CN118460647A - A preparation method of Patisiran - Google Patents

Abstract

The invention provides a preparation method of PATISIRAN. The PATISIRAN is a double-stranded RNA consisting of a complementarily paired sense strand and antisense strand; the preparation method comprises the following steps: mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment is capable of constituting a sense strand and the antisense strand substrate fragment is capable of constituting an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, and the head base and the tail base of the sense strand substrate fragment and the antisense strand substrate fragment are not connected with each other to form a double-stranded nucleotide structure containing the nicks; the bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form PATISIRAN. Can solve the problem of lower purity of PATISIRAN prepared in the prior art, and is suitable for the technical field of medicine biosynthesis.

Description

A preparation method of Patisiran

Technical Field

The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing Patisiran.

Background Art

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 widespread 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.

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

At present, the method for preparing Patisiran is a chemical method, using a solid phase carrier such as controlled microporous glass beads (Controlled Pore Glass, CPG) or polystyrene resin, and cyclically synthesizing by the phosphoramidite triester method to extend the oligonucleotide chain along the 3' to 5' direction. After the synthesis cycle is completed, the Patisiran 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, and the yield of the synthesis decreases with the increase of the synthesis chain length, and the impurities generated in the synthesis process, such as impurities with one more nucleotide than the target sequence (N+1 impurities) or impurities with one less nucleotide than the target sequence (N-1 impurities), will also increase with the increase of the synthesis chain length. When using the existing chemical method for solid phase synthesis of Patisiran, the synthesis length is 21nt, and the N+1 and N-1 impurities generated in the synthesis are more, which are not easy to remove through the purification process, and the final content of N+1 and N-1 impurities is 1-3%. Therefore, the cost of preparing Patisiran is high, and it is difficult to scale up production, so it is necessary to develop a more efficient Patisiran synthesis method.

Summary of the invention

The main purpose of the present invention is to provide a method for preparing Patisiran, so as to solve the problem of low purity of Patisiran prepared in the prior art.

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

Furthermore, the nucleotide sequence of the sense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 20.

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 5-12nt, more preferably 6-11nt; preferably, the length of the antisense strand substrate fragment is 2-14nt, more preferably 5-13nt.

Furthermore, 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, connecting the first sense chain substrate fragment and the second sense chain substrate fragment to form a sense chain under the catalytic action of RNA ligase, 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 Patisiran 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 Patisiran.

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

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 a hydroxyl 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.

Further, 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; preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 13; the nucleotide sequence of the second sense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of the third sense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 15;

Preferably, the nucleotide sequence of the first antisense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the second antisense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 17; and the nucleotide sequence of the third antisense strand substrate fragment is the nucleic acid sequence nucleotide sequence shown in SEQ ID NO: 16.

Furthermore, the preparation method comprises: mixing the first sense chain substrate fragment, the second sense chain substrate fragment, the third sense chain substrate fragment, the first antisense chain substrate fragment, the second antisense chain substrate fragment, the third antisense chain substrate fragment and the RNA ligase; under the catalytic action of the RNA ligase, the first sense chain substrate fragment, the second sense chain substrate fragment and the third sense chain substrate fragment are connected to form the sense chain, the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third antisense chain substrate fragment are connected to form the antisense chain, and the sense chain and the antisense chain form Patisiran through base complementary pairing.

Furthermore, 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; preferably, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM; 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.

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 Patisiran sense strand, and the antisense strand substrate fragments are connected to form the Patisiran antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method for preparing Patisiran by chemical synthesis, the product obtained by the preparation method of the present application has high purity, few impurities, simple preparation process, mild reaction conditions, low amount of organic reagents, reduced production costs, and is easy to achieve scale-up industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

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:

FIG. 1 shows a schematic diagram of an enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

FIG. 2 shows the electrophoresis results of the catalytic products of RNA ligases Ligase25 and Ligase11 according to Example 1 of the present invention.

FIG. 3 shows a graph showing the HPLC detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

FIG. 4 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

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.

Terminology explanation:

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

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

As mentioned in the background technology, the prior art uses a chemical synthesis method for the preparation of Patisiran, 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 the present application, the inventor attempts to develop a preparation method for Patisiran, and prepares Patisiran using enzyme-catalyzed synthesis, thus proposing a series of protection schemes of the present application.

In a first typical embodiment of the present application, a method for preparing Patisiran is provided, wherein Patisiran is a double-stranded RNA composed of a complementary sense strand and an antisense strand; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, so as to form a double-stranded nucleotide structure containing a nick; using RNA ligase to connect the bases at both ends of the nick by a phosphodiester bond to form Patisiran; 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 Patisiran; the RNA ligase is a polypeptide having SEQ ID An RNA ligase having the amino acid sequence shown in SEQ ID NO: 1; or an enzyme having 70% or more identity with the RNA ligase shown in SEQ ID NO: 1 and having activity of catalyzing the formation of phosphodiester bonds.

