A kind of preparation method of Vutrisiran

Abstract

The present invention provides a method for preparing Vutrisiran. The Vutrisiran is a double-stranded RNA composed of a complementary sense strand and an antisense strand; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, forming a double-stranded nucleotide structure containing a nick; and using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Vutrisiran. The method can solve the problem of low purity of Vutrisiran prepared in the prior art, and is applicable to the field of drug biosynthesis technology.

Description

Technical Field

[0001] The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing Vutrisiran. Background Art

[0002] In the prior art, diseases caused by gene expression and mutation cannot be cured by traditional small molecule drugs, and such diseases can only be treated by gene expression intervention. siRNA is a double-stranded RNA oligonucleotide with a length of 19-25nt, which can specifically bind to the mRNA of the same target gene, and by blocking the translation process, it can achieve the effect of hindering the expression of the target gene, that is, by mediating RNA interference to cause gene silencing. The research and development of siRNA drugs has brought huge development prospects to gene therapy, and has been widely used in the field of disease treatment. siRNA acts on mRNA, and the druggable target is significantly larger than that of traditional small molecule drugs whose action sites are proteins, and it can act on new targets by changing the sequence. Intervene in gene expression.

[0003] Vutrisiran is a gene-targeted siRNA-type double-stranded RNA drug developed by Alnylam for the treatment of hereditary transthyretin amyloidosis (hATTR). It was approved by the FDA for marketing in 2022 under the trade name Amvuttra.

[0004] The synthesis method of Vutrisiran mainly uses a chemical solid phase synthesis method for preparation. In the synthesis method, a solid phase carrier is used, and the synthesis is cyclically performed by the phosphoramidite triester method. After the synthesis cycle is completed, the Vutrisiran chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain the target product. In this preparation method, the yield of product synthesis decreases with the increase of the synthesis chain length. Impurities generated during the synthesis process, such as impurities with one more nucleotide than the target sequence (N+1 impurities) or impurities with one less nucleotide than the target sequence (N-1 impurities), will also increase with the increase of the synthesis chain length. It is not easy to remove, resulting in a complex purification process and low efficiency. As Vutrisiran is increasingly widely used in the treatment of hereditary transthyretin amyloidosis, its synthesis scale is limited by the limitations of the synthesis instrument, the cost is high, and it is difficult to scale up production. Therefore, it is necessary to develop a more efficient Vutrisiran synthesis method. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for preparing Vutrisiran to solve the problem of low purity of Vutrisiran prepared in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a method for preparing Vutrisiran is provided, wherein Vutrisiran is a double-stranded RNA composed of a complementary sense chain and an antisense chain; the preparation method comprises: mixing a sense chain substrate fragment, an antisense chain substrate fragment and an RNA ligase, wherein the sense chain substrate fragment can form a sense chain, and the antisense chain substrate fragment can form an antisense chain; the sense chain substrate fragment and the antisense chain substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense chain substrate fragment and the antisense chain substrate fragment are not connected to each other, so as to form a double-stranded nucleotide structure containing a nick; using RNA ligase to connect the bases at both ends of the nick by phosphodiester bonds to form Vutrisiran; the bases at both ends of the nick are the 5' end and the 3' end of different substrate fragments, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Vutrisiran; the RNA ligase is a polypeptide having SEQ ID A protein having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having 70% or more identity with the RNA ligase shown in SEQ ID NO: 1 and having activity of catalyzing the formation of phosphodiester bonds.

[0007] Furthermore, the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO: 20.

[0008] Furthermore, the sense strand substrate fragment includes 2 or more strands, and the antisense strand substrate fragment includes 2 or more strands; preferably, the length of the sense strand substrate fragment is 4-16 nt, more preferably 8-12 nt; preferably, the length of the antisense strand substrate fragment is 4-16 nt, more preferably 5-14 nt.

