Method for preparing sirna for treating non-arteritic anterior ischemic optic neuropathy

Abstract

Provided in the present application is a method for preparing an siRNA for treating non-arteritic anterior ischemic optic neuropathy. The siRNA is cosdosiran, which is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method comprises mixing sense strand substrates, antisense strand substrates and an RNA ligase, and using the RNA ligase to catalyze the ligation between the sense strand substrates and between the antisense strand substrates via phosphodiester bonds, so as to obtain the sense strand and the antisense strand, and thereby obtaining cosdosiran. The sense strand substrates can form the sense strand, and the antisense strand substrates can form the antisense strand. Compared with the preparation of cosdosiran by means of chemical synthesis, a product obtained by means of the preparation method of the present application has a relatively high purity and few impurities. Moreover, the reaction conditions are mild, thus facilitating the realization of industrial scaled-up production.

Description

Preparation method of siRNA for treating non-arterial ischemic optic neuropathy

[0001] This application is based on and claims priority to Chinese application CN application number 202410921741.7 filed on July 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of drug biosynthesis, and more specifically, to a method for preparing siRNA for treating non-arterial ischemic optic neuropathy. 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] Cosdosiran is a double-stranded siRNA drug co-developed with Quark and is currently in Phase 3 clinical trials. Cosdosiran can be used to treat non-arteritic anterior ischemic optic neuropathy (NAION).

[0005] The existing synthetic method for cosdosiran is a chemical solid-phase synthesis, using a solid support such as controlled-porous glass (CPG) or polystyrene resin. The oligonucleotide chain is extended along the 3' to 5' direction via a cyclic synthesis using a phosphoramidite method. After the synthesis cycle is complete, the cosdosiran chain is removed from the solid support by ammonolysis, followed by purification to obtain the pure product. This synthetic process requires expensive nucleic acid synthesizers, and the scale of synthesis is limited by the size of the synthesizer. Batch processes are on the order of kg, and increasing the synthesis throughput requires adding more equipment or more batches, making scale-up very costly. Furthermore, solid-phase synthesis is a cyclic method, and the yield decreases with increasing chain length. Impurities generated during the synthesis process, such as impurities 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, ultimately resulting in N+1 and N-1 impurity contents of 1-3%. Summary of the Invention

[0006] The main objective of this invention is to provide a method for preparing siRNA for treating non-arterial ischemic optic neuropathy, thereby solving the problem of low purity in the preparation of Cosdosiran in the prior art.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for preparing siRNA for treating non-arterial ischemic optic neuropathy is provided. The siRNA is Cosdosiran, which is a double-stranded RNA composed of complementary positive and negative strands. The preparation method includes: mixing a positive strand substrate, an antisense strand substrate, and an RNA ligase, wherein the positive strand substrate can form a positive strand, and the antisense strand substrate can form an antisense strand; the positive and antisense strand substrates are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrates are not linked to each other, forming a double-stranded nucleotide structure with a nick; using the RNA ligase to link the bases at both ends of the nick with phosphodiester bonds to form Cosdosiran; the bases at both ends of the nick are the 5' and 3' ends of different substrates, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; using the 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, thereby obtaining Cosdosiran.

[0008] Further, the RNA ligase is selected from RNA ligase family 2; the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2; or an enzyme that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2 and has catalytic activity for the formation of phosphodiester bonds.

[0009] Furthermore, the sense and antisense substrates are obtained by solid-phase synthesis or liquid-phase synthesis; preferably, the sense strand is the nucleic acid sequence shown in SEQ ID NO: 8, and the antisense strand is the nucleic acid sequence shown in SEQ ID NO: 9.

[0010] Furthermore, the justice chain consists of two or more justice chain substrates, the length of which is 3-16nt, more preferably 5-12nt; preferably, the antisense chain consists of two or more antisense chain substrates, the length of which is 3-16nt, more preferably 7-14nt.

[0011] Furthermore, the double-stranded RNA formed after annealing the sense and antisense substrates has three or more complementary base pairs; preferably, the ends of the double-stranded RNA are sticky ends; preferably, the sticky ends are 2-7 nt in length.

