A preparation method of Givosiran

Abstract

The present invention provides a preparation method of Givosiran. In the above method, the Givosiran is a double-stranded siRNA, which is composed of a sense strand and an antisense strand through complementary pairing; the method comprises: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, wherein the sense strand substrate can form a sense strand, and the antisense strand substrate can form an antisense strand; the sense strand substrate and the antisense strand substrate are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate and the antisense strand substrate 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 Givosiran. The method can solve the problem of low purity of Givosiran 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 Givosiran. Background Art

[0002] Small interfering RNA (siRNA) is a double-stranded RNA with a length of 19-25 nt. After entering the cell, siRNA can dissociate into single strands, in which the positive strand can specifically bind to the messenger RNA (mRNA) of the target gene through base matching, inducing a series of effects, and finally degrading the mRNA of the target gene, preventing the translation of mRNA to achieve the effect of hindering the expression of the target gene. In recent years, the research and development of siRNA drugs has received extensive attention. siRNA drugs act on mRNA, and the druggable targets are significantly larger than traditional small molecule drugs whose action sites are proteins; in addition, siRNA drugs can act on new targets by changing the sequence, and the research and development time is relatively short.

[0003] Givosiran is a siRNA-type double-stranded RNA drug developed by Alnylam for the treatment of acute hepatic porphyria (AHP) in adults. It was approved for marketing by the FDA in 2019 under the trade name GIVLAARI.

[0004] At present, the method for preparing Givosiran is a chemical method, using a solid phase carrier such as controlled pore glass beads (CPG) or polystyrene resin, and cyclic synthesis through the phosphoramidite triester method to extend the oligonucleotide chain from the 3' to 5' direction. After the synthesis cycle is completed, the Givosiran chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain a pure product. The solid phase synthesis method is a cyclic method. The yield of the synthesis decreases with the increase of the length of the synthesis chain, and the 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 length of the synthesis chain. This impurity is not easy to remove, resulting in a complicated purification process and low efficiency. Therefore, the cost of preparing Givosiran is high and it is difficult to scale up production, so it is necessary to develop a more efficient method for synthesizing Givosiran. Summary of the invention

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

[0006] In order to achieve the above object, according to the first aspect of the present invention, a method for preparing Givosiran is provided, wherein Givosiran is a double-stranded siRNA, which is composed of a sense strand and an antisense strand through complementary pairing; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase;

[0007] 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; using RNA ligase to connect the bases at both ends of the nick by a phosphodiester bond to form Givosiran; 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 Givosiran; the RNA ligase is an RNA ligase of RNA ligase family 1 and\or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is selected from: NO:3; the RNA ligase of RNA ligase family 2 is selected from: a protein having an amino acid sequence as shown in SEQ ID NO: 1 and\or SEQ ID NO: 2; or an enzyme that has more than 70% identity with any RNA ligase having an amino acid sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 3 and has activity in catalyzing phosphodiester bond formation.

[0008] Furthermore, the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO: 21, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO: 22.

[0009] 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 2-19 nt, more preferably 8-12 nt; preferably, the length of the antisense strand substrate fragment is 2-21 nt, more preferably 4-15 nt.

[0010] Furthermore, 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 fragment includes a first antisense chain substrate fragment and a second antisense chain 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, and under the catalytic action of RNA ligase, connecting 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 Givosiran 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 Givosiran.

[0011] Further, 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 chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 7, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9.

[0013] Further, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13.

[0014] Further, the sense chain substrate fragments and the antisense chain substrate fragments each include 3 fragments, the sense chain substrate fragments include a first sense chain substrate fragment, a second sense chain substrate fragment and a third sense chain substrate fragment; the antisense chain substrate fragments include a first antisense chain substrate fragment, a second antisense chain substrate fragment and a third antisense chain substrate fragment; preferably, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18.

[0015] Furthermore, the concentrations of the sense chain substrate fragment and the antisense chain substrate fragment are independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense chain substrate fragment, the antisense chain substrate fragment and the RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT; preferably, the reaction temperature of the preparation method is 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48h, 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 Givosiran sense strand, and the antisense strand substrate fragments are connected to form the Givosiran antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method for preparing Givosiran by chemical synthesis, the product obtained by the preparation method of the present application has high purity, few 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 structural diagram of the L96 group according to an embodiment of the present invention is shown.

