Method for preparing vutrisiran

Abstract

Provided is a method for preparing Vutrisiran. Vutrisiran is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method therefor comprises: mixing sense strand substrate fragments, antisense strand substrate fragments, and an RNA ligase, wherein the sense strand substrate fragments can form the sense strand, and the antisense strand substrate fragments can form the antisense strand. The sense strand substrate fragments and the antisense strand substrate fragments are linked by means of hydrogen bonds formed by base complementation, and terminal bases of the sense strand substrate fragments and the antisense strand substrate fragments are not linked to each other, to form a double-stranded nucleotide structure containing a nick. The bases at two ends of the nick are linked by means of a phosphodiester bond by using the RNA ligase to form Vutrisiran. The preparation method can solve the problem in the prior art that the purity of prepared Vutrisiran is relatively low.

Description

A method for preparing Vutrisiran

[0001] This application is based on and claims priority to Chinese application CN application number 2024109217313 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 Vutrisiran. 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] Vutrisiran is a gene-targeted therapy siRNA-based double-stranded RNA drug developed by Alnylam. It is used to treat hereditary transthyretin amyloidosis (hATTR) and was approved by the FDA in 2022 under the brand name Amvuttra.

[0005] Vutrisiran is primarily synthesized using 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 uvrisiran 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 uvrisiran is increasingly used in the treatment of hereditary transthyretin amyloidosis, its synthesis scale is limited by the limitations of synthetic equipment, resulting in high costs and hindering large-scale production. Therefore, there is a need to develop a more efficient method for uvrisiran synthesis. Summary of the Invention

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

[0007] To achieve the above objective, according to a first aspect of the present invention, a method for preparing Vutrisiran is provided, wherein the Vutrisiran 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 a positive strand, and the antisense strand substrate fragment can form an antisense strand; the positive strand substrate fragment and the antisense strand substrate fragment are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive strand substrate fragment and the antisense strand substrate fragment are not linked to each other, forming a double-stranded nucleotide structure containing a nick; using an RNA ligase to link the bases at both ends of the nick with phosphodiester bonds to form Vutrisiran; the bases at both ends of the nick are the 5' end and 3' end of different substrate fragments, respectively, with the 5' being a phosphate group and the 3' end being a hydroxyl group; using an RNA ligase to link the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form phosphodiester bonds to obtain Vutrisiran; the RNA ligase is a [SEQ ID NO] A protein having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 and having catalytic activity in forming phosphodiester bonds.

[0008] Furthermore, the nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO: 20.

[0009] 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 4-16 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 4-16 nt, more preferably 5-14 nt.

[0010] Furthermore, both the justice chain substrate fragment and the antisense chain substrate fragment include two segments. 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.

[0011] 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; under the catalysis of RNA ligase, the first and second sense strand substrate fragments are ligated to form a sense strand, and the first and second antisense strand substrate fragments are ligated to form an antisense strand; the sense and antisense strands form a virisiran through base complementarity pairing; preferably, both the sense and antisense strand substrate fragments consist of three fragments, the sense strand substrate fragments include a first sense strand substrate fragment, a second sense strand substrate fragment, and a third sense strand substrate fragment; the antisense strand substrate fragments include a first antisense strand substrate fragment, a second antisense strand substrate fragment, and a third antisense strand substrate fragment. The antisense substrate fragment; the preparation method includes: mixing a first sense substrate fragment, a second sense substrate fragment, a third sense substrate fragment, a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment; under the catalysis of RNA ligase, the first sense substrate fragment, the second sense substrate fragment, and the third sense substrate fragment are mixed and ligated to form a sense strand, and the first antisense substrate fragment, the second antisense substrate fragment, and the third sense substrate fragment are ligated to form an antisense strand, and the sense strand and antisense strand form Vutrisiran through base complementarity pairing; preferably, the sense substrate fragment and the antisense substrate fragment are annealed and then mixed with RNA ligase to obtain Vutrisiran.

[0012] Furthermore, both the sense and antisense substrate fragments comprise two segments. The 3' end of the first sense substrate fragment and the 5' end of the second sense substrate fragment are ligated together by RNA ligase to form the sense strand. Similarly, the 3' end of the first antisense substrate fragment and the 5' end of the second antisense substrate fragment are ligated together by RNA ligase to form the antisense strand. Preferably, the 5' end of the first sense substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense substrate fragment is a phosphate group, and the 3' end is an L96 group.