[0012] Furthermore, both the sense and antisense substrates comprise two substrates: the sense substrate comprises a first sense substrate and a second sense substrate, and the antisense substrate comprises a first antisense substrate and a second antisense substrate. The preparation method includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate; using RNA ligase to catalyze the ligation of the first and second sense substrates to form a sense strand; catalyzing the ligation of the first and second antisense substrates to form an antisense strand; and the sense and antisense strands complementaryly pairing to form Cosdosiran.

[0013] Further, the 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are ligated under the catalysis of RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated under the catalysis of RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate is an ia protecting group and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate is a phosphate group and the 3' end is a hydroxyl protecting group; preferably, the 5' end of the first antisense strand substrate is a hydroxyl protecting group and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate is a phosphate group and the 3' end is a hydroxyl protecting group.

[0014] Furthermore, the first sense strand substrate is the nucleic acid sequence GCCAGA or the nucleic acid sequence shown in SEQ ID NO: 10, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence AACUCCU.

[0015] Furthermore, the first antisense substrate is the nucleic acid sequence AGmGAmGUmUCm or the nucleic acid sequence shown in SEQ ID NO: 11, and the second antisense substrate is the nucleic acid sequence shown in SEQ ID NO: 7 or the nucleic acid sequence CmUGmGCm.

[0016] Furthermore, both the justice chain substrate and the antisense chain substrate include three substrates: the justice chain substrate includes the first justice chain substrate, the second justice chain substrate, and the third justice chain substrate; the antisense chain substrate includes the first antisense chain substrate, the second antisense chain substrate, and the third antisense chain substrate.

[0017] Furthermore, the first-strand substrate is the nucleic acid sequence GCCA; the second-strand substrate is the nucleic acid sequence GAUGUG; and the third-strand substrate is the nucleic acid sequence GAACUCCU.

[0018] Furthermore, the first antisense substrate is the nucleic acid sequence AGmGAmG; the second antisense substrate is the nucleic acid sequence UmUCmCACm; and the third antisense substrate is the nucleic acid sequence AUmUCmUGmGCm.

[0019] Furthermore, the concentrations of the sense and antisense substrates are 0.1-4.5 mM, more preferably 0.8-1.6 mM.

[0020] Further, the concentration of RNA ligase is 0.05-0.6 mg / mL, more preferably 0.2 mg / mL; preferably, the reaction system formed by mixing the sense substrate, antisense substrate, and RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the temperature of the enzyme-catalyzed reaction is 0-60℃, more preferably 4-37℃; preferably, the time of the enzyme-catalyzed reaction is 0.5-24 h, more preferably 16-24 h; preferably, the pH value of the enzyme-catalyzed reaction is 6.0-8.5; preferably, after the enzyme-catalyzed reaction, the product is purified and freeze-dried to obtain Cosdosiran.

[0021] By applying the technical solution of this invention and utilizing the above-described preparation method, RNA ligase catalyzes the ligation between sense and antisense substrates to form the sense strand of Cosdosiran, and catalyzes the ligation between antisense substrates to form the antisense strand of Cosdosiran. This allows for the biosynthetic preparation of this complex siRNA with multiple modifications. Compared to the chemical synthesis of Cosdosiran, the preparation method of this application yields a product with higher purity and fewer impurities, and the reaction conditions are mild, facilitating industrial-scale production. Attached Figure Description

[0022] 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:

[0023] Figure 1 shows a schematic diagram of the synthesis of Cosdosiran using RNA ligase according to the present invention;

[0024] Figure 2 shows the Urea-PAGE detection results of the catalytic products of ligase 25, ligase 48 and ligase 11 in Example 1 of the present invention;

[0025] Figure 3 shows the HPLC detection results of ligase 48 in Example 2 of the present invention. Detailed Implementation

[0026] 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.

[0027] Terminology Explanation:

[0028] N-1 impurities: Nucleic acid impurities that have a single nucleotide deletion compared to the target synthetic sequence.

[0029] N+1 impurities: Nucleic acid impurities that have an additional single nucleotide link compared to the target synthetic sequence.

[0030] As mentioned in the background section, existing technologies for preparing Cosdosiran employ chemical synthesis, which not only utilizes large amounts of organic solvents, contradicting the principles of green chemistry, but also incurs very high costs for scaling up the synthesis. Furthermore, the prepared product suffers from low purity and numerous impurities, particularly the presence of difficult-to-remove N+1 and N-1 impurities, affecting subsequent product purification. In this application, the inventors attempt to develop a method for preparing siRNA for treating non-arterial ischemic optic neuropathy, utilizing enzymatic catalysis to synthesize Cosdosiran, and thus propose a series of protective schemes for this application.