[0019] Figure 2 A schematic diagram of the enzyme-catalyzed ligation reaction according to Example 1 of the present invention is shown.

[0020] Figure 3 The electrophoresis result diagram of the catalytic products of RNA ligases Ligase25 and Ligase26 according to Example 1 of the present invention is shown.

[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 Givosiran, 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 preparation method for Givosiran, using enzyme-catalyzed synthesis to prepare Givosiran, 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 Givosiran is provided, wherein Givosiran 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; 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 mutually connected by bases; The bases at both ends of the nick are connected by hydrogen bonds formed by complementation, 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; the bases at both ends of the nick are connected by phosphodiester bonds using RNA ligase to form Givosiran; 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; RNA ligase is used 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 Givosiran;

[0029] The RNA ligase is an RNA ligase of RNA ligase family 1 and / or RNA ligase family 2; the RNA ligase of RNA ligase family 1 is selected from a protein having an amino acid sequence as shown in SEQ ID NO: 3; the RNA ligase of RNA ligase family 2 is selected from a protein having an amino acid sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or an enzyme having more than 70% identity with any RNA ligase having an amino acid sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 3, 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 in catalyzing the formation of phosphodiester bonds. 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.

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

[0031] In the above-mentioned preparation method, the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase are mixed to prepare Givosiran. 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 with a sticky end, and this double-stranded nucleotide with a sticky end continues to bind to other substrates to form a double-stranded nucleotide structure containing a nick; RNA ligase can recognize the specific recognition site of this double-stranded nucleotide containing a nick, connect the nick, and thus prepare the target product Givosiran.

[0032] In a preferred embodiment, the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO:21, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO:22.

[0033] SEQ ID NO: 21: CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm.

[0034] SEQ ID NO: 22: UmsAfsAfGfAmUfGmAfGmAfCmAf CmUfCmUfUmUfCmUfGmsGmsUm.

[0035] 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 ribonucleotide in "sAm", "sGm" and the like indicates thio modification of the 5' phosphate of the ribonucleotide.

[0036] SEQ ID NO: 1 (Ligase-25, Vibrio phage NT-1):

[0037] MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF。

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

[0039] MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY。

[0040] SEQ ID NO:3(Ligase 42,Escherichia phage JN02):

[0041] MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY。

[0042] SEQ ID NO:4(Ligase 11,Thermococcus):

[0043] MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD。

[0044] SEQ ID NO:5(Ligase 20,Archaea):

[0045] MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

[0046] SEQ ID NO:6(Ligase 32,bacteria):

[0047] MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFFSKKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAE EYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVVRVKKIETYKMVSHFERMGLNIHIHDDIELGNGYWKITFKRVYDDATKEIRELWNGHAFVD。

[0048] 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.

[0049] 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.

[0050] 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).

[0051] 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:

[0052] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0053] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0054] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0055] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0056] 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.

[0057] In the present application, only the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 3, or an enzyme with more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 3, can be used to catalyze the formation of a phosphodiester bond between the phosphate group and the hydroxyl group of the substrate in the present application to obtain the product Givosiran. In the relevant experiments of the present application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 3 that can synthesize Givosiran by screening from a large number of enzymes. However, the negative results that accounted for a large proportion in the experiments showed that most RNA ligases were difficult to catalyze the synthesis of Givosiran, including but not limited to the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 6. In the present application specification, only SEQ ID NO: 4 to SEQ ID NO: 6 are used as examples to reflect this type of RNA ligase that has no catalytic activity in the synthesis of Givosiran.

[0058] In a preferred embodiment, 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 2-19 nt, more preferably 8-12 nt; preferably, the length of the antisense strand substrate fragment is 2-21 nt, more preferably 4-15 nt.

[0059] 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 fragment includes a first antisense chain substrate fragment and a second antisense chain 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 RNA ligase, connecting 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 Givosiran 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 Givosiran.

[0060] 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 Givosiran with a complete double-stranded structure.

[0061] In a preferred embodiment, 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.

[0062] In a preferred embodiment, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 7, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8. Preferably, the nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 10, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9.

[0063] In a preferred embodiment, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12. Preferably, the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13.

