Double-stranded circular sirna assembly for gene silencing

Abstract

A double-stranded circular siRNA assembly, which is assembled from one circular sense strand (circSS strand) and one or more antisense strands (AS strands). The cyclization elements in the circular sense strand (circSS strand) comprise a key motif, a cyclization promoter and a lock motif. By relying on complementary base pairing interaction between the cyclization motifs, the 3' end and 5' end of an RNA sequence are very close to each other in terms of space and are ligated via an RNA ligase to complete cyclization of a target RNA. Further provided is the use of the double-stranded circular siRNA assembly in inhibiting the expression of a target gene in a subject.

Description

Double-stranded circular siRNA assemblies for gene silencing Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a double-stranded circular siRNA assembly and its preparation method. Background Technology

[0002] RNA interference, or “RNAi,” is a term originally coined by Fire and colleagues to describe the observed ability of double-stranded RNAi (dsRNA) to block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes & Development 15, 188-200). Short dsRNAs guide gene-specific posttranscriptional silencing in many organisms, including vertebrates, and have provided a novel tool for studying gene function. RNA interference (RNAi)-based drug development requires double-stranded RNA (dsRNA) molecules with favorable gene-silencing properties. For three-dimensional siRNA duplex assemblies, the biomedical field continues to require novel and improved designs to realize and enhance the therapeutic potential of RNAi therapeutics, such as increasing their potency, metabolic stability, and reducing off-target effects. Summary of the Invention

[0003] This invention provides a double-stranded circular siRNA assembly, which is assembled from a circular sense strand (circSS strand) and one or more antisense strands (AS strands). The circularization elements in the circular sense strand (circSS strand) include a key motif, a circularization promoter, and a locking motif. Through complementary base pairing interactions between the circularization motifs, the 3′ and 5′ ends of the RNA sequence are spatially very close, and then ligated using RNA ligase to complete the circularization of the target RNA. This invention also provides the use of the double-stranded circular siRNA assembly in inhibiting the expression of target genes in subjects.

[0004] On the one hand, the present invention provides a double-stranded circular siRNA.

[0005] On the other hand, the invention provides a method for preparing double-stranded circular siRNA.

[0006] On the other hand, the invention provides a pharmaceutical composition comprising the double-stranded circular siRNA described in this invention and a pharmaceutically acceptable excipient.

[0007] On the other hand, the invention provides a double-stranded circular siRNA, which has the use in treating or preventing diseases, or in the preparation of drugs for treating or preventing diseases.

[0008] On the other hand, the invention provides a circular sense strand (circSS strand) for preparing double-stranded circular siRNA.

[0009] In one embodiment, a double-stranded circular siRNA is provided, which is assembled from a circular sense strand (circSS strand) and one or more antisense strands (AS strands).

[0010] The circular sense chain (circSS chain) includes: a circular region, one or more antisense chain (AS chain) complementary regions (target regions), and one or more spacer regions (linker regions);

[0011] The one or more antisense strands (AS strands) independently bind to the complementary region of the antisense strand (AS strand) of the circular sense strand (SS strand) in a complementary pairing manner, and are capable of independently generating the function of interfering RNA.

[0012] In some implementations, the circSS chain comprises the following structure from the 5′ end to the 3′ end:

[0013] (1) Cyclic promoter: -GGGA- noncomplementary region -X1X2X3X4-;

[0014] (2) Locking primitive;

[0015] (3) First antisense chain complementary region;

[0016] (4) The gap between the complementary regions of the antisense chain;

[0017] (5) The complementary region of the second antisense chain;

[0018] (6) The gap between the antisense chain complementary region and the key primitive;

[0019] (7) Key primitive;

[0020] The 5′ and 3′ ends of the meaningful chain (circSS chain) are connected to form a loop;

[0021] Optionally, insert -(the gap between the antisense chain complements - the nth antisense chain complement) after the 3' end of the second antisense chain (AS chain) complement region. n-2 - sequences, where n = 3-5;

[0022] The complementary regions of any antisense chain (AS chain) are either the same or different.

