Method for preparing lumasiran

Abstract

Provided is a method for preparing Lumasiran. Lumasiran is a double-stranded RNA composed of a sense strand and an antisense strand by means of complementary pairing. The method comprises: mixing sense strand substrate fragments, antisense strand substrate fragments, and an RNA ligase, wherein the sense strand substrate fragments can form a sense strand, and the antisense strand substrate fragments can form an antisense strand; the sense strand substrate fragments and the antisense strand substrate fragments are connected by means of hydrogen bonds formed by base complementation, and bases at heads and tails of the sense strand substrate fragments and the antisense strand substrate fragments are not connected to each other, thereby forming a double-stranded nucleotide structure containing a nick; and the bases at both ends of the nick are connected by means of a phosphodiester bond by using an RNA ligase to form Lumasiran. The method can solve the problem in the prior art of relatively low purity of prepared Lumasiran.

Description

A method for preparing Lumasilan

[0001] This application is based on and claims priority to Chinese application CN application number 202410921759.7 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 Lumasilan. 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] Lumasiran is a gene-targeted therapy siRNA-based double-stranded RNA drug developed by Alnylam for the treatment of primary hyperoxaluria type I (PH I). It was approved by the FDA in 2020. Lumasiran was the first drug approved for the treatment of PH1. Phase 3 clinical data showed that Lumasiran treatment significantly reduced oxalate production in the liver, promoting the resolution of the intrinsic pathophysiological problems of PH1.

[0005] The synthesis of lumasiran 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, the lumasiran 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 lumasiran is increasingly 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 lumasiran synthesis method. Summary of the Invention

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

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

[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 Lumasilan; 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 Lumasilan; the RNA ligase includes RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 includes: having SEQ ID NO: 3 and ... RNA ligases with the amino acid sequence shown in NO:5; RNA ligases of RNA ligase family 2 are selected from one or more of the RNA ligases with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4; or enzymes that have more than 70% identity with any of the RNA ligases shown in SEQ ID NO:1 to SEQ ID NO:5 and have catalytic activity in forming phosphodiester bonds.

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

[0010] 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 5-14 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 4-16 nt, more preferably 7-12 nt.

[0011] Further, both the positive and negative substrate fragments comprise two segments: the positive substrate fragment includes a first positive substrate fragment and a second positive substrate fragment, and the negative substrate fragment includes a first negative substrate fragment and a second negative substrate fragment. Preferably, the nucleotide sequence of the first positive substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second positive substrate fragment is AmAmAmUmAmUmAm; the nucleotide sequence of the first negative substrate fragment is SEQ ID NO: 11, and the nucleotide sequence of the second negative substrate fragment is AfAmAmGmUmCmsCmsAm; preferably, the nucleotide sequence of the first positive substrate fragment is GmsAmsCmUmUmUmCfAmUf, and the nucleotide sequence of the second positive substrate fragment is SEQ ID NO: 16; the nucleotide sequence of the first negative substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second negative substrate fragment is AmAmGmUmCmsCmsAm.

[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] Furthermore, when both the sense and antisense substrate fragments consist of two fragments, 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 and second sense substrate fragments are ligated to form the sense strand; the first and second antisense substrate fragments are ligated to form the antisense strand; and the sense and antisense strands form Lumasilan through base complementarity pairing. Preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain Lumasilan.

[0014] Furthermore, 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 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 GmsAmsCmUmUm; the nucleotide sequence of the second positive substrate fragment is UmCfAmUfCfCfUmGm; and the nucleotide sequence of the third positive substrate fragment is GmAmAmAmUmAmUmAm. Preferably, the nucleotide sequence of the first negative substrate fragment is SEQ ID. NO: 22; The nucleotide sequence of the second antisense substrate fragment is AmUfGmAfAmAmGm; The nucleotide sequence of the third antisense substrate fragment is UmCmsCmsAm; Preferably, 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 substrate fragment, the second antisense substrate fragment, the third antisense substrate fragment and RNA ligase; under the catalysis of 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 a positive strand, the first antisense substrate fragment, the second antisense substrate fragment and the third antisense substrate fragment are ligated to form an antisense strand, and the positive strand and the antisense strand form Lumasilan through complementary base pairing.

[0015] 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.

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

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

[0018] Figure 1 shows a schematic diagram of the enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

[0019] Figure 2 shows the electrophoretic results of the RNA ligases Ligase25, Ligase26, Ligase31 and Ligase41 catalyzed by the RNA ligases according to Example 1 of the present invention.

[0020] Figure 3 shows a schematic diagram of the structure of L96 according to an embodiment of the present invention.

[0021] Figure 4 shows the HPLC detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention.

