm; The nucleotide sequence of the second positive-strand substrate fragment is AmGfAmGfUmGfUm; The nucleotide sequence of the third positive-sense substrate fragment is CfUmCfAmUmCmUmUmAm; The nucleotide sequence of the first antisense substrate fragment is UfGmsGmsUm; The nucleotide sequence of the second antisense substrate fragment is CmUfCmUfUmUf-Cm; The nucleotide sequence of the third antisense substrate fragment is SEQ ID NO:

20.

12. The preparation method according to any one of claims 1-2, 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.

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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