[0023] In some embodiments, the cyclic region of the circSS chain has the following structure:

[0024] Where -GGGA- is the non-complementary region -X1X2X3X4- As a cyclic promoter, the GGGA- and -X1X2X3X4- are fully or partially complementary, so that the cyclic promoter forms a stem-loop structure; the lock primitive and the key primitive are fully complementary to form a pairing structure of at least 3 bp (base pairs);

[0025] Preferably, the GGGA- and the -X1X2X3X4- are completely complementary or at least three complementary pairs, and more preferably there is only a mismatch between G and X3.

[0026] In some implementations, the non-complementary region of the circular promoter is 3-15 nt in length; the non-complementary region sequence is not complementary to GGGA-, -X1X2X3X4-, or the target RNA.

[0027] In some embodiments, the ratio of C to A bases in the non-complementary region of the cyclized promoter is 66%-100%.

[0028] In some implementations, the non-complementary region in the cyclopromoted promoter is a -CCAAC- sequence from the 5' end to the 3' end.

[0029] In some implementations, the cyclic promoter is the sequence SEQ ID NO.40GGGACCAACUCUC from the 5′ end to the 3′ end (the RNA sequence corresponding to SEQ ID NO.1).

[0030] In some implementations, the lock primitive and the key primitive complement each other to form a pairing structure of 3-15 bp, preferably a pairing structure of 3-9 bp.

[0031] In some implementations, the locking motif consists of 1-5 repeating UAG units from the 5' end to the 3' end, and the key motif, which is completely complementary to the locking motif, consists of 1-5 repeating CUA units from the 5' end to the 3' end; preferably, the locking motif from the 5' end to the 3' end is the sequence of SEQ ID NO.41 UAGUAG (the RNA sequence corresponding to SEQ ID NO.2), and the key motif from the 5' end to the 3' end is the sequence of SEQ ID NO.42 CUACUA (the RNA sequence corresponding to SEQ ID NO.8).

[0032] In some implementations, the linker region sequence between the complementary regions of the antisense strands, from the 5′ end to the 3′ end, is the sequence of SEQ ID NO.43UUACCGGUUUGAAGGC or SEQ ID NO.44UUACCGGU.

[0033] In some implementations, the linker region sequence between the antisense complement region and the key primitive is the sequence of SEQ ID NO.45 UUGACAUCUUACGUGG or SEQ ID NO.46 UUGACAUC.

[0034] In some implementations, the length of the circular sense chain (circSS chain) is 28 nt or more, preferably 50 nt or more; the length of the antisense chain (AS chain) is 19-28 nt.

[0035] In some implementations, delivery molecules are further conjugated to the cyclic sense strand (circSS strand) or the antisense strand (AS strand).

[0036] In some embodiments, in the antisense strand (AS strand) with the delivery molecule conjugated, the delivery molecule is conjugated to the 5' or 3' end of the antisense strand (AS strand), and the sequence of the antisense strand (AS strand) is partially or completely complementary to the sequence of the complementary region (target region) of the antisense strand (AS strand).

[0037] In some embodiments, in a circular sense strand (circSS) with a delivery molecule conjugated, the delivery molecule is conjugated to the 5' or 3' end of the complementary strand of the circular sense strand (circSS); the complementary strand of the circular sense strand (circSS) is partially or completely complementary to the non-target region of the circSS, and the complementary strand of the circular sense strand (circSS) is selected from modified or unmodified RNA or single-stranded DNA, preferably 5-30 nt in length, more preferably 19 nt or 21 nt.

[0038] In some embodiments, in a circ-sense chain (circSS chain) with a delivery molecule attached, the delivery molecule is attached to the circ-sense chain at a non-target region of the circ-sense chain.

[0039] In some embodiments, the delivery molecule is selected from targeted lipids, monosaccharides, peptides, antibodies, antibody fragments, or molecules that facilitate permeation of cell membranes; preferred delivery molecules are selected from ASGPR ligands, C... 16 or its derivatives.

[0040] In some embodiments, the ASGPR ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; preferably... One or more of LICA-1, LICA-2, LICA-3, or L96;

[0041] The structural formula of LICA-1 is:

[0042] The structural formula of LICA-2 is:

[0043] The structural formula of LICA-3 is:

[0044] The structural formula of L96 is:

[0045] The C 16 Or its derivatives are selected from 2'-oxo-hexadecyl.