[0064] Givosiran can be prepared using the above preparation method and the substrate fragments shown in SEQ ID NO: 7-SEQ ID NO: 10 or SEQ ID NO: 11-SEQ ID NO: 14. However, it should be noted that the selection of substrate fragments is not limited to the substrate fragments shown in SEQ ID NO: 7-10 or SEQ ID NO: 11-14. All substrate fragments 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 Givosiran but is not limited to the different connection positions of the substrate fragments. The above preparation has a good connection effect for the connection of the sense chain sequence and the antisense chain sequence of Givosiran. The number of sense chain substrate fragments or antisense chain substrate fragments includes but is not limited to 2, 3, 4 or even more.

[0065] SEQ ID NO: 7: CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCf.

[0066] SEQ ID NO: 8: AmUmCmUmUmAm.

[0067] SEQ ID NO: 9: CmUfCmUfUmUfCmUfGmsGmsUm.

[0068] SEQ ID NO: 10: UmsAfsAfGfAmUfGmAfGmAfCmAf.

[0069] SEQ ID NO: 11: CmsAmsGmAmAmAmGfAmGfUm.

[0070] SEQ ID NO: 12: GfUmCfUmCfAmUmCmUmUmAm.

[0071] SEQ ID NO: 13: UfUmUfCmUfGmsGmsUm.

[0072] SEQ ID NO: 14: UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm.

[0073] In a preferred embodiment, the sense chain substrate fragments and the antisense chain substrate fragments each include 3 fragments, the sense chain substrate fragments include a first sense chain substrate fragment, a second sense chain substrate fragment and a third sense chain substrate fragment; the antisense chain substrate fragments include a first antisense chain substrate fragment, a second antisense chain substrate fragment and a third antisense chain substrate fragment; preferably, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18.

[0074] SEQ ID NO: 15: CmsAmsGmAmAm.

[0075] SEQ ID NO: 16: AmGfAmGfUmGfUm.

[0076] SEQ ID NO: 17: CfUmCfAmUmCmUmUmAm.

[0077] SEQ ID NO: 18: UfGmsGmsUm.

[0078] SEQ ID NO: 19: CmUfCmUfUmUfCm.

[0079] SEQ ID NO: 20: UmsAfsAfGfAmUfGmAfGmAfCmAf.

[0080] In a preferred embodiment, the concentrations of the sense chain substrate fragment and the antisense chain substrate fragment are independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense chain substrate fragment, the antisense chain substrate fragment and RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT; preferably, the reaction temperature of the preparation method is 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48h, more preferably 12-24h.

[0081] 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.

[0082] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0083] Embodiment 1:

[0084] Based on the sense strand and antisense strand of the Givosiran sequence (SEQ ID NO: 21 and SEQ ID NO: 22), four single-stranded RNA fragments (length: nt) were designed as shown in Table 1:

[0085] Table 1

[0086] .

[0087] Among them, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, s before the ribonucleotide in the sequence of "sAm", "sGm" etc. indicates thio modification of the 5' phosphate of the ribonucleotide, L96 indicates that the 3' end of the sense strand is modified with L96 group, and the structure is as follows Figure 1 As shown, the wavy line in the figure represents the base connected to the L96 group.

[0088] Substrate 1 has 2' methoxy modifications at ribonucleotide positions 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14, 2' fluorine modifications at ribonucleotide positions 7, 9, 11, 13 and 15, and thio modifications at the 5' phosphates on ribonucleotide positions 2 and 3.

[0089] The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 2 have 2' methoxy modifications.

[0090] Substrate 3 has 2' methoxy modifications at ribonucleotides 1, 3, 5, 7, 9, 10 and 11, 2' fluorine modifications at ribonucleotides 2, 4, 6 and 8, and thio modifications at the 5' phosphates on ribonucleotides 10 and 11.

[0091] Substrate 4 has 2' methoxy modifications at ribonucleotides 1, 5, 7, 9 and 11, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10 and 12, and thio modifications at the 5' phosphates on ribonucleotides 2 and 3.

[0092] The above four single-stranded RNA fragments were prepared using a solid phase synthesis method.

[0093] The four single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture with a final concentration of 2.5mM (each substrate was 2.5mM), annealed, and the annealed RNA fragment mixture was obtained. The annealed RNA fragment mixture was subjected to an enzyme-catalyzed ligation reaction. The reaction system was set to 10μL. The reaction system included reaction buffer (50mM Tris-HCl, pH7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT), and RNA ligases Ligase 25, Ligase 26, Ligase 11, Ligase 20, and Ligase 32 were added respectively. The reaction system was reacted at 16°C for 16h. The obtained reaction system was inactivated by 80°C for 5min, and the precipitate was removed by centrifugation at 12000rpm. The schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 2 shown.

