Method for preparing patisiran

Abstract

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

Description

A method for preparing Patisiran

[0001] This application is based on and claims priority to Chinese application CN application number 2024109217277 filed on July 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of pharmaceutical biosynthesis, and more specifically, to a method for preparing Patisiran. Background Technology

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

[0004] Patisiran, developed by Alnylam, is a double-stranded siRNA drug used to treat hereditary transthyretin amyloidosis (hATTR). It was approved by the FDA in 2018 under the brand name ONPATTRO. Patisiran contains two 21-nt RNA single strands, which anneal to form a 19-bp double strand. This double-stranded RNA is encapsulated in lipid nanoparticles for delivery to hepatocytes. hATTR is a rare disease (affecting approximately 50,000 people). Patisiran works by specifically binding to a genetically conserved sequence in the 3' untranslated region of the transthyretin (TTR) gene mRNA. This binding leads to mRNA degradation, subsequently reducing serum TTR protein levels and tissue TTR protein deposition.

[0005] Currently, the preparation of Patisiran is a chemical method using a solid-phase support such as controlled-porous glass (CPG) or polystyrene resin. The oligonucleotide chain is extended along the 3' to 5' direction via a phosphoramidite cyclic synthesis. After the synthesis cycle is complete, the Patisiran chain is removed from the solid-phase support by ammonolysis, followed by purification to obtain the pure product. This solid-phase synthesis method is cyclical, and the yield decreases with increasing chain length. Furthermore, impurities generated during the synthesis process, such as those with one more nucleotide than the target sequence (N+1 impurity) or one less nucleotide than the target sequence (N-1 impurity), also increase with increasing chain length. When using the existing chemical solid-phase synthesis of Patisiran, the synthesis length is 21 nt, and a large number of N+1 and N-1 impurities are generated during the synthesis, which are difficult to remove during purification, ultimately resulting in a final N+1 and N-1 impurity content of 1-3%. Therefore, the preparation cost of Patisiran is high, making large-scale production difficult. Thus, a more efficient Patisiran synthesis method needs to be developed. Summary of the Invention

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

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

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

[0009] Furthermore, the positive chain substrate fragment includes two or more segments, and the negative chain substrate fragment includes two or more segments; preferably, the length of the positive chain substrate fragment is 5-12 nt, more preferably 6-11 nt; preferably, the length of the negative chain substrate fragment is 2-14 nt, more preferably 5-13 nt.

[0010] Further, 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; the preparation method includes: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, and the second antisense strand substrate fragment; under the catalysis of RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are ligated to form a sense strand; the first antisense strand substrate fragment and the second antisense strand substrate fragment are ligated to form an antisense strand; the sense strand and the antisense strand form Patisiran through base complementarity pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain Patisiran.

[0011] Further, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 5, and the nucleotide sequence of the second positive strand substrate fragment is SEQ ID NO: 6; preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is GUUmACdTdT; preferably, the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second positive strand substrate fragment is AUmUmCmCmAUmdTdT; preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 11.

[0012] Further, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group; preferably, the 5' end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0013] Further, the sense substrate fragment includes a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; the antisense substrate fragment includes a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment; preferably, the nucleotide sequence of the first sense substrate fragment is the nucleic acid sequence nucleotide sequence shown in GUmAACmCm; the nucleotide sequence of the second sense substrate fragment is the nucleic acid sequence nucleotide sequence shown in AAGAGUmA; and the nucleotide sequence of the third sense substrate fragment is the nucleic acid sequence nucleotide sequence shown in UmUmCmCmAUmdTdT.

[0014] Preferably, the nucleotide sequence of the first antisense substrate fragment is the nucleic acid sequence nucleotide sequence shown in UmACdTdT; the nucleotide sequence of the second antisense substrate fragment is the nucleic acid sequence nucleotide sequence shown in UCUUGGU; and the nucleotide sequence of the third antisense substrate fragment is the nucleic acid sequence nucleotide sequence shown in AUGGAAUmAC.

[0015] Further, the preparation method includes: 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, a third antisense strand substrate fragment, and the RNA ligase; under the catalysis of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment, and the third sense strand substrate fragment are ligated to form the sense strand, and the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment are ligated to form the antisense strand, and the sense strand and the antisense strand form Patisiran through complementary base pairing.