[0013] Further, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 5, and the nucleotide sequence of the second positive strand substrate fragment is CmCmAmAmGmAm; the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is AfUmCmCmCmAmsUmsCm. Preferably, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 9, and the sequence of the second positive strand substrate fragment is SEQ ID NO: 10; the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is AmAfUmCmCmCmAmsUmsCm.

[0014] Furthermore, both the sense and antisense substrate fragments comprise three segments: the first sense substrate fragment has the nucleotide sequence UmsGmsGmGmAmUm; the second sense substrate fragment has the nucleotide sequence UfUmCfAfUfGmUmAmAm; and the third sense substrate fragment has the nucleotide sequence CmCmAmAmGmAm. Preferably, the first antisense substrate fragment has the nucleotide sequence CmCmAmsUmsCm; the second antisense substrate fragment has the nucleotide sequence CmAmUmGmAfAmAfUmCm; and the third antisense substrate fragment has the nucleotide sequence UmsCfsUmUmGmGfUmUmAf.

[0015] Furthermore, the concentrations of the sense and antisense substrate fragments are each independently selected from 0.1 to 4.5 mM; preferably, 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.

[0016] Furthermore, 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.

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

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

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

[0020] Figure 2 shows the electrophoresis results of the RNA ligase Ligase 25 and Ligase 11 catalyzed products according to Example 1 of the present invention.

[0021] Figure 3 shows a schematic diagram of the structure of L96 according to an embodiment 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 uvurisiran 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 uvurisiran using enzymatic catalytic synthesis, thus proposing a series of protective solutions.

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

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

[0031] The aforementioned sense and antisense substrates can be synthesized chemically via solid-phase or liquid-phase methods.

[0032] In the above preparation method, Vutrisiran 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 double-stranded nucleotide with sticky ends. This double-stranded nucleotide with sticky ends can then bind to other substrates to form a double-stranded nucleotide with nicks. The RNA ligase can recognize the nicks in this double-stranded nucleotide and link the nicks with phosphodiester bonds to obtain the target product Vutrisiran. Preferably, the positive and negative substrate fragments are annealed and then mixed with RNA ligase to obtain Vutrisiran.

[0033] 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 Vutrisiran with a complete double-stranded structure.

[0034] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 28, and the nucleotide sequence of the antisense strand is SEQ ID NO: 29.

[0035] SEQ ID NO: 19:

[0036] UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm.

[0037] SEQ ID NO: 20:

[0038] UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAmAfUmCmCmCmAmsUmsCm.

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

[0040] In a preferred embodiment, the RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; or has more than 70% identity with any RNA ligase having the amino acid sequence shown in SEQ ID NO: 1, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more), and is an enzyme that catalyzes the formation of phosphodiester bonds.

[0041] 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.

[0042] SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

[0043] SEQ ID NO: 2: (Ligase 11, Thermococcus):

[0044] SEQ ID NO: 3: (Ligase 20, Archaea):

[0045] SEQ ID NO: 4: (Ligase 32, bacteria):

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

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

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

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

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

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

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

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

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

[0055] In this application, only the RNA ligase shown in SEQ ID NO: 1, or an enzyme with more than 70% identity with the RNA ligase shown in SEQ ID NO: 1, 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 Vutrisiran. In the relevant experiments of this application, the inventors obtained the aforementioned RNA ligase shown in SEQ ID NO: 1, which is capable of synthesizing Vutrisiran, 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 Vutrisiran, including but not limited to the RNA ligases shown in SEQ ID NO: 2 to SEQ ID NO: 4. In this application specification, only SEQ ID NO: 2 to SEQ ID NO: 4 are used as examples to illustrate this type of RNA ligase that does not have the activity of catalyzing the synthesis of Vutrisiran.

[0056] In a preferred embodiment, the positive chain substrate fragment includes two or more substrate fragments, and the negative chain substrate includes two or more substrate fragments; preferably, the length of the positive chain substrate fragment is 4-16 nt, more preferably 8-12 nt; preferably, the length of the negative chain substrate fragment is 4-16 nt, more preferably 5-14 nt.