[0094] The products catalyzed by RNA ligase Ligase 25, Ligase 26, Ligase 11, Ligase 20 and Ligase 32 were detected by SDS-PAGE. The electrophoresis results of the products catalyzed by RNA ligase Ligase25 and Ligase26 are shown in the figure below. Figure 3 As shown, Figure 3 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 26. 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.

[0095] After activity screening of six RNA ligases, it was found that Ligase25 had the best ligation effect and could convert most substrates into Givosiran.

[0096] The sense strand of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21), and the antisense strand is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22). The reaction results are shown in Table 2.

[0097] Table 2

[0098] .

[0099] Remark:

[0100] 1) Reaction conditions: substrate fragment 100 μM, ATP 10 eq, MgCl2 100 eq, DTT 10 eq (1 eq = 100 μM), 0.2 mg / mL enzyme, 1891 V, 50 mM Tris-HCl pH 7.5, 16 °C, 16 h;

[0101] 2) ++ means 25~50% (excluding the endpoint value of 50%), and +++ means 50~75%.

[0102] 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).

[0103] Embodiment 2:

[0104] Ligase 25, Ligase 26 and Ligase 42 capable of catalyzing the synthesis of Givosiran in Example 1 were used to perform enzyme-catalyzed ligation reactions using the annealed substrate fragments, and the reaction conditions were as follows: the reaction system was set to 50 μL, and reaction buffer (50 mM Tris-HCl, pH 7.5), adenosinetriphosphate (ATP), MgCl2, dithiothreitol (DTT) and RNA ligase were added to the reactor in sequence, and the reaction was carried out at 16°C for 16 hours.

[0105] After the reaction, the protein was inactivated by heating at 80℃ for 5 minutes and the supernatant was collected by centrifugation. The HPLC results of the product catalyzed by Ligase 25 were as follows: Figure 4 As shown, the yield is measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.

[0106] Table 3

[0107] .

[0108] Remark:

[0109] 1) Reaction conditions: substrate fragment 800 μM, ATP 4 eq, MgCl2 100 eq, DTT 10 eq (1 eq = 800 μM), 0.2 mg / mL enzyme, 236 V, 50 mM Tris-HCl pH 7.5, 16 °C, 16 h;

[0110] 2) ND means no product formation was detected, ++ means 50~70% (excluding the endpoint value of 70%), and +++ means 70~80%.

[0111] The molecular weight of the sense strand product identified by LC-MS was 8732.04, and the molecular weight of the antisense strand product was 7560.03. The theoretical value of the sense strand product was 8732.04±8, and the theoretical value of the antisense strand product was 7560.03±8, indicating that Ligase 25, Ligase 26, and Ligase 42 were linked to generate Givosiran. The LC-MS detection results of Ligase 25 are shown in the figure below. Figure 5 shown.

[0112] Example 3

[0113] The annealed substrate fragments and ligase Ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 10 mL, and reaction buffer (50mM Tris-HCl, pH7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT) and RNA ligase were added to the reactor in sequence, and the reaction was carried out at 16°C for 16 hours. After the overnight reaction, the protein was inactivated by heating at 50°C for 15 minutes, and the supernatant was obtained by centrifugation and purified using a Nano-Q column, NaCl gradient elution, membrane package (molecular weight cutoff 1kDa) desalting, and freeze-dried in a freeze dryer to obtain dry powder. The calculated yield was 66.18%, and the purity (HPLC detection) was 91.88%.

[0114] Example 4

[0115] Four single-stranded RNA fragments (length: nt) were designed based on the sequence of Givosiran as shown in Table 4.

[0116] Table 4

[0117] .

[0118] Among them, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in the sequences such as "sAm" and "sGm" represents thio modification of the 5' phosphate of the ribonucleotide.

[0119] Substrate 5 has 2' methoxy modifications at ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8 and 10, 2' fluorine modifications at ribonucleotides at positions 7 and 9, and thio modifications at the 5' phosphates at positions 2 and 3.

[0120] The ribonucleotides at positions 2, 4, 6, 7, 8, 9, 10 and 11 of substrate 6 have 2' methoxy modifications, and the nucleotides at positions 1, 3 and 5 have 2' fluorine modifications. The 5' end is modified with an L96 group.