[0016] Furthermore, the reaction system formed by mixing the sense substrate fragment, the antisense substrate fragment, and the RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the concentrations of the sense substrate fragment and the antisense substrate fragment are each independently selected from 0.1-4.5 mM; preferably, the reaction temperature of the preparation method is 10-40℃, more preferably 15-30℃; preferably, the reaction time of the preparation method is 2-48 h, more preferably 12-24 h.

[0017] By applying the technical solution of this invention and utilizing the above-described preparation method, under the catalysis of RNA ligase, the sense strand substrate fragments are ligated to form the Patisiran sense strand, and the antisense strand substrate fragments are ligated to form the Patisiran antisense strand, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with the chemical synthesis method for preparing Patisiran, the preparation method of this application yields a product with high purity, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production. Attached Figure Description

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

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

[0020] Figure 2 shows the electrophoresis results of the RNA ligase Ligase25 and Ligase11 catalyzed products according to Example 1 of the present invention.

[0021] Figure 3 shows the HPLC detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention.

[0022] Figure 4 shows the LC-MS detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention. Detailed Implementation

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0024] Terminology Explanation:

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

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

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

[0028] In a first typical embodiment of this application, a method for preparing Patisiran is provided. Patisiran is a double-stranded RNA composed of complementary positive and negative strands. The preparation method includes: mixing a positive strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the positive strand substrate fragment can form a positive strand, and the antisense strand substrate fragment can form an antisense strand; the positive and antisense strand substrate fragments are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrate fragments are not interconnected, forming a double-stranded nucleotide structure with a nick; using RNA ligase to link the bases at both ends of the nick with phosphodiester bonds to form Patisiran; the bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with the 5' being a phosphate group and the 3' being a hydroxyl group; using 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 phosphodiester bonds, obtaining Patisiran; the RNA ligase is a [SEQ ID NO] RNA ligases with the amino acid sequence shown in SEQ ID NO: 1; or enzymes that have more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 and have catalytic activity in forming phosphodiester bonds.

[0029] In the above preparation method, the positive-sense substrate fragment is two or more nucleotide sequences that can form the positive-sense chain. That is, multiple nucleotide sequences of the positive-sense substrate fragment can be spliced ​​together to form a sequence identical to the positive-sense chain. The difference between the positive-sense substrate fragment and the positive-sense chain is that there are nicks between the positive-sense substrate fragments, and they are not linked by phosphodiester bonds. Similarly, the antisense substrate fragment and the antisense chain have the above characteristics. Using RNA ligase, two or more positive-sense substrate fragments or antisense substrate fragments are linked by phosphodiester bonds to obtain the positive and antisense chains of Patisiran.

[0030] In the above preparation method, Patisiran can be prepared by mixing the sense and antisense substrate fragments with RNA ligase. In this preparation method, the sense and antisense substrate fragments can complement each other to form a double-stranded nucleotide with sticky ends. This double-stranded nucleotide with sticky ends can then bind to other substrate fragments to form a double-stranded nucleotide structure with nicks. The RNA ligase of this application can recognize the nicks in this double-stranded structure and link the nicks with phosphodiester bonds to prepare the target product Patisiran.

[0031] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO: 20.

[0032] SEQ ID NO: 19: GUMAACmCmAAGAGUmAUmUmCmCmAUmdTdT.

[0033] SEQ ID NO: 20: AUGGAAUmACUCUUGGUUmACdTdT.

[0034] In this application, the m following A, C, G or U indicates the 2' methoxy modification of the ribonucleotide, and the d before T indicates that the nucleotide is the deoxyribonucleotide thymine.

[0035] In a preferred embodiment, the RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having more than 70% identity with the RNA ligase shown in SEQ ID NO: 1, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more), and having catalytic activity for the formation of phosphodiester bonds.

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

[0037] SEQ ID NO: 2 (Ligase 11, Thermococcus):

[0038] SEQ ID NO: 3 (Ligase 20, Archaea):

[0039] SEQ ID NO: 4 (Ligase 32, bacteria):

[0040] In this application, "identity" refers to the "identity" between amino acid sequences or nucleotide sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0041] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) of identicality and function are highly likely to have the same active site, active pocket, active mechanism, and protein structure as the proteins provided by the above sequences.