[0022] 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

[0023] 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.

[0024] Terminology Explanation:

[0025] N+1 impurities: Nucleic acid impurities that have an additional single nucleotide link compared to the target synthetic sequence.

[0026] N-1 impurities: Nucleic acid impurities that have a single nucleotide deletion compared to the target synthetic sequence.

[0027] As mentioned in the background section, existing technologies for preparing Lumasilan employ chemical synthesis methods, which are not only complex and costly but also generate numerous N+1 and N-1 impurities, affecting subsequent product purification. In this application, the inventors attempt to develop a method for preparing Lumasilan using enzyme-catalyzed synthesis, and thus propose a series of protective solutions.

[0028] In a first typical embodiment of this application, a method for preparing Lumasilan is provided. Lumasilan is a double-stranded RNA 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 the positive strand, and the antisense strand substrate fragment can form the 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 strand with notches. Nucleotide structure; using RNA ligase to link the bases at both ends of the notch with phosphodiester bonds to form Lumasilan; 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; using RNA ligase to link the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the notch to form phosphodiester bonds to obtain Lumasilan; the RNA ligase includes RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is selected from: having SEQ ID NO: 10 ... One or more of the RNA ligases having the amino acid sequences shown in SEQ ID NO: 3 and / or SEQ ID NO: 5; the RNA ligases of RNA ligase family 2 are selected from: RNA ligases having the amino acid sequences shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or having more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, 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.

[0029] 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 Lumasilan.

[0030] In the above preparation method, Lumasilan 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 complement each other to form a nucleotide with sticky ends. This double-stranded nucleotide with sticky ends can further bind with other substrates to form a double-stranded nucleotide structure with a notch. RNA ligase can recognize this notch and connect the notch with a phosphodiester bond to prepare the target product Lumasilan. Preferably, the positive and negative substrate fragments are annealed and then mixed with RNA ligase to obtain Lumasilan.

[0031] In the above preparation method, it is preferable to first mix and anneal the sense and antisense substrate fragments. 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 Lumasilan with a complete double-stranded structure.

[0032] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO: 24.

[0033] SEQ ID NO: 23:

[0034] GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm.

[0035] SEQ ID NO: 24:

[0036] UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm.

[0037] 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, sCm, etc., indicates thiomodification of the 5' phosphate of the ribonucleotide.

[0038] 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.

[0039] SEQ ID NO: 1 (Ligase-25, Vibrio phage NT-1):

[0040] SEQ ID NO: 2 (Ligase 26, Escherichia phage AR1):

[0041] SEQ ID NO: 3 (Ligase 31, Vibrio phage VH12019):

[0042] SEQ ID NO: 4 (Ligase 41, Vibrio phage VH7D):

[0043] SEQ ID NO: 5 (Ligase 42, Escherichia phage JN02):

[0044] SEQ ID NO: 6 (Ligase 11, Thermococcus):

[0045] SEQ ID NO: 7 (Ligase 20, Archaea):

[0046] SEQ ID NO: 8 (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 RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, or enzymes with more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, 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 Lumasilan. In the relevant experiments of this application, the inventors obtained the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 8 that are capable of synthesizing Lumasilan 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 Lumasilan, including but not limited to the RNA ligases shown in SEQ ID NO: 6 to SEQ ID NO: 8. In this application specification, only SEQ ID NO: 6 to SEQ ID NO: 8 are used as examples to illustrate this type of RNA ligase that does not have the activity of catalyzing the synthesis of Lumasilan.

[0057] In a preferred embodiment, the positive chain substrate fragment includes two or more substrate fragments, and the negative chain substrate fragment includes two or more substrate fragments; preferably, the length of the positive chain substrate fragment is 5-14 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 4-16 nt, more preferably 7-12 nt.

[0058] In a preferred embodiment, both the justice chain substrate fragment and the antisense chain substrate fragment include two fragments. The justice chain substrate fragment includes a first justice chain substrate fragment and a second justice chain substrate fragment, and the antisense chain substrate fragment includes a first antisense chain substrate fragment and a second antisense chain substrate fragment.

[0059] In a preferred embodiment, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate fragment is AmAmAmUmAmUmAm; 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 AfAmAmGmUmCmsCmsAm.

[0060] Preferably, the nucleotide sequence of the first sense substrate fragment is GmsAmsCmUmUmUmCfAmUf, the nucleotide sequence of the second sense substrate fragment is SEQ ID NO: 14; the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate fragment is AmAmGmUmCmsCmsAm.