[0009] Further, the sense strand substrate fragment and the antisense strand substrate fragment each include 2, the sense strand substrate fragment includes a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment includes a first antisense strand substrate fragment and a second antisense strand substrate fragment;

[0010] The preparation method comprises: mixing a first sense chain substrate fragment, a second sense chain substrate fragment, a first antisense chain substrate fragment and a second antisense chain substrate fragment; under the catalytic action of RNA ligase, the first sense chain substrate fragment and the second sense chain substrate fragment are connected to form a sense chain, the first antisense chain substrate fragment and the second antisense chain substrate fragment are connected to form an antisense chain, and the sense chain and the antisense chain form Vutrisiran through base complementary pairing; preferably, the sense chain substrate fragment and the antisense chain substrate fragment each include 3, the sense chain substrate fragment includes the first sense chain substrate fragment, the second sense chain substrate fragment and the third sense chain substrate fragment; the antisense chain substrate fragment includes the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third Antisense strand substrate fragment; the preparation method comprises: mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a third sense strand substrate fragment, a first antisense strand substrate fragment, a second antisense strand substrate fragment and a third antisense strand substrate fragment; under the catalytic action of RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are mixed and connected to form a sense strand, the first antisense strand substrate fragment, the second antisense strand substrate fragment and the third sense strand substrate fragment are connected to form an antisense strand, and the sense strand and the antisense strand form Vutrisiran through base complementary pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain Vutrisiran.

[0011] Further, the sense chain substrate fragment and the antisense chain substrate fragment each include two strands, the 3' end of the first sense chain substrate fragment and the 5' end of the second sense chain substrate fragment are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate fragment and the 5' end of the second antisense chain substrate fragment are connected under the catalysis of RNA ligase to form an antisense chain; preferably, the 5' end of the first sense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate fragment is a phosphate group, and the 3' end is an L96 group; preferably, the 5' end of the first antisense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0012] Further, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 5, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 6; the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8, and the second antisense strand substrate is the nucleotide sequence shown in the nucleotide sequence of the sequence fragment of SEQ ID NO: 7. Preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11.

[0013] Furthermore, the sense chain substrate fragment and the antisense chain substrate fragment each include 3 fragments, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16.

[0014] Furthermore, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1 to 4.5 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT.

[0015] 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 2-48h, more preferably 12-24h.

[0016] By applying the technical solution of the present invention and utilizing the above-mentioned preparation method, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form the Vutrisiran sense strand, and the antisense strand substrate fragments are connected to form the Vutrisiran antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method for preparing Vutrisiran by chemical synthesis, the product obtained by the preparation method of the present application has high purity, less impurities, a simple preparation process, mild reaction conditions, low organic reagent dosage, reduced production costs, and is easy to achieve scale-up industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

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

[0019] Figure 2 The electrophoresis results of the products catalyzed by RNA ligases Ligase 25 and Ligase 11 according to Example 1 of the present invention are shown.

[0020] Figure 3 FIG. 4 is a schematic diagram showing the structure of L96 according to an embodiment of the present invention.

[0021] Figure 4 The figure shows the HPLC detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

[0022] Figure 5 The figure shows the LC-MS detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] Terminology explanation:

[0025] N+1 impurity: A nucleic acid impurity that has a single nucleotide extra attached compared to the target synthetic sequence.

[0026] N-1 impurity: A nucleic acid impurity that has a single nucleotide missing compared to the target synthetic sequence.

[0027] As mentioned in the background technology, the prior art uses a chemical synthesis method for the preparation of Vutrisiran, which is not only complicated and costly, but also produces a large number of N+1 and N-1 impurities, which affect the subsequent purification of the product. In this application, the inventor attempts to develop a method for preparing Vutrisiran, using enzyme-catalyzed synthesis to prepare Vutrisiran, and thus proposes a series of protection schemes of this application.

[0028] In a first typical embodiment of the present application, a method for preparing Vutrisiran is provided, which is a double-stranded RNA composed of a complementary sense chain and an antisense chain; the preparation method comprises: mixing a sense chain substrate fragment, an antisense chain substrate fragment and an RNA ligase, wherein the sense chain substrate fragment can form a sense chain, and the antisense chain substrate fragment can form an antisense chain; the sense chain substrate fragment and the antisense chain substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense chain substrate fragment and the antisense chain substrate fragment are not connected to each other, so as to form a double-stranded nucleotide structure containing a nick; using RNA ligase to connect the bases at both ends of the nick by phosphodiester bonds to form Vutrisiran; the bases at both ends of the nick are the 5' end and the 3' end of different substrate fragments, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Vutrisiran; the RNA ligase is a polypeptide having SEQ ID A protein having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having 70% or more identity with the RNA ligase shown in SEQ ID NO: 1 and having activity of catalyzing the formation of phosphodiester bonds.