[0031] In a first typical embodiment of this application, a method for preparing siRNA for treating non-arterial ischemic optic neuropathy is provided. The siRNA is Cosdosiran, which is a double-stranded RNA composed of complementary positive and negative strands. The method includes: mixing a positive strand substrate, an antisense strand substrate, and an RNA ligase, wherein the positive strand substrate can form a positive strand, and the antisense strand substrate can form an antisense strand; the positive and antisense strand substrates are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrates are not linked to each other, 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 Cosdosiran; the bases at both ends of the nick are the 5' and 3' ends of different substrates, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; using RNA ligase to link the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form phosphodiester bonds, thereby obtaining Cosdosiran.

[0032] In a preferred embodiment, the RNA ligase is selected from RNA ligase family 2; the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2; or an enzyme having more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having catalytic activity in forming phosphodiester bonds. The notched double-stranded nucleotide structure formed by the above-mentioned sense substrate and antisense substrate has sticky ends.

[0033] In the above preparation method, the positive strand substrate consists of two or more nucleotide sequences that can form the positive strand. That is, multiple nucleotide sequences of the positive strand substrate can be spliced ​​together to form a sequence identical to the positive strand sequence. The difference between the positive strand and the positive strand is that the positive strand substrate has notches and is not linked by phosphodiester bonds. Similarly, RNA ligase is used to link two or more antisense strand substrates by phosphodiester bonds to obtain the antisense strand of Cosdosiran.

[0034] In the above preparation method, the sense and antisense substrates and RNA ligase are mixed together and prepared directly in a one-pot method to obtain Cosdosiran. In the one-pot ligation, the sense and antisense substrates can perform complementary base pairing to form a double-stranded structure. After recognizing this double-stranded structure, the RNA ligase ligates the nicks in the double-stranded structure to obtain the target product Cosdosiran.

[0035] Any method capable of RNA synthesis is applicable to this application. In a preferred embodiment, the sense and antisense substrates are obtained by solid-phase or liquid-phase synthesis. In a preferred embodiment, the sense strand is the nucleic acid sequence shown in SEQ ID NO: 8, and the antisense strand is the nucleic acid sequence shown in SEQ ID NO: 9.

[0036] SEQ ID NO: 8:

[0037] GCCAGAAUGUGGAACUCCU.

[0038] SEQ ID NO: 9:

[0039] AGmGAmGUmUCmCACmAUmUCmUGmGCm.

[0040] In this application, the m following A, C, G or U indicates a 2' methoxy modification of the ribonucleotide; the C at position 18 in SEQ ID NO: 8 is an L-type cytosine deoxyribonucleotide.

[0041] Cosdosiran has a sense strand length of 19 nt and an antisense strand length of 19 nt. By dividing the sense / antisense strands into several segments of different lengths based on their ligation efficiency and then ligating them via an enzymatic reaction, a product with high purity can be obtained. In a preferred embodiment, the sense strand consists of two or more sense strands of substrate with a length of 3-16 nt, more preferably 4-12 nt; preferably, the antisense strand consists of two or more antisense strands of substrate with a length of 3-16 nt, more preferably 5-14 nt.

[0042] In the one-pot synthesis of Cosdosiran, as shown in Figure 1, the sense and antisense substrates can undergo complementary base pairing to form a double-stranded structure. RNA ligase recognizes the notched double-stranded structure formed by the complementary substrate pairing and catalyzes a phosphate dihydration reaction between the two single-stranded RNA fragments, generating a new phosphodiester bond and releasing one water molecule, thereby linking the sense and antisense substrates to obtain Cosdosiran. In a preferred embodiment, the double-stranded RNA formed after annealing of the sense and antisense substrates contains three or more complementary base combinations. In a preferred embodiment, the double-stranded RNA ends are sticky ends; the sticky ends are 2-7 nt in length.