[0061] Lumasilan can be prepared using the above-described preparation method and substrates. However, it should be noted that the choice of substrate is not limited to those described above; any substrate capable of forming both the sense and antisense strands can be used in the above preparation method. The above preparation method is applicable to the preparation of Lumasilan but is not limited to different substrate connection positions. The above preparation method demonstrates good ligation effects for both the sense and antisense strand sequences of Lumasilan. The number of sense or antisense substrates can include, but is not limited to, 2, 3, 4, or even more.

[0062] SEQ ID NO: 9: GmsAmsCmUmUmUmCfAmUfCfCfUmGmGm.

[0063] SEQ ID NO: 12: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGm.

[0064] SEQ ID NO: 14: CfCfUmGmGmAmAmAmUmAmUmAm.

[0065] SEQ ID NO: 16: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAf.

[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 by 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 by 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 L96 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, the preparation method includes: mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a first antisense strand substrate fragment, and a second antisense strand substrate fragment; using RNA ligase to catalyze the ligation of the first sense strand substrate fragment and the second sense strand substrate fragment to form a sense strand; catalyzing the ligation of the first antisense strand substrate fragment and the second antisense strand substrate fragment to form an antisense strand; and the sense strand and the antisense strand forming Lumasilan through complementary base pairing.

[0068] 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 GmsAmsCmUmUm; the nucleotide sequence of the second positive substrate fragment is UmCfAmUfCfCfUmGm; and the nucleotide sequence of the third positive substrate fragment is GmAmAmAmUmAmUmAm. Preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 22; the nucleotide sequence of the second antisense substrate fragment is AmUfGmAfAmAmGm; and the nucleotide sequence of the third antisense substrate fragment is UmCmsCmsAm.

[0069] SEQ ID NO: 13: UmsAfsUmAmUmUfUmCfCfAmGmGm.

[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 26, Ligase 31, Ligase 41, Ligase 42, 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 subjected to inactivation of the ligases at 80 °C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. Figure 1 shows a schematic diagram of the enzyme-catalyzed ligation reaction.

[0075] The products catalyzed by RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase 11, Ligase 20, and Ligase 32 were analyzed by Urea-PAGE. The electrophoresis results of the products catalyzed by Ligase 25, Ligase 26, Ligase 31, and Ligase 41 are shown in Figure 2. In Figure 2, lane M represents the RNA molecule marker, lane 1 represents the reaction system of Ligase 25, lane 2 represents the reaction system of Ligase 26, lane 3 represents the reaction system of Ligase 31, and lane 4 represents the reaction system of Ligase 41. 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 the figure, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, s in sequences such as "sAm" and "sGm" indicates thio modification 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 3, where the wavy line represents the base connected to the L96 group.

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

[0080] The ribonucleotides at positions 1-7 of substrate 2 have a 2' methoxy group modification.

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

[0082] The ribonucleotides at positions 1, 3, 4, 5, 6, 9, 11, 12, 13, and 15 of substrate 4 have a 2' methoxy group, and the ribonucleotides at positions 2, 7, 8, 10, and 14 have a 2' fluorine group. The 5' phosphate group on the 2nd and 3rd ribonucleotides has a thio group.

[0083] The positive chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the negative chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUf GmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

[0084] Table 2

[0085] In the representation of product yield, ND indicates no product generation was detected, "++" 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, 10 eq DTT (1 eq = 800 μM). The RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, and Ligase 42, which have high catalytic activity as described in Example 1, were added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. After the reaction, the protein was inactivated by heating at 80 °C for 5 min, and the precipitate was removed by centrifugation.

[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 50-70% (excluding the 70% endpoint), "+++" indicates a yield of 70-80%, and "++++" indicates a yield >80%.

[0092] The molecular weight of the positive chain product was determined by LC-MS to be 8705.03 and the molecular weight of the antisense chain product was 7627.16. The theoretical values ​​for the positive chain product were 8705.03±8 and the theoretical values ​​for the antisense chain product were 7627.16±8, indicating that Ligase 25 was linked to form Lumasilan. The LC-MS 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 from substrates 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 resulting reaction system was heated at 50 °C for 15 min to inactivate the protein, centrifuged at 12000 rpm to remove the precipitate, and the supernatant was purified using a Nano-Q column. The obtained product was subjected to gradient elution with NaCl, membrane desalting treatment (molecular weight cutoff 1 kDa), and lyophilized. The yield was calculated to be 87.53%, and the purity was 96.09%.

[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, with length in nt. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 9–14. The reaction system was set to 10 mL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 8 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 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 80.6%.

[0097] Four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0098] Table 4

[0099] 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 in sequences such as "sAm" and "sGm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0100] The ribonucleotides at positions 1, 2, 3, 4, 5, and 6 of substrate 5 have 2' methoxy modifications, the 5' phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications, and the ribonucleotides at positions 7 and 9 have 2' fluoride modifications.