[0046] In some embodiments, a phosphate ester mimic is included at the 5'-terminus of the antisense strand nucleotide sequence, selected from the group consisting of 5'-thiophosphate (5'-PS), 5'-dithiophosphate (5'-PS2), 5'-vinyl phosphate (5'-VP), 5'-methyl phosphate (5'-MePhos), or 5'-deoxy-5'-C-malonyl; preferably 5'-vinyl phosphate (5'-VP).

[0047] In some embodiments, the one or more antisense strands (AS strands) are modified or unmodified, and the cyclic sense strands (circSS strands) are modified or unmodified; the modifications are selected from internucleotide linkage modifications, nucleobase modifications, sugar modifications, and combinations thereof.

[0048] In some embodiments, the modification is selected from LNA, ENA, HNA, CeNA, 2'-methoxyalkyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-ON-methylacetamido (2-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modification, and combinations thereof.

[0049] In some implementations, the target gene of each antisense strand (AS strand) interference is an associated gene; preferably, each is independently selected from one or more of PCSK9, AGT, FXI or ANGPTL3; more preferably, at least two of PCSK9, AGT, FXI and ANGPTL3.

[0050] In some implementations, the antisense strand (AS strand) sequence of PCSK9 is selected from the RNA sequence shown in SEQ ID NO. 26, 27, 28, 29 or 47.

[0051] In some implementations, the antisense strand (AS strand) sequence of AGT is selected from the RNA sequence shown in SEQ ID NO. 33, 34, 35 or 48.

[0052] In some implementations, the antisense strand (AS strand) sequence of FXI is selected from the RNA sequence shown in SEQ ID NO. 36 or 49.

[0053] In some implementations, the antisense strand (AS strand) sequence of ANGPTL3 is selected from the RNA sequence shown in SEQ ID NO. 30, 31, 32 or 50.

[0054] In some embodiments, optionally, the nucleotides in the antisense strand are independently selected from modified or unmodified ones, and the modifications are selected from LNA, ENA, HNA, CeNA, 2'-methoxyalkyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-ON-methylacetamido (2-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications, and combinations thereof.

[0055] In some implementations, the circular sense chain (circSS chain) is selected from any one of the sequences shown in SEQ ID NO.20-25.

[0056] In some implementations, the antisense chain is selected from any one of the sequences shown in SEQ ID NO.26-36, 47-50.

[0057] In some implementations, the assembly of the circular sense chain (circSS chain) and the antisense chain (AS chain) is selected from any one of the assemblies CON-1 to CON-19 shown in Table 4.

[0058] In another embodiment, a method for preparing double-stranded circular siRNA is provided, comprising the following steps:

[0059] Step 1. Connect the circular promoter, locking motif, one or more antisense strand (AS strand) complementary regions, one or more spacer regions (linker regions) and key motif in sequence to obtain a DNA template strand with circular components connected.

[0060] Step 2: Transcribe the DNA template strand in vitro to obtain a linear RNA strand for circularization;

[0061] Step 3: Convert the 5′ triphosphate of the RNA sequence obtained in Step 2 into a monophosphate to obtain the precursor strand for the circular sense strand (circSS strand);

[0062] Step 4: Place the circular RNA precursor strand in a buffer solution and anneal to form a precursor strand of circular sense strand (circSS strand) in a circular intermediate state;

[0063] Step 5: The RNA precursor strand in the circular intermediate state described in Step 4 is treated with RNA ligase to connect the 5′ and 3′ ends to obtain a covalently closed circular sense strand (circSS strand).

[0064] Step 6: Purify the cyclic sense chain (circSS chain) obtained in Step 5;

[0065] Step 7: Prepare one or more antisense chains (AS chains) using a solid-state synthesis method;

[0066] Step 8: The circular sense strand (circSS strand) and one or more antisense strands (AS strand) are self-assembled into a double-stranded circular siRNA in the reaction system.

[0067] In some embodiments, the method for preparing the double-stranded circular siRNA includes the following steps:

[0068] By replacing steps 1 to 3 of the aforementioned method for preparing double-stranded circular siRNA with a solid-phase synthesis method, a linear RNA strand for circularization is obtained;

[0069] Step 4: Place the circular RNA precursor strand in a buffer solution and anneal to form a precursor strand of circular sense strand (circSS strand) in a circular intermediate state;

[0070] Step 5: The RNA precursor strand in the circular intermediate state described in Step 4 is treated with RNA ligase to connect the 5′ and 3′ ends to obtain a covalently closed circular sense strand (circSS strand).