[0057] In a preferred embodiment, both the sense and antisense substrate fragments comprise two fragments. The sense substrate fragment includes a first sense substrate fragment and a second sense substrate fragment, and the antisense substrate includes a first antisense substrate fragment and a second antisense substrate fragment. The preparation method includes: mixing the first sense substrate fragment, the second sense substrate fragment, the first antisense substrate fragment, and the second antisense substrate fragment; using RNA ligase to catalyze the ligation of the first sense substrate fragment and the second sense substrate fragment to form a sense strand; catalyzing the ligation of the first antisense substrate fragment and the second antisense substrate fragment to form an antisense strand; and the sense and antisense strands forming Vutrisiran through complementary base pairing. Preferably, both the sense and antisense substrate fragments comprise three segments: a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. The preparation method includes: mixing the first, second, and third sense substrate fragments, the first and second antisense substrate fragments, and the third antisense substrate fragment; catalyzing with RNA ligase, the first, second, and third sense substrate fragments are ligated to form the sense strand; the first, second, and third antisense substrate fragments are ligated to form the antisense strand; and the sense and antisense strands form Vutrisiran through base complementarity pairing. Preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain Vutrisiran.

[0058] In a preferred embodiment, both the sense and antisense substrate fragments comprise two segments. The 3' end of the first sense substrate fragment and the 5' end of the second sense substrate fragment are ligated together by RNA ligase to form a sense strand. Similarly, the 3' end of the first antisense substrate fragment and the 5' end of the second antisense substrate fragment are ligated together by RNA ligase to form an antisense strand. Preferably, in the above preparation method, the 5' end of the first sense substrate fragment is a 5' hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense substrate fragment is a phosphate group, and the 3' end is an L96 group; the 5' end of the first antisense substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; and the 5' end of the second antisense substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0059] In a preferred embodiment, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 5, and the nucleotide sequence of the second sense substrate fragment is CmCmAmAmGmAm; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense substrate fragment is AfUmCmCmCmAmsUmsCm.

[0060] 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 SEQ ID NO: 10; preferably, 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 AmAfUmCmCmCmAmsUmsCm.

[0061] Using the above-described preparation method and substrate fragments, uvurisiran can be prepared. However, it should be noted that the choice of substrate is not limited to the aforementioned substrate fragments; 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 uvurisiran but is not limited to different substrate connection positions. The above preparation method shows good ligation effects for both the sense and antisense strand sequences of uvurisiran. The number of sense or antisense substrates can include, but is not limited to, 2, 3, 4, or even more.

[0062] SEQ ID NO: 5: UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAm.

[0063] SEQ ID NO: 7: AfUmCmCmCmAmsUmsCm.

[0064] SEQ ID NO: 8: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAm.

[0065] SEQ ID NO: 9: UmsGmsGmGmAmUmUfUmCfAf.

[0066] SEQ ID NO: 10: UfGmUmAmAmCmCmAmAmGmAm.

[0067] SEQ ID NO: 12: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAf.

[0068] In a preferred embodiment, both the sense and antisense substrate fragments comprise three segments: the first sense substrate fragment has the nucleotide sequence UmsGmsGmGmAmUm; the second sense substrate fragment has the nucleotide sequence UfUmCfAfUfGmUmAmAm; and the third sense substrate fragment has the nucleotide sequence CmCmAmAmGmAm. Preferably, the first antisense substrate fragment has the nucleotide sequence CmCmAmsUmsCm; the second antisense substrate fragment has the nucleotide sequence CmAmUmGmAfAmAfUmCm; and the third antisense substrate fragment has the nucleotide sequence UmsCfsUmUmGmGfUmUmAf.

[0069] In a preferred embodiment, the concentrations of the sense substrate fragment and the antisense substrate fragment are each independently selected from 0.1 to 4.5 mM; preferably, 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.

[0070] In a preferred embodiment, the reaction temperature of the preparation method is 10–40°C, more preferably 15–30°C; preferably, the reaction time of the preparation method is 2–48 h, more preferably 12–24 h.

[0071] The concentrations of the aforementioned sense and antisense substrate fragments are each selected from, but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mM; the reaction temperatures of the aforementioned preparation methods are, but not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 °C; and the reaction times of the aforementioned preparation methods are, but not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, or 48 h.

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

[0073] Example 1

[0074] Substrate fragments 1-4 were added to a clean reagent bottle in equimolar proportions and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrate fragments 1-4 are shown in Table 1, with length in nt. The substrate fragment mixture was annealed to obtain an RNA fragment mixture. The reaction system was set to 10 μL, including 100 μM RNA fragment mixture, 50 mM Tris-HCl, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM), and RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 at a final concentration of 0.2 mg / mL. 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. A schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

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

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

[0077] Table 1

[0078] In these sequences, "m" after A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide, "f" indicates a 2' fluorine modification of the ribonucleotide, and "sGm," "sUm," etc., indicate a thiolation modification of the 5' phosphate group of the ribonucleotide. "L96" indicates that the 3' end of the positive strand is modified with an L96 group. The structure of the L96 group is shown in Figure 3, where the wavy line represents the base linked to the L96 group.