[0043] Any RNA ligase capable of recognizing notched double-stranded structures formed by complementary substrate pairing and catalyzing the formation of phosphodiester bonds between phosphate and hydroxyl groups is suitable for this application. In a preferred embodiment, the RNA ligase is selected from RNA ligase family 2. RNA ligase family 2 can catalyze the formation of phosphodiester bonds and can link multiple RNA molecules into relatively long chains. Ligase family 2 exhibits high activity for the ligation of double-stranded RNA and can also perform ligation reactions of single-stranded RNA. Appropriate enzymes can be selected for the reaction as needed.

[0044] In a preferred embodiment, the RNA ligase comprises one or more of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2; or has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2, 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, even more than 99.9%), and is an enzyme with catalytic activity for the formation of phosphodiester bonds. SEQ ID NO: 1: (Lyase 48, Escherichia phage JN02)

[0045] SEQ ID NO: 2: (Ligase 25, Vibrio phage NT-1)

[0046] SEQ ID NO: 3: (Ligase 11, Thermococcus)

[0047] SEQ ID NO: 4: (Lyse 20, Archea)

[0048] SEQ ID NO: 5: (Ligase 32, bacteria)

[0049] In this application, "identity" refers to the "identity" between amino acid sequences or nucleic acid sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid or nucleic acid sequences. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0050] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (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 have an active site, active pocket, active mechanism, protein structure, etc., which are highly likely to be the same as the protein provided by sequence a).

[0051] 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).

[0052] 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:

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

[0054] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

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

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

[0057] 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.

[0058] This application utilizes the RNA ligase shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an enzyme with more than 70% identity to any of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2, to catalyze the formation of phosphodiester bonds between the substrates in this application, yielding the product Cosdosiran. In the relevant experiments of this application, the inventors screened from a large number of enzymes (50 types) and obtained the aforementioned RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2 that are capable of synthesizing Cosdosiran. A large proportion (up to 70%) of the negative results in the experiments showed that most RNA ligases are unable to catalyze the synthesis of Cosdosiran, including but not limited to the RNA ligases shown in SEQ ID NO: 3 to SEQ ID NO: 5. This application specification only uses SEQ ID NO: 3 to SEQ ID NO: 5 as examples to illustrate this type of RNA ligase without Cosdosiran synthesis activity.

[0059] In a preferred embodiment, both the sense and antisense substrates comprise two substrates. The sense substrate comprises a first sense substrate and a second sense substrate, and the antisense substrate comprises a first antisense substrate and a second antisense substrate. The preparation method includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate; using RNA ligase to catalyze the ligation of the first sense substrate and the second sense substrate to form a sense strand; catalyzing the ligation of the first antisense substrate and the second antisense substrate to form an antisense strand; and the sense and antisense strands complementaryly pairing to form Cosdosiran.

[0060] In a preferred embodiment, the 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated by RNA ligase to form an antisense strand; preferably, the 5' end of the first sense strand substrate is an ia protecting group and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate is a phosphate group and the 3' end is a hydroxyl protecting group; preferably, the 5' end of the first antisense strand substrate is a hydroxyl protecting group and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate is a phosphate group and the 3' end is a hydroxyl protecting group.

[0061] In a preferred embodiment, the first positive strand substrate is the nucleic acid sequence GCCAGA or the nucleic acid sequence shown in SEQ ID NO: 10, and the second positive strand substrate is the nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence AACUCCU.

[0062] In a preferred embodiment, the first antisense substrate is the nucleic acid sequence AGmGAmGUmUCm or the nucleic acid sequence shown in SEQ ID NO: 11, and the second antisense substrate is the nucleic acid sequence shown in SEQ ID NO: 7 or the nucleic acid sequence CmUGmGCm.

[0063] Cosdosiran can be prepared using the above-described preparation method and the substrates shown in GCCAGA, AACUCCU, AGmGAmGUmUCm, CmUGmGCm, SEQ ID NOs: 6-7, and SEQ ID NOs: 10-11. However, it should be noted that the choice of substrates is not limited to those shown in GCCAGA, AACUCCU, AGmGAmGUmUCm, CmUGmGCm, SEQ ID NOs: 6-7, and SEQ ID NOs: 10-11. Substrates capable of forming both the sense and antisense strands can be used in the above preparation method. The above preparation method is applicable to the preparation of Cosdosiran but is not limited to different substrate connection positions. The above preparation method shows good connection effects for both the sense and antisense strand sequences of Cosdosiran. The number of sense or antisense strand substrates includes, but is not limited to, 2, 3, 4, or even more. Among them, the C at position 12 of SEQ ID NO:6 and the C at position 6 of AACUCCU are L-type cytosine deoxyribonucleotides.