[0042] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0043] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substitution are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0044] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0045] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0046] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0047] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0048] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0049] In this application, only the RNA ligase shown in SEQ ID NO: 1, or an enzyme with more than 70% identity to the RNA ligase shown in SEQ ID NO: 1, can catalyze the formation of phosphodiester bonds between the phosphate and hydroxyl groups of the substrate to obtain the product Patisiran. In the relevant experiments of this application, the inventors obtained the aforementioned RNA ligase shown in SEQ ID NO: 1, which is capable of synthesizing Patisiran, by screening a large number of enzymes. However, the large proportion of negative results in the experiments showed that most RNA ligases are difficult to catalyze the synthesis of Patisiran, including but not limited to the RNA ligases shown in SEQ ID NO: 2 to SEQ ID NO: 4. In this application specification, only SEQ ID NO: 2 to SEQ ID NO: 4 are used as examples to illustrate this type of RNA ligase that does not have the activity to catalyze the synthesis of Patisiran.

[0050] In a preferred embodiment, the positive chain substrate fragment comprises two or more segments, and the negative chain substrate fragment comprises two or more segments; preferably, the length of the positive chain substrate fragment is 3-15 nt, more preferably 6-11 nt, and even more preferably 8-10 nt; preferably, the length of the negative chain substrate fragment is 2-14 nt, more preferably 5-13 nt, and even more preferably 7-10 nt.

[0051] In a preferred embodiment, both the sense and antisense substrate fragments comprise two fragments. The sense substrate fragment includes a first sense substrate fragment and a second sense substrate fragment, and the antisense substrate fragment includes a first antisense substrate fragment and a second antisense substrate fragment. The preparation method includes: mixing the first sense substrate fragment, the second sense substrate fragment, the first antisense substrate fragment, and the second antisense substrate fragment; catalyzing the ligation of the first and second sense substrate fragments to form a sense strand under the catalysis of RNA ligase; catalyzing the ligation of the first and second antisense substrate fragments to form an antisense strand; and forming Patisiran through base complementarity pairing between the sense and antisense strands. Preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain Patisiran.

[0052] In the above preparation method, the sense and antisense substrate fragments are first mixed and annealed. The sense and antisense substrate fragments can form a double-stranded RNA structure through base complementarity, and this double-stranded RNA structure contains nicks between the different substrate fragments. The annealed reaction system is then mixed with RNA ligase, which uses phosphodiester bonds to connect the phosphate and hydroxyl groups on both sides of the nick, repairing the nick and thus obtaining the target product Patisiran with a complete double-stranded structure.

[0053] In a preferred embodiment, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 5, and the nucleotide sequence of the second sense substrate fragment is SEQ ID NO: 6; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense substrate fragment is GUUmACdTdT.

[0054] In a preferred embodiment, the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate fragment is AUmUmCmCmAUmdTdT sequence; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate fragment is SEQ ID NO: 11.

[0055] Patisiran can be prepared using the above-described 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 substrate fragments is not limited to those shown in SEQ ID NOs: 5-8 or SEQ ID NOs: 9-12. Substrate fragments that can be combined to form both the positive and negative strands can be used in the above preparation method. The above preparation method is applicable to the preparation of Patisiran but is not limited to different substrate fragment connection positions. The above preparation method has good connection effects on both the positive and negative strand sequences of Patisiran. The number of positive or negative strand substrate fragments includes, but is not limited to, 2, 3, 4, or even more.

[0056] SEQ ID NO: 5: GUMAACmCmAAGA.

[0057] SEQ ID NO: 6: GUMAUmUmCmCmAUmdTdT.

[0058] SEQ ID NO: 8: AUGGAAUmACUCUUG.

[0059] SEQ ID NO: 9: GUMAACmCmAAGAGUm.

[0060] SEQ ID NO: 11: UUGGUUmACdTdT.

[0061] SEQ ID NO: 12: AUGGAAUmACUC.

[0062] In a preferred embodiment, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group; preferably, the 5' end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

[0063] In a preferred embodiment, both the sense and antisense substrate fragments comprise three segments: a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the nucleotide sequence of the first sense substrate fragment is GUmAACmCm; the nucleotide sequence of the second sense substrate fragment is AAGAGUmA; and the nucleotide sequence of the third sense substrate fragment is UmUmCmCmAUmdTdT. Preferably, the nucleotide sequence of the first antisense substrate fragment is UmACdTdT; the nucleotide sequence of the second antisense substrate fragment is UCUUGGU; and the nucleotide sequence of the third antisense substrate fragment is AUGGAAUmAC.

[0064] In a preferred embodiment, the above preparation method includes: 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, a third antisense strand substrate fragment, and an RNA ligase; under the catalysis of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment, and the third sense strand substrate fragment are ligated to form the sense strand; the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment are ligated to form the antisense strand; and the sense strand and the antisense strand form Patisiran through complementary base pairing.