[0101] The ribonucleotides at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 of substrate 6 have 2' methoxy modifications, and the ribonucleotides at positions 1 and 2 have 2' fluorine modifications.

[0102] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, and 7 of substrate 7 have 2' methoxy modifications, the ribonucleotides at positions 6 and 7 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 6 and 7 has 5' thio modifications.

[0103] The ribonucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, and 15 of substrate 8 have 2' methoxy modifications, the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.

[0104] The positive chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the negative chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUf GmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

[0105] Example 5

[0106] 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, with length in nt. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 9–14. The reaction system was set to 10 mL. The reaction system 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 8 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 catalyzed by Ligase 25 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 76.1%.

[0107] Six single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0108] Table 5

[0109] 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 in sequences such as "sAm" and "sGm" indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0110] The ribonucleotides at positions 1, 2, 3, 4 and 5 of substrate 9 have 2' methoxy modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.

[0111] The ribonucleotides at positions 1, 3, 7, and 8 of substrate 10 have 2' methoxy modifications, and the ribonucleotides at positions 2, 4, 5, and 6 have 2' fluorine modifications.

[0112] The ribonucleotides at positions 1-8 of substrate 11 have 2' methoxy modifications.

[0113] The ribonucleotides at positions 1, 2, 3, and 4 of substrate 12 have 2' methoxy modifications, and the 5' phosphate of the ribonucleotides at positions 3 and 4 has 5' thio modifications.

[0114] The ribonucleotides at positions 1, 3, 5, 6 and 7 of substrate 13 have 2' methoxy modifications, and the ribonucleotides at positions 2 and 4 have 2' fluorine modifications.

[0115] The ribonucleotides at positions 1, 3, 4, 5, 7, 8, 10, 11, and 12 of substrate 14 have 2' methoxy modifications, the ribonucleotides at positions 2, 6, 8, and 9 have 2' fluorine modifications, and the 5' phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.

[0116] The positive chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the negative chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUf GmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

[0117] Comparative Example 1

[0118] The average yield of the full-length Lumasilan product synthesized by solid-phase synthesis was 29.6%, with N+1 and N-1 impurities accounting for a total of 1.45%.

[0119] The yield of Lumasilan prepared using the enzyme-linked method of this invention was 71.91%, and the yield of the substrate used in solid-phase synthesis was 42.2% of the overall process yield. Multiplying these yields, the overall yield was 30.35%, 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 process of Lumasilan synthesis by solid-phase synthesis.

[0120] 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, Lumasilan is formed by catalyzing a single-stranded RNA fragment designed based on the Lumasilan sequence using RNA ligase, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with chemical synthesis methods, the preparation method of this application yields products with high purity, produces fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production.

[0121] 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 Lumasilan, characterized in that, The Lumasilan is a double-stranded siRNA composed of complementary sense and antisense strands, and 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 Lumasilan; 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 Lumasilan.

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

3. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the sense strand is SEQ ID NO: 14, and the nucleotide sequence of the antisense strand is SEQ ID NO:

15.

4. The preparation method according to claim 1, 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 5-14 nt; The antisense substrate fragment is 4-16 nt in length.

5. The preparation method according to claim 4, 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.

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

7. The preparation method according to claim 5, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is GmsAmsCmUmUmUmCfAmUf, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO: 14; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate fragment is AmAmGmUmCmsCmsAm.

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 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.

10. The preparation method according to claim 5, characterized in that, When both the sense substrate fragment and the antisense substrate fragment comprise two segments, the preparation method includes: 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 are mixed. 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, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are ligated to form the antisense strand. The sense strand and the antisense strand form the Lumasilan through complementary base pairing.

11. The preparation method according to claim 4, 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.

12. The preparation method according to claim 11, characterized in that, The nucleotide sequence of the first positive-strand substrate fragment is GmsAmsCmUmUm; The nucleotide sequence of the second positive-strand substrate fragment is UmCfAmUfCfCfUmGm; The nucleotide sequence of the third-positive strand substrate fragment is GmAmAmAmUmAmUmAm; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 22; The nucleotide sequence of the second antisense substrate fragment is AmUfGmAfAmAmGm; The nucleotide sequence of the third antisense substrate fragment is UmCmsCmsAm.

13. The preparation method according to claim 12, characterized in that, The preparation method includes: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment, and the RNA ligase; Under the catalysis of RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment, and the third sense strand substrate fragment are linked to form the sense strand, and the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment are linked to form the antisense strand. The sense strand and the antisense strand form the Lumasilan through complementary base pairing.

14. 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.

15. 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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