[0029] In the above preparation method, the sense strand substrate fragment is a nucleotide sequence that can form two or more sense strands, that is, multiple nucleotide sequences of the sense strand substrate fragment can be spliced ​​to form a sequence identical to the sense strand sequence, and the difference from the sense strand is that there are nicks between the sense strand substrate fragments and they are not connected by phosphodiester bonds. Similarly, the antisense strand substrate fragment and the antisense strand have the above characteristics. Two or more sense strand substrate fragments or antisense strand substrate fragments are connected by phosphodiester bonds using RNA ligase to obtain the sense strand and antisense strand of Vutrisiran.

[0030] The sense strand substrate and antisense strand substrate can be synthesized by solid phase chemical synthesis or liquid phase chemical synthesis.

[0031] In the above-mentioned preparation method, the sense strand substrate fragment and the antisense strand substrate fragment are mixed with RNA ligase to prepare Vutrisiran. In this preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementary paired to form a double-stranded nucleotide containing a sticky end, and the double-stranded nucleotide containing a sticky end continues to bind to other substrates to form a double-stranded nucleotide containing a nick, and RNA ligase can recognize the nick of such a double-stranded nucleotide and connect the nick with a phosphodiester bond, thereby preparing the target product Vutrisiran; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and mixed with RNA ligase to obtain Vutrisiran.

[0032] In the above preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be mixed and annealed first, and the sense strand substrate fragment and the antisense strand substrate fragment can form a double-stranded RNA structure through base complementary pairing, and there are nicks between different substrate fragments in the double-stranded RNA structure. Then, the annealed reaction system is mixed with RNA ligase, and the RNA ligase is used to connect the phosphate groups and hydroxyl groups on both sides of the nick with phosphodiester bonds to repair the nick, thereby obtaining the target product Vutrisiran with a complete double-stranded structure.

[0033] In a preferred embodiment, the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO:28, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO:29.

[0034] SEQ ID NO: 19:

[0035] UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm.

[0036] SEQ ID NO: 20:

[0037] UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAmAfUmCmCmCmAmsUmsCm.

[0038] In the present 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 "sGm", "sUm" and the like indicates thio modification of the 5' phosphate of the ribonucleotide.

[0039] In a preferred embodiment, the RNA ligase is an RNA ligase having an amino acid sequence as shown in SEQ ID NO: 1; or an enzyme having more than 70% identity with any RNA ligase of the amino acid sequence as shown in SEQ ID NO: 1, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (such as 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 activity of catalyzing phosphodiester bond formation.

[0040] The above-mentioned RNA ligases are all able to recognize the double-stranded structure with nicks formed by complementary pairing of substrate fragments, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

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

[0042] MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGT LDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.

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

[0044] MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD。

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

[0046] MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH。

[0047] SEQ ID NO: 4: (Ligase 32, bacteria):

[0048] MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAE EYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.

[0049] Identity in this application 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.

[0050] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (for example, 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) identity and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are likely to be the same as the proteins provided by the above sequences.

[0051] As used herein, the amino acid residues are abbreviated 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).

[0052] Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0053] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0054] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0055] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0056] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0057] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62" scoring matrix in the prior art.

[0058] In the present application, the phosphate group and the hydroxyl group of the substrate in the present application can only be catalyzed by 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 to form a phosphodiester bond to obtain the product Vutrisiran. In the relevant experiments of the present application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 that can synthesize Vutrisiran by screening from a large number of enzymes. The negative results, which accounted for a large proportion of the experiments, showed that most RNA ligases were 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 the present application specification, only SEQ ID NO: 2 to SEQ ID NO: 4 are used as examples to reflect this type of RNA ligase that has no catalytic activity in Vutrisiran synthesis.

[0059] In a preferred embodiment, the sense chain substrate fragment includes 2 or more substrate fragments, and the antisense chain substrate includes 2 or more substrate fragments; preferably, the length of the sense chain substrate fragment is 4-16nt, more preferably 8-12nt; preferably, the length of the antisense chain substrate fragment is 4-16nt, more preferably 5-14nt.