[0121] Substrate 7 has 2' methoxy modifications at ribonucleotides at positions 2, 4, 6, 7 and 8, 2' fluorine modifications at ribonucleotides at positions 1, 3 and 5, and thio modifications at the 5' phosphates at positions 7 and 8.

[0122] Substrate 8 has 2' methoxy modifications at ribonucleotides 1, 5, 7, 9, 11, 13 and 15, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10, 12 and 14, and thio modifications at the 5' phosphates on ribonucleotides 2 and 3.

[0123] The annealed substrate fragment and ligase Ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 10 mL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 25, 50 mM Tris-HCl pH 7.5 were added to the reactor in sequence, and the reaction was carried out at 16 ° C for 16 h. After the reaction, the protein was inactivated by heating at 80 ° C for 5 min, and the supernatant was taken by centrifugation. The sample was sent for HPLC detection, and the yield 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 yield was +++ (the product peak accounted for 75.2%).

[0124] The sense strand of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21), and the antisense strand is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).

[0125] Example 5

[0126] The six single-stranded RNA fragments (length: nt) shown in Table 5 were designed based on the sequence of Givosiran.

[0127] Table 5

[0128] .

[0129] Among them, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in the sequences such as "sAm" and "sGm" represents thio modification of the 5' phosphate of the ribonucleotide.

[0130] Substrate 9 has 2' methoxy modifications at ribonucleotide positions 1, 2, 3, 4 and 5, and thio modifications at the 5' phosphates on ribonucleotide positions 2 and 3.

[0131] The ribonucleotides at positions 1, 3, 5 and 7 of substrate 10 have 2' methoxy modifications, and the nucleotides at positions 2, 4 and 6 have 2' fluorine modifications.

[0132] The ribonucleotides at positions 2, 4, 5, 6, 7, 8 and 9 of substrate 11 have 2' methoxy modifications, and the nucleotides at positions 1 and 3 have 2' fluorine modifications.

[0133] The substrate 12 has 2' methoxy modifications on the 2nd, 3rd and 4th ribonucleotides, 2' fluorine modifications on the 1st nucleotide, and thio modifications on the 5' phosphates on the 3rd and 4th ribonucleotides.

[0134] The ribonucleotides at positions 1, 3, 5 and 7 of substrate 13 have 2' methoxy modifications, and the nucleotides at positions 2, 4 and 6 have 2' fluorine modifications.

[0135] Substrate 14 has 2' methoxy modifications at ribonucleotides 1, 5, 7, 9 and 11, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10 and 12, and thio modifications at the 5' phosphates on ribonucleotides 2 and 3.

[0136] The sense strand of the prepared Givosiran is CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21), and the antisense strand is UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).

[0137] The annealed substrate fragment and ligase Ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 10 mL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 25, 50 mM Tris-HCl pH 7.5 were added to the reactor in sequence, and the reaction was carried out at 16 ° C for 16 h. After the reaction, the protein was inactivated by heating at 80 ° C for 5 min, and the supernatant was taken by centrifugation. 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 yield was +++ (the product peak accounted for 70.3%).

[0138] Comparative Example 1

[0139] The average yield of full-length Givosiran product using solid phase synthesis was 32.9%, and the total proportion of N+1 and N-1 impurities was 1.52%.

[0140] The yield of the Givosiran product prepared by the enzyme-linked method of the present invention is 87.53%, the average solid-phase synthesis yield of the substrate used is 39.1%, and the yield of the overall process is 34.2%, which is higher than the average yield of the product obtained by the solid-phase synthesis method, and the total proportion of N+1 and N-1 impurities is 0.41%, which is lower than the proportion of such impurities in the process of synthesizing Givosiran by solid-phase synthesis.

[0141] 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 Givosiran are catalyzed by RNA ligase to form Givosiran, 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.

[0142] 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 Givosiran, characterized in that: The Givosiran is a double-stranded siRNA, which is composed of a sense strand and an antisense strand through base complementary pairing; 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 Givosiran; 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 Givosiran; The RNA ligase is one or more RNA ligases of RNA ligase family 1 and / or RNA ligase family 2; The RNA ligase of RNA ligase family 1 is a protein having an amino acid sequence shown in SEQ ID NO: 3; The RNA ligase of RNA ligase family 2 is a protein having an amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; 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 2-19 nt; The length of the antisense strand substrate fragment is 2-21 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 strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment and the second antisense strand substrate fragment, and under the catalytic action of the RNA ligase, connecting the first sense strand substrate fragment and the second sense strand substrate fragment to form the sense strand; Under the catalytic action of the RNA ligase, the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to connect to form the antisense strand; The sense strand and the antisense strand form the Givosiran through complementary base pairing.