[0065] In a preferred embodiment, the concentrations of the sense and antisense substrate fragments are each preferably 2.5-10 mM; preferably, the reaction system formed by mixing the sense and antisense substrate fragments and RNA ligase further includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the reaction temperature of the preparation method is 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48 h, more preferably 12-24 h.

[0066] The concentrations of the aforementioned sense and antisense substrate fragments are each selected from, but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mM; the reaction temperatures of the aforementioned preparation methods are, but not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 °C; and the reaction times of the aforementioned preparation methods are, but not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, or 48 h.

[0067] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0068] Example 1

[0069] The four single-stranded RNA fragments based on Patisiran sequences are shown in Table 1, with length units in nt.

[0070] Table 1

[0071] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and the 'd' before T indicates that the sugar in the nucleotide is deoxyribose.

[0072] The ribonucleotides at positions 2, 5, and 6 of substrate 1 have 2' methoxy groups modified.

[0073] The ribonucleotides at positions 2, 4, 5, 6, 7, and 9 of substrate 2 have a 2' methoxy group, and positions 10 and 11 are thymine containing deoxyribose.

[0074] The ribonucleotide at position 3 of substrate 3 has a 2' methoxy group, and positions 6 and 7 are thymine containing deoxyribose.

[0075] The 7th ribonucleotide of substrate 4 has a 2' methoxy group modification.

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

[0077] Four single-stranded RNA fragments were mixed in equimolar proportions to obtain a substrate mixture with a final concentration of 2.5 mM (2.5 mM for each substrate). The mixture was then annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzymatic ligation reaction in a 10 μL volume. The reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, and dithiothreitol (DTT). RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 were added at concentrations of 0.2 mg / mL, respectively. The reaction system was incubated at 16 °C for 16 h. The resulting reaction system was then subjected to 80 °C for 5 min to inactivate the ligases, and the precipitate was removed by centrifugation at 12000 rpm. A schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

[0078] The products catalyzed by RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 were analyzed by SDS-PAGE. Figure 2 shows the electrophoresis results of the products catalyzed by Ligase 25 and Ligase 11. In Figure 2, lane M represents the RNA molecule marker, lane 1 represents the Ligase 25 reaction system, and lane 2 represents the Ligase 11 reaction system. The yield was estimated based on the grayscale analysis of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0079] The sense strand of the prepared Patisiran is GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT (SEQ ID NO: 19), and the antisense strand is AUGGAAUmACUCUUGGUUmACdTdT (SEQ ID NO: 20).

[0080] Table 2

[0081] Remark:

[0082] 1) Reaction conditions: 100 μM substrate fragment, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM), 0.2 mg / mL of selected RNA ligase, 50 mM Tris-HCl, pH 7.5, reaction at 16℃ for 16 h;

[0083] 2)++ indicates 25% to 50%.

[0084] The formula for calculating yield is: product gray value / (product gray value + substrate gray value).

[0085] Example 2

[0086] The reaction conditions in this embodiment are as follows: the reaction system is set to 50 μL, and the reaction system includes reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT) and RNA ligase Ligase 25, and the reaction is carried out at 16°C for 16 h.

[0087] After the reaction, the protein was inactivated by heating at 80°C for 5 min, and the supernatant was collected by centrifugation. The samples were then analyzed by HPLC and LC-MS. Pat-strand 1 represents the sense strand, and Pat-strand 2 represents the antisense strand. The yield was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample. The results are shown in Table 3. (The text repeats itself here, so the translation reflects that.)

[0088] Table 3

[0089] Remark:

[0090] 1) Reaction conditions: 800 μM substrate fragment, 4 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 800 μM), 0.2 mg / mL RNA ligase, 387 V, 50 mM Tris-HCl, pH 7.5, 16 °C, 16 h;

[0091] 2)++ indicates 50-70% (excluding the 70% endpoint value).

[0092] The molecular weight of the positive-sense product was determined by LC-MS to be 6761.0, and the molecular weight of the antisense product was 6656.9. The theoretical values ​​for the positive-sense product were 6761.0±8 and the theoretical values ​​for the antisense product were 6656.9±8, indicating that Ligase 25 was linked to form Patisiran. The LC-MS detection results for Ligase 8 are shown in Figure 4.