[0060] In a preferred embodiment, the sense chain substrate fragment and the antisense chain substrate fragment each include 2, the sense chain substrate fragment includes a first sense chain substrate fragment and a second sense chain substrate fragment, and the antisense chain substrate includes a first antisense chain substrate fragment and a second antisense chain substrate fragment; the preparation method includes: mixing the first sense chain substrate fragment, the second sense chain substrate fragment, the first antisense chain substrate fragment and the second antisense chain substrate fragment, using RNA ligase to catalyze the connection of the first sense chain substrate fragment and the second sense chain substrate fragment to form a sense chain, catalyzing the connection of the first antisense chain substrate fragment and the second antisense chain substrate fragment to form an antisense chain, and the sense chain and the antisense chain form Vutrisiran through base complementary pairing. Preferably, the sense chain substrate fragment and the antisense chain substrate fragment each include 3, the sense chain substrate fragment includes the first sense chain substrate fragment, the second sense chain substrate fragment and the third sense chain substrate fragment; the antisense chain substrate fragment includes the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third antisense chain substrate fragment; the preparation method comprises: mixing the first sense chain substrate fragment, the second sense chain substrate fragment, the third sense chain substrate fragment, the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third antisense chain substrate fragment, under the catalytic action of RNA ligase, the first sense chain substrate fragment, the second sense chain substrate fragment and the third sense chain substrate fragment are mixed and connected to form the sense chain, the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third sense chain substrate fragment are connected to form the antisense chain, and the sense chain and the antisense chain form Vutrisiran through base complementary pairing; preferably, the sense chain substrate fragment and the antisense chain substrate fragment are annealed and then mixed with RNA ligase to obtain Vutrisiran.

[0061] In a preferred embodiment, the sense chain substrate fragment and the antisense chain substrate fragment each include 2 strands, the 3' end of the first sense chain substrate fragment and the 5' end of the second sense chain substrate fragment are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate fragment and the 5' end of the second antisense chain substrate fragment are connected under the catalysis of RNA ligase to form an antisense chain; preferably, in the above preparation method, the 5' end of the first sense chain substrate fragment is a 5' hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate fragment is a phosphate group, and the 3' end is an L96 group; the 5' end of the first antisense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0062] In a preferred embodiment, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 5, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 6; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 7.

[0063] In a preferred embodiment, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO:9, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO:10; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO:12, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO:11.

[0064] Vutrisiran can be prepared using the above preparation method and the substrate fragments shown in SEQ ID NO: 5-SEQ ID NO: 8 or SEQ ID NO: 9-SEQ ID NO: 12. However, it should be noted that the selection of substrates is not limited to the substrate fragments shown in SEQ ID NOs: 5-8 or SEQ ID NOs: 9-12. All substrates that can be combined to form a sense chain and an antisense chain can be applied to the above preparation method. The above preparation method is suitable for the preparation of Vutrisiran but is not limited to the difference in substrate connection positions. The above preparation has a good connection effect for the connection of the sense chain sequence and the antisense chain sequence of Vutrisiran. The number of sense chain substrates or antisense chain substrates includes but is not limited to 2, 3, 4 or even more.

[0065] SEQ ID NO: 5: UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAm.

[0066] SEQ ID NO: 6: CmCmAmAmGmAmL96.

[0067] SEQ ID NO: 7: AfUmCmCmCmAmsUmsCm.

[0068] SEQ ID NO: 8: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAm.

[0069] SEQ ID NO: 9: UmsGmsGmGmAmUmUfUmCfAf.

[0070] SEQ ID NO: 10: UfGmUmAmAmCmCmAmAmGmAm.

[0071] SEQ ID NO: 11: AmAfUmCmCmCmAmsUmsCm.

[0072] SEQ ID NO: 12: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAf.

[0073] In a preferred embodiment, the sense chain substrate fragment and the antisense chain substrate fragment each include 3 fragments, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16.

[0074] SEQ ID NO: 13: UmsGmsGmGmAmUm.

[0075] SEQ ID NO: 14: UfUmCfAfUfGmUmAmAm.

[0076] SEQ ID NO: 15: CmCmAmAmGmAm.