[0071] Step 6: Purify the cyclic sense chain (circSS chain) obtained in Step 5;

[0072] Step 7: Prepare one or more antisense chains (AS chains) using a solid-state synthesis method;

[0073] Step 8: The circular sense strand (circSS strand) and one or more antisense strands (AS strand) are self-assembled into a double-stranded circular siRNA in the reaction system.

[0074] In some embodiments, the RNA ligase is selected from T4 RNA ligase 1 or T4 RNA ligase 2.

[0075] In another embodiment, a pharmaceutical composition is provided comprising the aforementioned double-stranded circular siRNA and a pharmaceutically acceptable excipient.

[0076] In another embodiment, the aforementioned double-stranded circular siRNA is provided for use in the treatment or prevention of diseases, or for use in the preparation of medicaments for the treatment or prevention of diseases.

[0077] In some embodiments, the disease is a disease related to the PCSK9, AGT, FXI, or ANGPTL3 genes; preferably, the disease is treated by downregulating the PCSK9, AGT, FXI, or ANGPTL3 genes or their expression.

[0078] In some implementations, the disease is a cardiovascular disease, preferably dyslipidemia, hypertension, or venous thrombosis.

[0079] In some implementations, the double-stranded circular siRNA has comparable gene silencing efficiency and improved stability compared to linear siRNA.

[0080] In another embodiment, a circ-SS chain is provided, wherein the 5′ to 3′ ends include the following structure:

[0081] 1) Cyclic promoter: -GGGA- non-complementary region -X1X2X3X4-;

[0082] 2) Locking primitive;

[0083] 3) The complementary region of the first antisense chain;

[0084] 4) The gap between the complementary regions of the antisense chain;

[0085] 5) The complementary region of the second antisense chain;

[0086] 6) The gap between the antisense chain complement region and the key primitive;

[0087] 7) Key primitive;

[0088] The 5′ and 3′ ends of a sense chain (circSS chain) are connected to form a loop;

[0089] Optionally, insert (the gap between the antisense chain complements - the nth antisense chain complement) after the 3' end of the second antisense chain (AS chain) complement region. n-2 sequence, n=3-5;

[0090] The complementary regions of any antisense chain (AS chain) are either the same or different.

[0091] Each of the components has the definition as described in any of the aforementioned implementations.

[0092] In some implementations, the circular sense chain (circSS chain) is any of the sequences described in SEQ ID NO. 20-25.

[0093] In some implementations, the circular sense strand (circSS strand) is obtained by circularizing any of the linear RNA strands in SEQ ID NO.14-19. Attached Figure Description

[0094] Figure 1 is a schematic diagram of linear single-stranded RNA.

[0095] Figure 2 shows the PAGE gel images of the denatured linear single chains and cyclic single chains.

[0096] Figure 3 shows an 8% non-denaturing PAGE gel image of the circRNA assembly.

[0097] Figure 4 shows the transfection of Hep3B cells with circRNA assemblies targeting different sites.

[0098] Figure 5 shows the transfection of Hep3B cells with circRNA assemblies that assemble different complementary strands.

[0099] Figure 6 shows the transfection of Hep3B cells with multiple concentrations of circRNA assemblies.

[0100] Figure 7 shows the circRNA assemblies free-uptake mouse primary liver cells.

[0101] Figure 8 shows the transfection of Hep3B cells with modified circRNA assemblies.

[0102] Figure 9 shows the free-uptake mouse primary liver cells of the modified circRNA assembly.

[0103] Figure 10 shows the transfection of mouse primary liver cells with a dual-target circRNA assembly.

[0104] Figure 11 shows mice subcutaneously administered the modified circRNA assembly.

[0105] Figure 12 shows the structure of the circulated region of a circSS chain. Detailed Implementation

[0106] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0107] Example 1: Preparation of double-stranded circular siRNA

[0108] Step 1: Construction of dsDNA template

[0109] dsDNA was prepared using the circularization components and target RNA described in Table 1. The circularization components included a circularization promoter, a locking motif, and a key motif. Two complementary ssDNA molecules were contained in equimolar concentrations in the reaction system (40 μL), using a 1×TE·Mg solution. 2+ Buffer solution (10mM Tris-HCl, 0.1mM EDTA, 5mM Mg) 2+ (pH 7.6). The reaction system was incubated at 95°C for 5 minutes and cooled to 25°C at a rate of 1°C / min to obtain the dsDNA template of SEQ ID NO:9-13.