[0093] Example 3

[0094] The annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation under the following conditions: the reaction system was 10 mL, containing reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT), and RNA ligase. The reaction was carried out at 16°C for 16 h. After the overnight reaction, the protein was inactivated by heating at 50°C for 10-20 min. The supernatant was collected by centrifugation, purified using a Nano-Q column, eluted with a NaCl gradient, desalted by membrane coating (molecular weight cutoff 1 kDa), and lyophilized to obtain a dry powder. The calculated yield was 66.18%, and the purity (HPLC detection) was 90.18%.

[0095] Example 4

[0096] The four single-stranded RNA fragments based on Patisiran sequences are shown in Table 4, with length units in nt.

[0097] Table 4

[0098] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and the 'd' before T indicates that the sugar in the nucleotide is deoxyribose.

[0099] The ribonucleotides at positions 2, 5, 6, and 12 of substrate 5 have a 2' methoxy group modification.

[0100] The ribonucleotides at positions 2, 3, 4, 5, and 7 of substrate 6 have a 2' methoxy group, and positions 8 and 9 are thymine containing deoxyribose.

[0101] The ribonucleotide at position 6 of substrate 7 has a 2' methoxy group, and positions 9 and 10 are thymine containing deoxyribose.

[0102] The 7th ribonucleotide of substrate 8 has a 2' methoxy group modification.

[0103] The above four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0104] The sense strand of the prepared Patisiran is GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT (SEQ ID NO: 19), and the antisense strand is AUGGAAUmACUCUUGGUUmACdTdT (SEQ ID NO: 20).

[0105] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 50 μL, and the following were added sequentially to the reactor: 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 8, 50 mM Tris-HCl, pH 7.5, 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 collected by centrifugation. The results were analyzed by HPLC, and the yield was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the proportion of the target peak in the sample was 71.3%, i.e., the yield was +++.

[0106] Example 5

[0107] The six single-stranded RNA fragments based on Patisiran sequences are shown in Table 5, with length units in nt.

[0108] Table 5

[0109] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and the 'd' before T indicates that the sugar in the nucleotide is deoxyribose.

[0110] The ribonucleotides at positions 2, 5, and 6 of substrate 9 have 2' methoxy groups modified.

[0111] The 6th ribonucleotide of substrate 10 has a 2' methoxy group modification.

[0112] The ribonucleotides at positions 1, 2, 3, 4 and 6 of substrate 11 have a 2' methoxy group, and positions 7 and 8 are thymine containing deoxyribose.

[0113] The first ribonucleotide of substrate 12 has a 2' methoxy group, and the fourth and fifth positions are thymine containing deoxyribose.

[0114] The 7th ribonucleotide of substrate 14 has a 2' methoxy group modification.

[0115] The above six single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0116] The sense strand of the prepared Patisiran is GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT (SEQ ID NO: 19), and the antisense strand is AUGGAAUmACUCUUGGUUmACdTdT (SEQ ID NO: 20).

[0117] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 50 μL, and the following were added sequentially to the reactor: 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), 0.2 mg / mL Ligase 25, 239V 50 mM Tris-HCl, pH 7.5. The reaction was carried out at 16℃ for 16 h. After the reaction, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The results were analyzed by HPLC, and the yield was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the proportion of the target peak in the sample was 75.6%, i.e., the yield was +++.

[0118] Comparative Example 1

[0119] The average yield of the full-length Patisiran product synthesized by solid-phase synthesis was 27.9%, with N+1 and N-1 impurities accounting for a total of 1.51%.

[0120] The yield of Patisiran product prepared using the enzyme-linked method of this invention was 63.17%, while the average yield of the substrates used in solid-phase synthesis was 45.2%. Multiplying these yields, the overall yield of the process was 28.6%, which is slightly higher than the average yield of the product obtained by solid-phase synthesis. Furthermore, the total proportion of N+1 and N-1 impurities was 0.39%, which is lower than the proportion of such impurities in the process of synthesizing Patisiran by solid-phase synthesis.