[0077] SEQ ID NO: 16: CmCmAmsUmsCm.

[0078] SEQ ID NO: 17: CmAmUmGmAfAmAfUmCm.

[0079] SEQ ID NO: 18: UmsCfsUmUmGmGfUmUmAf.

[0080] In a preferred embodiment, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1 to 4.5 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT.

[0081] 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 2-48h, more preferably 12-24h.

[0082] The concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are each selected from, including 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 temperature of the preparation method includes but is not limited to 10, 15, 16, 20, 25, 30, 35 or 40°C; the reaction time of the preparation method includes but is not limited to 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45 or 48 h.

[0083] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0084] Example 1

[0085] Add substrate fragments 1 to 4 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5 mM. The sequences of substrate fragments 1 to 4 are shown in Table 1, and the length unit is nt. After the substrate fragment mixture is annealed, an RNA fragment mixture is obtained. The reaction system is set to 10 μL, and the reaction system includes 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 are added at a final concentration of 0.2 mg / mL. The reaction system was reacted at 16 ° C for 16 hours. The obtained reaction system was inactivated by 80 ° C for 5 minutes, and the precipitate was removed by centrifugation at 12000 rpm. The schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1 shown.

[0086] The products catalyzed by RNA ligase Ligase 25, Ligase 11, Ligase 20 and Ligase 32 were subjected to SDS-PAGE detection. The electrophoresis results of the products catalyzed by RNA ligase Ligase 25 and Ligase 11 are shown in the figure below. Figure 2 As shown, Figure 2 Lane M represents the RNA molecule standard (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 results of the target band in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0087] Four single-stranded RNA fragments were prepared using a solid phase synthesis method.

[0088] Table 1

[0089] .

[0090] Among them, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, and s in the sequences of "sGm", "sUm" and the like indicates thio modification of the 5' phosphate of the ribonucleotide. L96 indicates that the 3' end of the sense strand is modified with the L96 group. The structure of the L96 group is as follows Figure 3 As shown, the wavy line indicates the base to which the L96 group is connected.

[0091] Substrate 1 has 2' methoxy modifications at ribonucleotide positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14 and 15, and 2' fluorine modifications at ribonucleotide positions 7, 9, 10 and 11. The 5' phosphates on ribonucleotide positions 2 and 3 have thio modifications.

[0092] The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 2 have 2' methoxy modifications.

[0093] The ribonucleotides at positions 2, 3, 4, 5, 6, 7 and 8 of substrate 3 have 2' methoxy modifications, and the ribonucleotide at position 1 has a 2' fluorine modification. The 5' phosphates at positions 7 and 8 have thio modifications.

[0094] Substrate 4 has 2' methoxy modifications at ribonucleotides 1, 3, 4, 5, 7, 8, 10, 11, 12, 13 and 15, and 2' fluorine modifications at ribonucleotides 2, 6, 9 and 14. The 5' phosphates on ribonucleotides 2 and 3 have thio modifications.

[0095] The sense strand of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm (SEQ ID NO: 19), and the antisense strand is UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAmAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[0096] Table 2

[0097] .

[0098] In the expression of product yield in Table 2, “++” means the yield is 25-50% (excluding the endpoint value of 50%).

[0099] The grayscale data of the product and substrate are obtained by grayscale analysis of the Urea-PAGE gel electrophoresis result image. The yield calculation formula in this embodiment is: yield = product grayscale data / (product grayscale data + substrate grayscale data).

[0100] Example 2

[0101] Add the substrate fragments 1 to 4 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5mM. After the substrate mixture is annealed, a mixture of RNA fragments of substrates 1 to 4 is obtained. The reaction system is set to 50μL, and the reaction system includes 800μM RNA fragment mixture, 50mM Tris-HCl, 4eq ATP, 100eq MgCl2, 10eq DTT (1eq=800μM), and the RNA ligase Ligase 25 with high catalytic activity in Example 1 is added at a final concentration of 0.2mg / mL. The reaction system is reacted at 16℃ for 16h. After the reaction is completed, heat at 80℃ for 5min to inactivate the protein, and centrifuge to remove the precipitate.

[0102] The product catalyzed by Ligase 25 was detected by HPLC and LC-MS. Figure 4 The yield was calculated based on the statistical results of the target peak area in the HPLC results, and the results are shown in Table 3.