[0110] Table 1. DNA sequences for preparing dsDNA templates

[0111] Note:

[0112] 1. SEQ ID NO.9-13 is a complementary double strand, where G, C, A, and T represent nucleotides with guanine, cytosine, adenine, and thymine as bases, respectively.

[0113] 2. Use underscores to indicate the corresponding target RNA sequences in Table 1.

[0114] Step 2: Using dsDNA as a template, 5' triphosphorylated linear ssRNA is transcribed in vitro.

[0115] The reaction mixture (20 μL) contained 500 ng of linear dsDNA template obtained in step one, 4 μL of T7 transcription 5× buffer, 6 μL of rNTPs (25 mM ATP, CTP, UTP, and GTP), 2 μL of enzyme mixture (T7), and nuclease-free water. The reaction mixture was incubated at 37°C for 4 hours to overnight. 1 μL of RQ1 RNase-Free DNase was added to the reaction mixture, and incubation continued at 37°C for 30 minutes to remove the template dsDNA. Finally, the reaction mixture was purified using the RNA Clean & Concentrator kit (RCC) to obtain 5' triphosphorylated linear precursor ssRNA.

[0116] Step 3: Convert 5' triphosphorylated linear ssRNA into monophosphorylated form.

[0117] The reaction system (10 μL) contained 4 μM 5' triphosphorylated linear ssRNA, 0.1 U Apyrase, 20 U RiboLock RNase inhibitor, and nuclease-free water. The reaction solution was 1×Apyrase reaction buffer (20 mM 4-morpholinoethanesulfonic acid (MES), 50 mM NaCl, 5 mM CaCl2, 1 mM dl-dithiothreitol (DTT), and 0.05% HCl). 20 (pH 7.5). The reaction system was incubated at 37°C for 1-2 hours and then inactivated at 65°C for 20 minutes to obtain monophosphorylated linear ssRNA (SEQ ID NO: 14-18) corresponding to step one, which has the sequence unit structure shown in Figure 1 from the 5'-3' end.

[0118] Step 4: Annealing of linear ssRNA

[0119] The reaction system (40 μL) contained 1 μM-10 μM monophosphorylated linear ssRNA (SEQ ID NO:14-18), and the solution used was 1×TE·Mg 2+ Buffer solution (10mM Tris-HCl, 0.1mM EDTA, 5mM Mg) 2+ (pH 7.6). The reaction system was incubated at 75°C for 5 minutes and cooled to 25°C at a rate of 0.5 or 1°C / min to obtain the RNA precursor strand in a circular intermediate state.

[0120] Alternatively, a nucleic acid synthesizer can be used to directly synthesize a long 5' monophosphorylated linear ssRNA chain (SEQ ID NO:14-19). The synthesized amount is dissolved and diluted according to the amount synthesized, and the reaction system is the same as above, followed by annealing.

[0121] Table 2 Linear Single-Stranded RNA Sequences

[0122] Note: G, C, A, and U represent nucleotides with guanine, cytosine, adenine, and uracil as bases, respectively. m indicates that the nucleotide adjacent to the left is 2'-methoxy modified; f indicates that the nucleotide adjacent to the left is 2'-fluorine modified; s indicates that the two adjacent nucleotides are linked by a single phosphate thiophosphate bond.

[0123] Step 5: T4Rnl2 is used to ligate the circular intermediate RNA precursor strand to obtain circular ssRNA.

[0124] The reaction system (80 μL) contained 0.5 μM–5 μM of RNA precursor strand in the circular intermediate state, 10 U T4 Rnl2, 40 U RiboLock RNase inhibitor, and DEPC water. The reaction solution was prepared in 1×T4 Rnl2 reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM dl-dithiothreitol (DTT), and 400 μM adenosine triphosphate (ATP), pH 7.5). The reaction system was incubated at 37 °C for 2 hours.

[0125] Table 3. Circulated RNA Sequences

[0126] Note: … indicates that the RNA is circularized by linking to the terminal or initial base, forming a complementary pair as shown in Figure 12. m indicates that the nucleotide adjacent to the left is a 2'-methoxy modified nucleotide; f indicates that the nucleotide adjacent to the left is a 2'-fluorine modified nucleotide; s indicates that the two adjacent nucleotides on both sides are linked by a phosphate thiophosphate bond.