[0121] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the preparation method of this application, four short substrate fragments (less than 21 nt in length) are first synthesized, and then full-length Patisiran is synthesized by enzymatic ligation, thereby realizing the preparation of this siRNA drug by biosynthesis. Due to the shortening of the length of the synthesized fragments, the N+1 and N-1 impurities generated during the synthesis process are correspondingly reduced; the ligation efficiency of N+1 and N-1 impurities in the substrate fragments is reduced, further reducing the N+1 and N-1 impurities in the full-length product; in addition, the chain lengths of N+1 and N-1 impurities in the substrate fragments differ significantly from those in the full-length product, making them easy to remove during purification, and ultimately the content of N+1 and N-1 impurities is <0.5%. Compared with chemical synthesis methods, the preparation method of this application yields products with high purity, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent usage, reduces production costs, and facilitates large-scale industrial production.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing Patisiran, characterized in that, Patisiran is a double-stranded RNA composed of complementary sense and antisense strands; The preparation method includes: A positive sense substrate fragment, an antisense substrate fragment, and an RNA ligase are mixed, wherein the positive sense substrate fragment is capable of forming the positive sense strand, and the antisense substrate fragment is capable of forming the antisense strand; The positive and negative substrate fragments are connected by hydrogen bonds formed by complementary bases. The head and tail bases of the positive and negative substrate fragments are not connected to each other, forming a double-stranded nucleotide structure with notches. The bases at both ends of the notch are linked by phosphodiester bonds using the RNA ligase to form the Patisiran; The bases at both ends of the notch are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; The 5' phosphate group and the 3' hydroxyl group upstream and downstream of the notch are linked using the RNA ligase to form the phosphodiester bond, thereby obtaining Patisiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is an RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; Or an RNA ligase with more than 70% identity to the amino acid sequence shown in SEQ ID NO: 1, and an enzyme with catalytic activity in forming phosphodiester bonds.

3. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the sense strand is SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is SEQ ID NO:

20.

4. The preparation method according to any one of claims 1-3, characterized in that, The justice chain substrate fragment includes two or more fragments, and the antisense chain substrate fragment includes two or more fragments; The length of the substrate segment of the justice chain is 5-12 nt; The antisense substrate fragment has a length of 2-14 nt.

5. The preparation method according to claim 4, characterized in that, The justice chain substrate segment includes a first justice chain substrate segment and a second justice chain substrate segment, and the antisense chain substrate segment includes a first antisense chain substrate segment and a second antisense chain substrate segment; The preparation method includes: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, and the second antisense strand substrate fragment; under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are ligated to form the sense strand; the first antisense strand substrate fragment and the second antisense strand substrate fragment are ligated to form the antisense strand; and the sense strand and the antisense strand form the Patisiran through complementary base pairing.

6. The preparation method according to claim 5, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO: 5, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO: 6; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 8, and the nucleotide sequence of the second antisense substrate fragment is GUUmACdTdT.

7. The preparation method according to claim 6, characterized in that, The nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment is AUmUmCmCmAUmdTdT. The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate fragment is SEQ ID NO:

11.

8. The preparation method according to claim 5, characterized in that, The 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated together under the catalysis of the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated together under the catalysis of the RNA ligase to form the antisense strand.

9. The preparation method according to claim 8, characterized in that, The first positive-chain substrate fragment has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second positive-chain substrate fragment has a phosphate group at its 5' end and a hydroxyl group at its 3' end. The first antisense substrate fragment has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second antisense substrate fragment has a phosphate group at its 5' end and a hydroxyl group at its 3' end.

10. The preparation method according to claim 4, characterized in that, The substrate segment of the justice chain includes a first justice chain substrate segment, a second justice chain substrate segment, and a third justice chain substrate segment; The antisense substrate fragment includes a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment.

11. The preparation method according to claim 10, characterized in that, The nucleotide sequence of the first positive-strand substrate fragment is GUmAACmCm; The nucleotide sequence of the second positive-strand substrate fragment is AAGAGUmA; The nucleotide sequence of the third positive-strand substrate fragment is UmUmCmCmAUmdTdT; The nucleotide sequence of the first antisense substrate fragment is UmACdTdT; The nucleotide sequence of the second antisense substrate fragment is UCUUGGU; The nucleotide sequence of the third antisense substrate fragment is AUGGAAUmAC.

12. The preparation method according to claim 10, characterized in that, The preparation method includes: The first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment, and the RNA ligase are mixed; Under the catalysis of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment, and the third sense strand substrate fragment are linked to form the sense strand, and the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the third antisense strand substrate fragment are linked to form the antisense strand. The sense strand and the antisense strand form the Patisiran through complementary base pairing.

13. The preparation method according to any one of claims 1-3, characterized in that, 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. The concentrations of the sense substrate fragment and the antisense substrate fragment are each independently selected from 0.1-4.5 mM.

14. The preparation method according to claim 1, characterized in that, The reaction temperature of the preparation method is 10-40℃; The reaction time for the preparation method is 2-48 hours.

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