[0064] SEQ ID NO: 6:

[0065] AUGUGGAACUCCU.

[0066] SEQ ID NO: 7:

[0067] CACmAUmUCmUGmGCm.

[0068] SEQ ID NO: 10:

[0069] GCCAGAAUGUGG.

[0070] SEQ ID NO: 11:

[0071] AGmGAmGUmUCmCACmAUmU.

[0072] In a preferred embodiment, both the justice chain substrate and the antisense chain substrate include three substrates: the justice chain substrate includes a first justice chain substrate, a second justice chain substrate, and a third justice chain substrate; the antisense chain substrate includes a first antisense chain substrate, a second antisense chain substrate, and a third antisense chain substrate.

[0073] In a preferred embodiment, the first positive strand substrate is the nucleic acid sequence GCCA; the second positive strand substrate is the nucleic acid sequence GAUGUG; and the third positive strand substrate is the nucleic acid sequence GAACUCCU.

[0074] In a preferred embodiment, the first antisense substrate is the nucleic acid sequence AGmGAmG; the second antisense substrate is the nucleic acid sequence UmUCmCACm; and the third antisense substrate is the nucleic acid sequence AUmUCmUGmGCm. In GAACUCCU, the C at position 7 is an L-type cytosine deoxyribonucleotide.

[0075] In a preferred embodiment, the concentrations of the sense and antisense substrates are 0.1-4.5 mM, more preferably 0.8-1.6 mM.

[0076] In a preferred embodiment, the concentration of RNA ligase is 0.05-0.6 mg / mL, more preferably 0.2 mg / L; preferably, the reaction system formed by mixing the sense substrate, antisense substrate, and RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the temperature of the enzyme-catalyzed reaction is 0-60°C, more preferably 4-37°C; preferably, the reaction time is 0.5-24 h, more preferably 16-24 h; preferably, the pH value of the enzyme-catalyzed reaction is 6.0-8.5; preferably, after the enzyme-catalyzed reaction, the product is purified and freeze-dried to obtain Cosdosiran.

[0077] 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.

[0078] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0079] Example 1

[0080] The following four single-stranded RNA fragments were designed based on the Cosdosiran sequence:

[0081] The four single-stranded RNA fragments were prepared using a solid-phase synthesis method. The 'm' following A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and the C at position 12 in SEQ ID NO:6 is an L-type cytosine deoxyribonucleotide.

[0082] Cosdosiran-1 has an ia protecting group added to its 5' end; Cosdosiran-2 has a deoxyribonucleotide at position 12, which is cytosine (C); Cosdosiran-3 has 2' methoxy groups on its ribonucleotides at positions 3, 5, 7, 9, and 11; and Cosdosiran-4 has 2' methoxy groups on its ribonucleotides at positions 2, 4, 6, and 8.

[0083] The prepared Cosdosiran has a sense strand of 5'-R-GCCAGAAUGUGGAACUCCU-3' (SEQ ID NO:8) and an antisense strand of 5'-AGmGAmGUmUCmCACmAUmUCmUGmGCm-3' (SEQ ID NO:9). The 5' end of the sense strand is protected by an ia group (a 3'5' reverse-linked deoxyribose).

[0084] Four single-stranded RNA fragments were mixed in equimolar proportions to obtain a substrate mixture, which was then annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzymatic ligation reaction under the following conditions: 100 μM substrate fragment, 10 eq ATP, 100 eq MgCl2, 10 eq DTT, and RNA ligase at a final concentration of 0.2 mg / mL were added sequentially to a 10 μL reactor. The reaction was carried out at 4814V, 50 mM Tris-HCl, and pH 7.5, and incubated at 16℃ for 16 h. After the reaction, the protein was inactivated by heating at 80℃ for 5 min. The supernatant was collected by centrifugation, and the Urea-PAGE results are shown in Figure 2. In Figure 2, lane M represents the RNA molecule marker, lane 1 represents the reaction system of ligase 25, lanes 2 and 3 represent the reaction systems of ligase 48, and lane 4 represents the reaction system of ligase 11. The yield was estimated based on the grayscale analysis of the product bands in the Urea-PAGE results. The final yield results are shown in the table below.