[0079] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, and 15 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, 2, 3, 4, 5, and 6 of substrate 2 have a 2' methoxy group modification.

[0081] The ribonucleotides at positions 2, 3, 4, 5, 6, 7, and 8 of substrate 3 have a 2' methoxy group, and the ribonucleotide at position 1 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, 7, 8, 10, 11, 12, 13, and 15 of substrate 4 have a 2' methoxy group, and the ribonucleotides at positions 2, 6, 9, and 14 have a 2' fluorine group. The 5' phosphate group on the ribonucleotides at positions 2 and 3 has a thio group.

[0083] The positive chain of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmC mAmAmGmAm (SEQ ID NO: 19), and the negative chain is UmsCfsUmUmGmGfUmUmAfCmAmUmGmA fAmAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[0084] Table 2

[0085] In the representation of product yield in Table 2, "++" indicates a yield of 25% to 50% (excluding the 50% endpoint).

[0086] The grayscale data of the product and substrate were obtained by grayscale analysis of the Urea-PAGE gel electrophoresis results image. In this example, the yield calculation formula is: Yield = Product grayscale data / (Product grayscale data + Substrate grayscale data).

[0087] Example 2

[0088] Substrate fragments 1-4 were added to a clean reagent bottle in equimolar proportions and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrates 1-4. The reaction system was set to 50 μL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 100 eq MgCl2, and 10 eq DTT (1 eq = 800 μM). Ligase 25, the RNA ligase with high catalytic activity shown in Example 1, was added to each ligase to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. After the reaction, the protein was inactivated by heating at 80 °C for 5 min, and the precipitate was removed by centrifugation.

[0089] The product obtained by Ligase 25 catalysis was detected by HPLC and LC-MS, as shown in Figure 4. The yield was calculated based on the statistical results of the target peak area in the HPLC results, as shown in Table 3.

[0090] Table 3

[0091] In the representation of product yield, "++" indicates a yield of 50-90% (excluding the 90% endpoint).

[0092] The molecular weight of the positive-sense product was determined by LC-MS to be 8784.08, and the molecular weight of the antisense product was 7553.13. The theoretical values ​​for the positive-sense product were 8784.06±8 and the theoretical values ​​for the antisense product were 7552.13±8, indicating that Ligase 25 was linked to generate Vutrisiran. The detection results are shown in Figure 5.

[0093] Example 3

[0094] Substrate 1–4 were added to a clean reagent bottle in equimolar proportions and mixed thoroughly to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The substrate mixture was annealed to obtain a mixture of RNA fragments of substrate 1–4. The reaction system was set to 10 mL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), and RNA ligase Ligase 25 to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was heated at 50℃ for 10-20 min to inactivate the protein, and the precipitate was removed by centrifugation at 12000 rpm. The supernatant was purified using a Nano-Q column, and the obtained product was eluted with NaCl gradient, desalted by membrane coating (molecular weight cutoff of 1 kDa), and then lyophilized. The yield was calculated to be 71.91% and the purity was 98.39%.

[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 50 μL and included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was inactivated by ligase at 80℃ for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product obtained by Ligase 25 catalysis was 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 proportion of the target peak in the sample was 90.4%, that is, the yield was +++.

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

[0100] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, and 8 of substrate 5 have a 2' methoxy group, and the ribonucleotides at positions 7, 9, and 10 have a 2' fluorine group. The 5' phosphate group at positions 2 and 3 has a thio group.

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

[0102] The ribonucleotides at positions 1, 3, 4, 5, 6, 7, and 8 of substrate 7 have a 2' methoxy group, and the ribonucleotide at position 2 has a 2' fluorine group. The 5' phosphate groups at positions 8 and 9 have a thio group.

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

[0104] The positive chain of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmC mAmAmGmAm (SEQ ID NO: 19), and the negative chain is UmsCfsUmUmGmGfUmUmAfCmAmUmGmA fAmAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[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 5–8. The reaction system was set to 50 μL. 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 25 to a final concentration of 0.2 mg / mL. The reaction system was incubated at 16 °C for 16 h. The obtained reaction system was inactivated by ligase at 80℃ for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product obtained by Ligase 25 catalysis was detected by HPLC. The enzyme activity was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the proportion of the target peak in the sample was 93.1%, that is, the yield was +++.