[0103] Table 3

[0104] .

[0105] In the expression of product yield, “++” means the yield is 50~90% (excluding the 90% endpoint).

[0106] The molecular weight of the sense chain product was 8784.08 and the molecular weight of the antisense chain product was 7553.13 by LC-MS. The theoretical values ​​of the sense chain product were 8784.06±8 and 7552.13±8, respectively, indicating that Ligase 25 ligated Vutrisiran. The test results are shown in the figure below. Figure 5 shown.

[0107] Example 3

[0108] Substrates 1 to 4 were added to a clean reagent bottle in an equimolar ratio and mixed to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. After the substrate mixture was annealed, a mixture of RNA fragments of substrates 1 to 4 was obtained. The reaction system was set to 10 mL, and 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 was added at a final concentration of 0.2 mg / mL. The reaction system was reacted at 16 ° C for 16 h. The obtained reaction system was heated at 50 ° C 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 gradient eluted with NaCl, desalted by membrane package (molecular weight cutoff 1 kDa), and the product was lyophilized. After calculation, the yield was 71.91% and the purity was 98.39%.

[0109] Example 4

[0110] Add substrates 5 to 8 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5mM. The sequences of substrates 5 to 8 are shown in Table 4, and the length unit is nt. After the substrate mixture is annealed, the RNA fragment mixture of substrates 9 to 14 is obtained. The reaction system is set to 50μL, and the reaction system includes 800μM RNA fragment mixture, 50mM Tris-HCl, 4eq ATP, 12.5eq MgCl2, 1.25eq DTT (1eq=800μM); add RNA ligase Ligase 25 with a final concentration of 0.2mg / mL. The reaction system is reacted at 16℃ for 16h. The obtained reaction system was inactivated by 80°C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The enzyme activity was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 90.4% in the sample, that is, the yield was +++.

[0111] Four single-stranded RNA fragments were prepared using a solid phase synthesis method.

[0112] Table 4

[0113] .

[0114] The 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 the s in the sequences "sGm", "sUm" etc. indicates a thio modification of the 5' phosphate of the ribonucleotide.

[0115] Substrate 5 has 2' methoxy modifications at ribonucleotide positions 1, 2, 3, 4, 5, 6 and 8, and 2' fluorine modifications at ribonucleotide positions 7, 9 and 10. The 5' phosphates on ribonucleotide positions 2 and 3 have thio modifications.

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

[0117] The ribonucleotides at positions 1, 3, 4, 5, 6, 7 and 8 of substrate 7 have 2' methoxy modifications, and the ribonucleotide at position 2 has a 2' fluorine modification. The 5' phosphates at positions 8 and 9 have thio modifications.

[0118] Substrate 8 has 2' methoxy modifications at ribonucleotides 1, 3, 4, 5, 7, 8, 10, 11, 12 and 13, and 2' fluorine modifications at ribonucleotides 2, 6, 9 and 14. The 5' phosphates on ribonucleotides 2 and 3 have thio modifications.

[0119] The sense strand of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm (SEQ ID NO: 19), and the antisense strand is UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAmAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[0120] Example 5

[0121] Add substrates 9 to 14 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5 mM. The sequences of substrates 9 to 14 are shown in Table 5, and the length unit is nt. After the substrate mixture is annealed, a mixture of RNA fragments of substrates 5 to 8 is obtained. The reaction system is set to 50 μL. The reaction system includes 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 is added at a final concentration of 0.2 mg / mL. The reaction system is reacted at 16 ° C for 16 hours. The obtained reaction system was inactivated by 80°C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The enzyme activity was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 93.1% in the sample, that is, the yield was +++.

[0122] Six single-stranded RNA fragments were prepared using solid phase synthesis.

[0123] Table 5

[0124] .

[0125] The 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 the s in the sequences "sGm", "sUm" etc. indicates a thio modification of the 5' phosphate of the ribonucleotide.

[0126] The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 9 have 2' methoxy modifications, and the 5' phosphates on the ribonucleotides at positions 2 and 3 have thio modifications.

[0127] The ribonucleotides at positions 2, 6, 7, 8 and 9 of substrate 10 have 2' methoxy modifications, and the ribonucleotides at positions 1, 3, 4 and 5 have 2' fluorine modifications.