3. The preparation method according to claim 2, characterized in that: The 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are 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.

4. The preparation method according to claim 2, 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.

5. 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: 7, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9; SEQ ID NO: 7: CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCf; SEQ ID NO: 8: AmUmCmUmUmAm; SEQ ID NO: 9: CmUfCmUfUmUfCmUfGmsGmsUm; SEQ ID NO: 10: UmsAfsAfGfAmUfGmAfGmAfCmAf; The first sense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14, 2' fluorine modifications at ribonucleotides 7, 9, 11, 13 and 15, and thio modifications at 5' phosphates on ribonucleotides 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 1, 5, 7, 9 and 11, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10 and 12, and thio modifications at 5' phosphates on ribonucleotides 2 and 3; The second antisense strand substrate fragment has 2' methoxy modification at ribonucleotides 1, 3, 5, 7, 9, 10 and 11, 2' fluorine modification at ribonucleotides 2, 4, 6 and 8, and thio modification at 5' phosphate at ribonucleotides 10 and 11.

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: 11, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13; SEQ ID NO: 11: CmsAmsGmAmAmAmGfAmGfUm; SEQ ID NO: 12: GfUmCfUmCfAmUmCmUmUmAm; SEQ ID NO: 13: UfUmUfCmUfGmsGmsUm; SEQ ID NO: 14: UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm; The first sense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 2, 3, 4, 5, 6, 8 and 10, 2' fluorine modifications at ribonucleotides 7 and 9, and thio modifications at 5' phosphates at ribonucleotides 2 and 3; The second sense strand substrate fragment has 2' methoxy modification at ribonucleotides at positions 2, 4, 6, 7, 8, 9, 10 and 11, 2' fluorine modification at nucleotides at positions 1, 3 and 5, and a L96 group modification at the 5' end; The first antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 5, 7, 9, 11, 13 and 15, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10, 12 and 14, and thio modifications at 5' phosphates on ribonucleotides 2 and 3; The second antisense strand substrate fragment has 2' methoxy modification at ribonucleotides at positions 2, 4, 6, 7 and 8, 2' fluorine modification at ribonucleotides at positions 1, 3 and 5, and thio modification at 5' phosphate at ribonucleotides at positions 7 and 8.

7. The preparation method according to claim 1, characterized in that: The sense strand substrate fragment and the antisense strand substrate fragment each include 3 strands, 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.

8. The preparation method according to claim 7, characterized in that: The nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; The nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16; The nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20; The nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; The nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; SEQ ID NO: 15: CmsAmsGmAmAm; SEQ ID NO: 16: AmGfAmGfUmGfUm; SEQ ID NO: 17: CfUmCfAmUmCmUmUmAm; SEQ ID NO: 18: UfGmsGmsUm; SEQ ID NO: 19: CmUfCmUfUmUfCm; SEQ ID NO: 20: UmsAfsAfGfAmUfGmAfGmAfCmAf; The first sense strand substrate fragment has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4 and 5, and thio modifications on the 5' phosphates at positions 2 and 3; The ribonucleotides at positions 1, 3, 5 and 7 of the second sense strand substrate fragment have 2' methoxy modifications, and the nucleotides at positions 2, 4 and 6 have 2' fluorine modifications; The ribonucleotides at positions 2, 4, 5, 6, 7, 8 and 9 of the third sense strand substrate fragment have 2' methoxy modifications, and the nucleotides at positions 1 and 3 have 2' fluorine modifications; The first antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 5, 7, 9 and 11, 2' fluorine modifications at ribonucleotides 2, 3, 4, 6, 8, 10 and 12, and thio modifications at 5' phosphates on ribonucleotides 2 and 3; The second antisense strand substrate fragment has 2' methoxy modifications at ribonucleotides 1, 3, 5 and 7, and 2' fluorine modifications at nucleotides 2, 4 and 6; The ribonucleotides at positions 2, 3 and 4 of the third antisense strand substrate fragment have 2' methoxy modifications, the nucleotide at position 1 has 2' fluorine modification, and the 5' phosphates at positions 3 and 4 have thio modification.

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 0.1-4.5 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 2-48h.

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