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

In the above-mentioned preparation method, the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase are mixed to prepare Patisiran. 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 the double-stranded nucleotide with a sticky end continues to bind to other substrate fragments to form a double-stranded nucleotide structure containing a nick. The RNA ligase of the present application can recognize the nick of this double-stranded structure and connect the nick with a phosphodiester bond, thereby preparing the target product Patisiran.

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

SEQ ID NO: 19: GUMAACmCmAAGAGUmAUmUmCmCmAUmdTdT.

SEQ ID NO: 20: AUGGAAUmACUCUUGGUUmACdTdT.

In the present application, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, and d before T indicates that the nucleotide is a deoxyribonucleotide thymine.

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

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

MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSE KRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.

SEQ ID NO: 2 (Ligase 11, Thermococcus):

MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKV GELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.

SEQ ID NO: 3 (Ligase 20, Archaea):

MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGS GNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQV ASDYLRIMERAIRKYRIPWPDRELLVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYK NVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

SEQ ID NO: 4 (Ligase 32, bacteria):

MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDE LYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.

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.

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.

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).

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:

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

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

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

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

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.

In the present application, the phosphate group and the hydroxyl group of the substrate in the present application can only be catalyzed by the RNA ligase shown in SEQ ID NO: 1, or an enzyme with more than 70% identity with the RNA ligase shown in SEQ ID NO: 1 to form a phosphodiester bond to obtain the product Patisiran. In the relevant experiments of the present application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 that can synthesize Patisiran by screening from a large number of enzymes. The negative results, which accounted for a large proportion in the experiment, showed that most RNA ligases were 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 the present application specification, only SEQ ID NO: 2 to SEQ ID NO: 4 are used as examples to reflect this type of RNA ligase that has no catalytic activity in Patisiran synthesis.

In a preferred embodiment, the sense chain substrate fragment includes 2 or more strands, and the antisense chain substrate fragment includes 2 or more strands; preferably, the length of the sense chain substrate fragment is 3-15nt, more preferably 6-11nt, and further preferably 8-10nt; preferably, the length of the antisense chain substrate fragment is 2-14nt, more preferably 5-13nt, and further preferably 7-10nt.

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 Patisiran 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 Patisiran.

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 Patisiran with a complete double-stranded structure.

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

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

Patisiran can be prepared using the above preparation method and the substrate fragments shown in SEQ ID NO: 5-SEQ ID NO: 8 or SEQ ID NO: 9-SEQ ID NO: 12. However, it should be noted that the selection of substrate fragments is not limited to the substrate fragments shown in SEQ ID NOs: 5-8 or SEQ ID NOs: 9-12. 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 Patisiran 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 Patisiran. The number of sense chain substrate fragments or antisense chain substrate fragments includes but is not limited to 2, 3, 4 or even more.

SEQ ID NO: 5: GUMAACmCmAAGA.

SEQ ID NO: 6: GUMAUmUmCmCmAUmdTdT.

SEQ ID NO: 7: GUUmACdTdT.

SEQ ID NO: 8: AUGGAAUmACUCUUG.

SEQ ID NO: 9: GUMAACmCmAAGAGUm.

SEQ ID NO: 10: AUmUmCmCmAUmdTdT.

SEQ ID NO: 11: UUGGUUmACdTdT.

SEQ ID NO: 12: AUGGAAUmACUC.

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 a hydroxyl 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.

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: 13; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16.

SEQ ID NO: 13: GUmAACmCm.

SEQ ID NO: 14: AAGAGUmA.

SEQ ID NO: 15: UmUmCmCmAUmdTdT.

SEQ ID NO: 16: UmACdTdT.

SEQ ID NO: 17: UCUUGGU.

SEQ ID NO: 18: AUGGAAUmAC.

In a preferred embodiment, the preparation method comprises: mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a third sense strand substrate fragment, a first antisense strand substrate fragment, a second antisense strand substrate fragment, a third antisense strand substrate fragment and RNA ligase; under the catalytic action of RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are connected 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 connected to form the antisense strand, and the sense strand and the antisense strand form Patisiran through base complementary pairing;

In a preferred embodiment, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are preferably 2.5-10 mM respectively; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase also includes ATP, Tris-HCl, MgCl ₂ 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.

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 48h.

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

Example 1

Four single-stranded RNA fragments were designed based on the sequence of Patisiran as shown in Table 1, and the length unit is nt.

Table 1

.

Among them, the m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, and the d before T indicates that the sugar in the nucleotide is deoxyribose.

The ribonucleotides at positions 2, 5 and 6 of substrate 1 have 2' methoxy modifications.