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

[0110] The ribonucleotides at positions 1, 2, 3, 4, 5, and 6 of substrate 9 have a 2' methoxy group, and the 5' phosphate group at positions 2 and 3 has a thio group.

[0111] The ribonucleotides at positions 2, 6, 7, 8, and 9 of substrate 10 have a 2' methoxy modification, and the ribonucleotides at positions 1, 3, 4, and 5 have a 2' fluorine modification.

[0112] The ribonucleotides at positions 1, 2, 3, 4, 5, and 6 of substrate 11 have a 2' methoxy group modification.

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

[0114] The ribonucleotides at positions 1, 2, 3, 4, 6, 8, and 9 of substrate 13 have 2' methoxy modifications, and the ribonucleotides at positions 5 and 7 have 2' fluorine modifications.

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

[0116] The positive chain of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAmGmAm (SEQ ID NO: 19), and the negative chain is UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[0117] Comparative Example 1

[0118] The average yield of the full-length Vutrisiran product synthesized by solid-phase synthesis was 30.1%, with N+1 and N-1 impurities accounting for a total of 1.49%.

[0119] The yield of Vutrisiran prepared using the enzyme-linked method of this invention was 76.45%, while the yield of the substrate used in solid-phase synthesis was 41.5%, resulting in an overall yield of 31.7%, 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 0.37%, which is lower than the proportion of such impurities in the solid-phase synthesis of Vutrisiran.

[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, Vutrisiran is formed by catalyzing a single-stranded RNA fragment designed based on the Vutrisiran 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 Vutrisiran, characterized in that, The Vutrisiran is a double-stranded RNA composed of complementary sense and antisense strands. The preparation method includes: A positive sense substrate fragment, an antisense substrate fragment, and an RNA ligase are mixed, wherein the positive sense substrate fragment is capable of forming the positive sense strand, and the antisense substrate fragment is capable of forming the antisense strand; The positive and negative substrate fragments are connected by hydrogen bonds formed by complementary bases. The head and tail bases of the positive and negative substrate fragments are not connected to each other, forming a double-stranded nucleotide structure with notches. The bases at both ends of the notch are linked by phosphodiester bonds using the RNA ligase to form the Vutrisiran; 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 the Vutrisiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is a protein having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 and having 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: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO:

20.

4. The preparation method according to any one of claims 1-3, characterized in that, The justice chain substrate fragment includes two or more fragments, and the antisense chain substrate fragment includes two or more fragments; The length of the substrate segment of the justice chain is 4-16 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. The preparation method includes: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, and the second antisense strand substrate fragment; under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are ligated to form the sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are ligated to form the antisense strand, and the sense strand and the antisense strand form the Vutrisiran through complementary base pairing.

6. The preparation method according to claim 4, characterized in that, Both the justice chain substrate fragment and the antisense chain substrate fragment include three segments. The justice chain substrate fragment includes a first justice chain substrate fragment, a second justice chain substrate fragment, and a third justice chain substrate fragment. The antisense chain substrate fragment includes a first antisense chain substrate fragment, a second antisense chain substrate fragment, and a third antisense chain substrate fragment. The preparation method includes: mixing the first positive strand substrate fragment, the second positive strand substrate fragment, the third positive strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment; under the catalysis of the RNA ligase, the first positive strand substrate fragment, the second positive strand substrate fragment, and the third positive strand substrate fragment are mixed and ligated to form the positive strand, and the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third positive strand substrate fragment are ligated to form the antisense strand, and the positive strand and the antisense strand form the Vutrisiran through base complementary pairing.

7. The preparation method according to claim 5, characterized in that, Both the sense strand substrate fragment and the antisense strand substrate fragment comprise two fragments. 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.

8. The preparation method according to claim 7, 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.

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

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

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

11. The preparation method according to claim 6, characterized in that, The nucleotide sequence of the first positive-strand substrate fragment is UmsGmsGmGmAmUm; The nucleotide sequence of the second positive-strand substrate fragment is UfUmCfAfUfGmUmAmAm; The nucleotide sequence of the third positive-strand substrate fragment is CmCmAmAmGmAm; The nucleotide sequence of the first antisense substrate fragment is CmCmAmsUmsCm; The nucleotide sequence of the second antisense substrate fragment is CmAmUmGmAfAmAfUmCm; The nucleotide sequence of the third antisense substrate fragment is UmsCfsUmUmGmGfUmUmAf.

12. 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 to 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°C; The reaction time for the preparation method is 2–48 h.

Images