[0085] In Example 1, the yield was estimated based on the grayscale analysis results of the target band in the Urea-PAGE results. "None" indicates that no target product band was detected, "++" indicates a yield of 25-50% (excluding the 50% endpoint), "+++" indicates a yield of 50-90%, and "++++" indicates a yield >90%.

[0086] Grayscale data of the product and substrate were obtained through grayscale analysis of the Urea-PAGE gel electrophoresis results. In this example, the yield calculation formula is: Yield = Product grayscale data / (Product grayscale data + Substrate grayscale data). It was found that ligases 25 and 48 had good ligation performance, capable of converting most of the substrate into Cosdosiran.

[0087] Example 2:

[0088] Ligase 48, with good reactivity, was selected for enzymatic ligation using an annealed substrate fragment. The reaction conditions were as follows: 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, and 1.25 eq DTT were added sequentially to a 50 μL reactor, resulting in a final concentration of 0.2 mg / mL RNA ligase. The reaction was carried out at 239 V with 50 mM Tris-HCl at pH 7.5, and incubated 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 LC-MS. An example of the HPLC results for ligase 48 is shown in Figure 3.

[0089] The yield was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample, and the results are as follows:

[0090] In Example 2, the yield was calculated based on the statistical results of the target peak in the HPLC results, where ++ indicates 70-80%, +++ indicates 80-90%, and ++++ indicates >90%.

[0091] The molecular weight of the positive strand product was determined by LC-MS to be 6221.90 and the molecular weight of the negative strand product was 6160.99. The theoretical values ​​for the positive strand product and the negative strand product were 6221.89±8, indicating that ligase 48 ligated to generate Cosdosiran.

[0092] Comparative Example 1

[0093] The average yield of the full-length Cosdosiran product synthesized using solid-phase synthesis was 25.2%, with N+1 and N-1 impurities accounting for a total of 1.51%.

[0094] The Cosdosiran product produced using the enzymatic ligation method described in this invention achieved a yield of 68.73% in the enzymatic synthesis step, while the average yield of the substrates used in solid-phase synthesis was 41.3%. Multiplying these yields, the overall yield of the process was 28.39%, which is higher than the average yield of the full-length Cosdosiran product synthesized in solid-phase synthesis. Furthermore, the total proportion of N+1 and N-1 impurities in the enzymatically synthesized Cosdosiran product was 0.39%, which is lower than the proportion of such impurities in the full-length Cosdosiran product synthesized in solid-phase synthesis.

[0095] 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, RNA ligase catalyzes the ligation between sense strand substrates to form the sense strand of Cosdosiran, and catalyzes the ligation between antisense strand substrates to form the antisense strand of Cosdosiran, thereby realizing the preparation of this structurally complex siRNA with multiple modifications through biosynthesis. Compared with the preparation of Cosdosiran by chemical synthesis, the preparation method of this application yields products with higher purity, fewer impurities (N+1 and N-1 impurities <0.5%), lower pressure for subsequent final product purification, and milder reaction conditions without the use of large amounts of organic reagents, which can reduce production costs and facilitate industrial-scale production.

[0096] 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 siRNA for treating non-arterial ischemic optic neuropathy, characterized in that, The siRNA is Cosdosiran, which is a double-stranded RNA composed of complementary sense and antisense strands; The preparation method includes: A positive sense substrate, an antisense substrate, and an RNA ligase are mixed, wherein the positive sense substrate is capable of forming the positive sense strand, and the antisense substrate is capable of forming the antisense strand; The positive and negative substrates are connected by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and negative substrates 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 Cosdosiran; The bases at both ends of the notch are the 5' and 3' ends of different substrates, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; The phosphate group at the 5' end and the hydroxyl group at the 3' end of the notch are linked together using the RNA ligase to form the phosphodiester bond, thereby obtaining the Cosdosiran.

2. The preparation method according to claim 1, characterized in that, The RNA ligase is selected from RNA ligase family 2; The RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2; or It has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 or SEQ ID NO: 2 and has the activity of catalyzing the formation of the phosphodiester bond.