[0128] The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 11 have 2' methoxy modifications.

[0129] The ribonucleotides at positions 1, 2, 3, 4 and 5 of substrate 12 have 2' methoxy modifications, and the 5' phosphates on positions 4 and 5 have thio modifications.

[0130] 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.

[0131] The substrate 14 has 2' methoxy modifications at positions 1, 3, 4, 5, 7 and 8, 2' fluorine modifications at positions 2, 6 and 9, and thio modifications at the 5' phosphates on positions 2 and 3.

[0132] The sense strand of the prepared Vutrisiran is UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm (SEQ ID NO: 19), and the antisense strand is UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAmAfUmCmCmCmAmsUmsCm (SEQ ID NO: 20).

[0133] Comparative Example 1

[0134] The average yield of full-length Vutrisiran product using solid phase synthesis was 30.1%, and the total proportion of N+1 and N-1 impurities was 1.49%.

[0135] The yield of the Vutrisiran product prepared by the enzyme-linked method of the present invention is 76.45%, and the yield of the solid phase synthesis substrate is 41.5% of the overall process. The overall yield is 31.7%, which is higher than the average yield of the product obtained by the solid phase synthesis method. The total proportion of N+1 and N-1 impurities is 0.37%, which is lower than the proportion of such impurities in the process of synthesizing Vutrisiran by solid phase synthesis.

[0136] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the preparation method of the present application, single-stranded RNA fragments designed based on the sequence of Vutrisiran are catalyzed by RNA ligase to form Vutrisiran, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the chemical synthesis preparation method, the product obtained by the preparation method of the present application has high purity, less impurities, simple preparation process, mild reaction conditions, low amount of organic reagents, reduced production costs, and is easy to achieve scale-up industrial production.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing Vutrisiran, characterized in that: The Vutrisiran is a double-stranded RNA consisting of a complementary sense strand and an antisense strand; The preparation method comprises: Mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment can constitute the sense strand, and the antisense strand substrate fragment can constitute the antisense strand; Part of the sense strand substrate fragment and part of the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity to form a double-stranded nucleotide structure with sticky ends; The double-stranded nucleotide structure containing the sticky end continues to connect with the remaining part of the sense strand substrate fragment and the remaining part of the antisense strand substrate fragment through hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, forming a double-stranded nucleotide structure containing a nick; Using the RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form the Vutrisiran; The bases at both ends of the nick are the 5' end and 3' end of different substrate fragments, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; Using the RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form the phosphodiester bond, thereby obtaining the Vutrisiran; The RNA ligase is a protein having an amino acid sequence shown in SEQ ID NO: 1; The sense strand substrate fragments include 2 or 3 fragments, and the antisense strand substrate fragments include 2 or 3 fragments; The length of the positive strand substrate fragment is 4-16 nt; The length of the antisense strand substrate fragment is 4-16 nt.

2. The preparation method according to claim 1, characterized in that: The sense strand substrate fragments and the antisense strand substrate fragments each include two, the sense strand substrate fragments include a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragments include a first antisense strand substrate fragment and a second antisense strand substrate fragment; The preparation method comprises: mixing the first sense chain substrate fragment, the second sense chain substrate fragment, the first antisense chain substrate fragment and the second antisense chain substrate fragment; under the catalytic action of the RNA ligase, the first sense chain substrate fragment and the second sense chain substrate fragment are connected to form the sense chain, the first antisense chain substrate fragment and the second antisense chain substrate fragment are connected to form the antisense chain, and the sense chain and the antisense chain form the Vutrisiran through base complementary pairing.

3. The preparation method according to claim 1, characterized in that: The sense strand substrate fragments and the antisense strand substrate fragments each include three fragments, wherein 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 preparation method comprises: 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 and the third antisense strand substrate fragment; under the catalytic action of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are mixed and connected to form the sense strand, the first antisense strand substrate fragment, the second antisense strand substrate fragment and the third sense strand substrate fragment are connected to form the antisense strand, and the sense strand and the antisense strand form the Vutrisiran through base complementary pairing.

4. The preparation method according to claim 2, characterized in that: The sense strand substrate fragment and the antisense strand substrate fragment each include two strands, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are connected 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 connected under the catalysis of the RNA ligase to form the antisense strand.