The ribonucleotides at positions 2, 4, 5, 6, 7 and 9 of substrate 2 have 2' methoxy modifications, and positions 10 and 11 are thymines containing deoxyribose.

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

The ribonucleotide at position 7 of substrate 4 has a 2' methoxy modification.

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

The four single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture with a final concentration of 2.5 mM (each substrate was 2.5 mM), and annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzyme-catalyzed ligation reaction. The reaction system was set to 10 μL, and the reaction system included a reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl ₂ , dithiothreitol (DTT), and RNA ligases Ligase 25, Ligase 11, Ligase 20, and Ligase 32 at a concentration of 0.2 mg/mL were added. The reaction system was reacted at 16°C for 16 hours. The obtained reaction system was inactivated by 80°C for 5 minutes, and the precipitate was removed by centrifugation at 12000 rpm. The schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

The products catalyzed by RNA ligase Ligase 25, Ligase 11, Ligase 20 and Ligase 32 were detected by SDS-PAGE. The electrophoresis results of the products catalyzed by RNA ligase Ligase 25 and Ligase 11 are shown in Figure 2. In Figure 2, lane M represents the RNA molecule standard (marker), lane 1 represents the reaction system of Ligase 25, and lane 2 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis results of the target band in the Urea-PAGE results, and the final yield results are shown in Table 2.

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

Table 2

.

Remark:

1) Reaction conditions: substrate fragment 100 μM, ATP 10 eq, MgCl ₂ 100 eq, DTT 10 eq, (1 eq = 100 μM), final concentration 0.2 mg/mL of the selected RNA ligase, 50 mM Tris-HCl pH 7.5, 16°C for 16 h;

2) ++ means 25~50%.

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

Example 2

The reaction conditions of this example 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), MgCl ₂ , dithiothreitol (DTT) and RNA ligase Ligase 25, and the reaction is carried out at 16° C. 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 samples were sent for HPLC and LC-MS detection. Pat-strand1 represents the positive strand, Pat-strand2 represents the antisense strand, 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 are shown in Table 3. After the reaction, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The samples were sent for HPLC and LC-MS detection. Taking the results of Ligase 25 as an example, the HPLC results are shown in Figure 3.

Table 3

.

Remark:

1) Reaction conditions: substrate fragment 800 μM, ATP 4 eq, MgCl ₂ 100 eq, DTT 10 eq (1 eq = 800 μM), RNA ligase at a final concentration of 0.2 mg/mL, 387 V, 50 mM Tris-HCl pH 7.5, 16°C, 16 h;

2) ++ means 50~70% (excluding the endpoint value of 70%).

The molecular weight of the sense strand product was 6761.0 and the molecular weight of the antisense strand product was 6656.9 by LC-MS. The theoretical values of the sense strand product were 6761.0±8 and 6656.9±8, respectively, indicating that Patisiran was generated by ligase 25. The LC-MS detection result of Ligase 8 is shown in FIG4 .

Example 3

The annealed substrate fragments and ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 10 mL, and the reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl ₂ , dithiothreitol (DTT) and RNA ligase, 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 10-20 minutes, and the supernatant was obtained by centrifugation and purified using a Nano-Q column, NaCl gradient elution, membrane package (molecular weight cutoff 1 kDa) desalting, and freeze-dried in a freeze dryer to obtain dry powder. The calculated yield was 66.18%, and the purity (HPLC detection) was 90.18%.

Example 4

Four single-stranded RNA fragments were designed based on the sequence of Patisiran as shown in Table 4, and the length unit is nt.

Table 4

.

Among them, the m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, and the d before T indicates that the sugar in the nucleotide is deoxyribose.

The ribonucleotides at positions 2, 5, 6 and 12 of substrate 5 have 2' methoxy modifications.

The ribonucleotides at positions 2, 3, 4, 5 and 7 of substrate 6 have 2' methoxy modifications, and positions 8 and 9 are thymines containing deoxyribose.

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

The ribonucleotide at position 7 of substrate 8 has a 2’ methoxy modification.

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

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

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 50 μL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl ₂ , 1.25 eq DTT (1 eq = 800 μM), 0.2 mg/mL Ligase 8 at a final concentration, 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 target peak accounted for 71.3% in the sample, that is, the yield was +++.

Example 5

Six single-stranded RNA fragments were designed based on the sequence of Patisiran as shown in Table 5, and the length unit is nt.

Table 5

.

Among them, the m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, and the d before T indicates that the sugar in the nucleotide is deoxyribose.

The ribonucleotides at positions 2, 5 and 6 of substrate 9 have 2' methoxy modifications.

The ribonucleotide at position 6 of substrate 10 has a 2' methoxy modification.