3. The preparation method according to claim 1, characterized in that, The sense and antisense substrates are obtained by solid-phase synthesis or liquid-phase synthesis. Preferably, the sense strand is the nucleic acid sequence shown in SEQ ID NO: 8, and the antisense strand is the nucleic acid sequence shown in SEQ ID NO:

9.

4. The preparation method according to claim 1, characterized in that, The justice chain consists of two or more justice chain substrates, and the length of the justice chain substrate is 3-16nt. Preferably, the antisense chain consists of two or more antisense chain substrates, and the length of the antisense chain substrate is 3-16 nt.

5. The preparation method according to claim 3, characterized in that, The double-stranded RNA formed by annealing the sense and antisense substrates has three or more complementary base pairs. Preferably, the ends of the double-stranded RNA are sticky ends; Preferably, the length of the viscous end is 2-7 nt.

6. The preparation method according to claim 3, characterized in that, Both the justice chain substrate and the antisense chain substrate include two substrates. The justice chain substrate includes a first justice chain substrate and a second justice chain substrate. The antisense chain substrate includes a first antisense chain substrate and a second antisense chain substrate. The preparation method includes: mixing the first sense strand substrate, the second sense strand substrate, the first antisense strand substrate, and the second antisense strand substrate; using RNA ligase to catalyze the ligation of the first sense strand substrate and the second sense strand substrate to form the sense strand; catalyzing the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand; and the sense strand and the antisense strand complementaryly pairing to form the Cosdosiran.

7. The preparation method according to claim 6, characterized in that, The 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are ligated under the catalysis of the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated under the catalysis of the RNA ligase to form the antisense strand. Preferably, the 5' end of the first positive chain substrate is a protecting group (ia) and the 3' end is a hydroxyl group; the 5' end of the second positive chain substrate is a phosphate group and the 3' end is a hydroxyl protecting group. Preferably, the 5' end of the first antisense substrate is a hydroxyl protecting group and the 3' end is a hydroxyl group; the 5' end of the second antisense substrate is a phosphate group and the 3' end is a hydroxyl protecting group.

8. The preparation method according to claim 7, characterized in that, The first positive-strand substrate is the nucleic acid sequence GCCAGA or the nucleic acid sequence shown in SEQ ID NO: 10, and the second positive-strand substrate is the nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence AACUCCU.

9. The preparation method according to claim 7, characterized in that, The first antisense substrate is the nucleic acid sequence AGmGAmGUmUCm or the nucleic acid sequence shown in SEQ ID NO: 11, and the second antisense substrate is the nucleic acid sequence shown in SEQ ID NO: 7 or the nucleic acid sequence CmUGmGCm.

10. The preparation method according to claim 3, characterized in that, Both the justice chain substrate and the antisense chain substrate include three substrates. The base of the justice chain includes a first justice chain base, a second justice chain base, and a third justice chain base; The antisense substrate includes a first antisense substrate, a second antisense substrate, and a third antisense substrate.

11. The preparation method according to claim 10, characterized in that, The first positive chain substrate is the nucleic acid sequence GCCA; The second positive-signal substrate is the nucleic acid sequence GAAUG; The third positive chain substrate is the nucleic acid sequence GAACUCCU.

12. The preparation method according to claim 10, characterized in that, The first antisense substrate is the nucleic acid sequence AGmGAmG; The second antisense substrate is the nucleic acid sequence UmUCmCACm; The third antisense substrate is a nucleic acid sequence AUmUCmUGmGCm.

13. The preparation method according to any one of claims 1-5, characterized in that, The concentrations of the sense and antisense substrates are 0.1-4.5 mM, more preferably 0.8-1.6 mM.

14. The preparation method according to any one of claims 1-5, characterized in that, The concentration of the RNA ligase is 0.05-0.6 mg / mL, more preferably 0.2 mg / mL; Preferably, the reaction system formed by mixing the sense substrate, the antisense substrate, and the RNA ligase further includes ATP, Tris-HCl, MgCl2, and DTT. Preferably, the temperature of the enzyme-catalyzed reaction is 0-60°C, more preferably 4-37°C; Preferably, the enzyme-catalyzed reaction time is 0.5-24 h, more preferably 16-24 h; Preferably, the pH value of the enzyme-catalyzed reaction is 6.0-8.5; Preferably, after the enzyme-catalyzed reaction, the product is purified and freeze-dried to obtain the Cosdosiran.

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