5. The preparation method according to claim 4, characterized in that: 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; 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.

6. The preparation method according to claim 2, characterized in that: The nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 5, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 6; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 7; SEQ ID NO: 5: UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAm; SEQ ID NO: 6: CmCmAmAmGmAm; SEQ ID NO: 7: AfUmCmCmCmAmsUmsCm; SEQ ID NO: 8: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAfAm; The first sense strand substrate fragment has 2' methoxy modifications at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14 and 15, and 2' fluorine modifications at positions 7, 9, 10 and 11; and thio modifications at the 5' phosphates on positions 2 and 3; The ribonucleotides at positions 1, 2, 3, 4, 5, and 6 of the second sense strand substrate fragment have 2' methoxy modifications; The first antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 2, 3, 4, 5, 6, 7 and 8, 2' fluorine modifications at ribonucleotide 1, and thio modifications at 5' phosphates at ribonucleotides 7 and 8; The second antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 3, 4, 5, 7, 8, 10, 11, 12, 13 and 15, 2' fluorine modifications at ribonucleotides 2, 6, 9 and 14, and thio modifications at 5' phosphates at ribonucleotides 2 and 3.

7. The preparation method according to claim 2, characterized in that: The nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11; SEQ ID NO: 9: UmsGmsGmGmAmUmUfUmCfAf; SEQ ID NO: 10: UfGmUmAmAmCmCmAmAmGmAm; SEQ ID NO: 11: AmAfUmCmCmCmAmsUmsCm; SEQ ID NO: 12: UmsCfsUmUmGmGfUmUmAfCmAmUmGmAf; The first sense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 2, 3, 4, 5, 6 and 8, 2' fluorine modifications at ribonucleotides 7, 9 and 10, and thio modifications at 5' phosphates on ribonucleotides 2 and 3; The second sense strand substrate fragment has 2' methoxy modifications at ribonucleotides 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, and 2' fluorine modifications at ribonucleotides 1; The first antisense strand substrate fragment has 2' methoxy modifications at positions 1, 3, 4, 5, 6, 7 and 8, 2' fluorine modifications at position 2, and thio modifications at the 5' phosphates at positions 8 and 9; The second antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 3, 4, 5, 7, 8, 10, 11, 12 and 13, 2' fluorine modifications at ribonucleotides 2, 6, 9 and 14, and thio modifications at 5' phosphates at ribonucleotides 2 and 3.

8. The preparation method according to claim 3, characterized in that: The nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13; The nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; The nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; The nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; The nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16; SEQ ID NO: 13: UmsGmsGmGmAmUm; SEQ ID NO: 14: UfUmCfAfUfGmUmAmAm; SEQ ID NO: 15: CmCmAmAmGmAm; SEQ ID NO: 16: CmCmAmsUmsCm; SEQ ID NO: 17: CmAmUmGmAfAmAfUmCm; SEQ ID NO: 18: UmsCfsUmUmGmGfUmUmAf; The first sense strand substrate fragment has 2' methoxy modifications at ribonucleotides at positions 1, 2, 3, 4, 5 and 6, and thio modifications at 5' phosphates at positions 2 and 3; The second sense strand substrate fragment has 2' methoxy modification at ribonucleotides 2, 6, 7, 8 and 9, and 2' fluorine modification at ribonucleotides 1, 3, 4 and 5; The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of the third sense strand substrate fragment have 2' methoxy modifications; The first antisense strand substrate fragment has 2' methoxy modifications at positions 1, 3, 4, 5, 7 and 8, 2' fluorine modifications at positions 2, 6 and 9, and thio modifications at 5' phosphates at positions 2 and 3; The second antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides at positions 1, 2, 3, 4, 6, 8 and 9, and 2' fluorine modifications at positions 5 and 7; The ribonucleotides at positions 1, 2, 3, 4 and 5 of the third antisense strand substrate fragment have 2' methoxy modifications, and the 5' phosphates at positions 4 and 5 have thio modifications.

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

10. The preparation method according to claim 1, characterized in that: The reaction temperature of the preparation method is 10~40°C; The reaction time of the preparation method is 2 to 48 hours.

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