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

The first ribonucleotide of substrate 12 has a 2' methoxy modification, and the 4th and 5th positions are thymine containing deoxyribose.

The ribonucleotide at position 7 of substrate 14 has a 2' methoxy modification.

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

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

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 50 μL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl ₂ , 1.25 eq DTT (1 eq = 800 μM) were added to the reactor in sequence, and Ligase 25 with a final concentration of 0.2 mg/mL was added, 239V 50 mM Tris-HCl pH 7.5, and the reaction was carried out at 16 ° C for 16 hours. After the reaction, the protein was inactivated by heating at 80 ° C for 5 minutes, 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 target peak accounted for 75.6% in the sample, that is, the yield was +++.

Comparative Example 1

The average yield of the full-length Patisiran product using solid phase synthesis was 27.9%, and the total proportion of N+1 and N-1 impurities was 1.51%.

The yield of the Patisiran product prepared by the enzyme-linked method of the present invention is 63.17%, and the average solid-phase synthesis yield of the substrate used is 45.2%. The yield of the overall process is 28.6%, which is slightly higher than the average yield of the product obtained by the solid-phase synthesis method. The total proportion of N+1 and N-1 impurities is 0.39%, which is lower than the proportion of such impurities in the process of synthesizing Patisiran by solid-phase synthesis.

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, four short substrate fragments (less than 21 nt in length) are first synthesized, and then the full-length Patisiran is synthesized by enzyme ligation, thereby realizing the preparation of such siRNA drugs by biosynthesis. Since the length of the synthetic fragment is shortened, the N+1 and N-1 impurities generated in the synthesis process are also reduced accordingly; the connection efficiency of the N+1 and N-1 impurities in the substrate fragment is reduced, further reducing the N+1 and N-1 impurities in the full-length product; in addition, the chain length of the N+1 and N-1 impurities in the substrate fragment is quite different from that of the full-length product, and it is easy to remove them during the purification process, and the final content of the N+1 and N-1 impurities is <0.5%. Compared with the chemical synthesis preparation method, the preparation method of the present application has a high purity of the product, generates less impurities, has a simple preparation process, mild reaction conditions, low amount of organic reagents, reduces production costs, and is convenient for large-scale industrial production.

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 (13)

1. A method for preparing Patisiran, characterized in that the Patisiran is a double-stranded RNA consisting of a complementary sense strand and an antisense strand; The preparation method comprises: Mixing a sense strand substrate fragment, an antisense strand substrate fragment, and RNA ligase, wherein the sense strand substrate fragment can constitute the sense strand, and the antisense strand substrate fragment can constitute the 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 the RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form the Patisiran; The bases at both ends of the nick are the 5' end and 3' end of different substrate fragments, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; Using the RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form the phosphodiester bond, thereby obtaining the Patisiran; The RNA ligase is an RNA ligase having an amino acid sequence shown in SEQ ID NO: 1; Or an enzyme that has more than 70% identity with the RNA ligase of the amino acid sequence shown in SEQ ID NO: 1 and has activity of catalyzing the formation of phosphodiester bonds. 2. The preparation method according to claim 1, characterized in that the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO: 20. 3. The preparation method according to any one of claims 1 to 2, characterized in that the sense strand substrate fragments include 2 or more strands, and the antisense strand substrate fragments include 2 or more strands; The length of the positive strand substrate fragment is 5-12 nt; The length of the antisense strand substrate fragment is 2-14 nt. 4. The preparation method according to claim 3, characterized in that 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; under the catalytic action of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form the sense strand; catalyzing the connection of the first antisense strand substrate fragment and the second antisense strand substrate fragment to form the antisense strand; and the sense strand and the antisense strand form the Patisiran through base complementary pairing. 5. The preparation method according to claim 4, characterized in that the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 5, and the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 6; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 8, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 7. 6. The preparation method according to claim 5, characterized in that the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:12, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:11. 7. The preparation method according to claim 4 is characterized in that 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 the RNA ligase to form the 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 the RNA ligase to form the antisense chain. 8. The preparation method according to claim 7, 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 a hydroxyl 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. 9. The preparation method according to claim 3, characterized in that 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. 10. The preparation method according to claim 9, characterized in that the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13; The nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; The nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15; The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; The nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; The nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:16. 11. The preparation method according to claim 9, characterized in that the preparation method comprises: Mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment and the RNA ligase; Under the catalytic action of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are connected 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 connected to form the antisense strand, and the sense strand and the antisense strand form the Patisiran through base complementary pairing. 12. The preparation method according to any one of claims 1-2, characterized in that the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase further comprises ATP, Tris-HCl, MgCl ₂ and DTT; The concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM. 13. The preparation method according to claim 1, characterized in that the reaction temperature of the preparation method is 10-40°C; The reaction time of the preparation method is 2-48h.