[0127] Step 6: Purification of circular ssRNA

[0128] Samples were subjected to denaturing polyacrylamide gel electrophoresis (denaturing PAGE) containing 7M urea. The gel composition ranged from 4% to 10% depending on the length of the precursor RNA, with an acrylamide to bisacrylamide ratio ranging from 19:1 to 39:1. The gel was then subjected to Sybr Gold electrophoresis. TM After staining, the siRNA was circularly excised and homogenized using a UV gel imaging system. 4 mL of DEPC water or dissolution buffer (containing 100 mM Tris HCl, 100 mM EDTA, 500 mM NaCl, pH 8) was added, and the mixture was inverted overnight to dissolve the siRNA. The mixture was then lyophilized and concentrated.

[0129] Alternatively, linear RNA can be removed using enzymatic digestion. The reaction system (20 μL) consists of 500 ng of crude circular ssRNA, 2 U RNase R, and DEPC water. The reaction solution is 1×RNase R reaction buffer (20 mM Tris-HCl, 100 mM KCl, and 0.1 mM MgCl2, pH 8.0). The reaction system is incubated at 37°C for 30 minutes to overnight, followed by incubation at 70°C for 5 minutes. Finally, the reaction system is purified using the RNA Clean & Concentrator kit (RCC) to obtain circular ssRNA.

[0130] Step 7: Denaturing PAGE Analysis

[0131] Samples were subjected to denaturing polyacrylamide gel electrophoresis (denaturing PAGE) containing 7M urea. The gel composition ranged from 4% to 10% depending on the length of the precursor RNA, with an acrylamide to bisacrylamide ratio ranging from 19:1 to 39:1. The gel was then subjected to Sybr Gold electrophoresis. TM Post-staining was performed, followed by imaging using a UV gel imaging system.

[0132] The test results are shown in Figures 2A-2C. Figure 2A shows the circularization results after generating linear single-stranded RNA by the IVT method; Figure 2B shows the circularization results after directly synthesizing linear single-stranded RNA; Figure 2C shows the circularization results after directly synthesizing linearly modified single-stranded RNA; the marker is Low Range ssRNA ladder (NEB).

[0133] Step 8: Assemble complementary strands to construct circRNA

[0134] The reaction system (40 μL) contained circular ssRNA and the complementary strand from Table 3 added at the molar ratio. The solution used was 1×PBS·NaCl buffer (1 mM KH2PO4, 3 mM Na2HPO4, 165 mM NaCl, pH 7.6). The reaction system was incubated at 75 °C for 5 minutes and then cooled to 25 °C at a rate of 1 °C / min to obtain the circular RNA assembly.

[0135] Table 3 Complementary strand RNA sequences

[0136] Note: m indicates that the adjacent nucleotide on the left is 2'-methoxy modified; f indicates that the adjacent nucleotide on the left is 2'-fluorine modified; s indicates that the two adjacent nucleotides on both sides are linked by a thiophosphate bond; VP indicates that the nucleotide immediately adjacent to the right is phosphate-modified. L represents the ligand, L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolylHyp-(GalNAc-alkyl)3. UR indicates an unmodified RNA sequence. UD indicates an unmodified DNA sequence. MR indicates a modified RNA sequence.

[0137] Step 9: Non-denaturing PAGE analysis

[0138] circRNAs were electrophoresed using non-denaturing polyacrylamide gels (non-denaturing PAGE). The gel composition ranged from 4% to 10% depending on the circRNA length, with an acrylamide to bisacrylamide ratio ranging from 19:1 to 39:1. The gels were then subjected to Sybr Gold electrophoresis. TM Post-staining was performed, followed by imaging with a UV gel imager to assess whether the complementary strands were successfully assembled.

[0139] Table 4 circRNA assemblies

[0140] Note: The circular and complementary strands in the table are annealed into circRNA assemblies according to step 8 of the preparation of double-stranded circular siRNA in Example 1.

[0141] The test results are shown in Figures 3A-3E. Figure 3A shows the results of circularizing and assembling complementary strands of linear single-stranded RNA generated by the IVT method; Figures 3B, 3C, and 3D show the results of circularizing and assembling complementary strands of directly synthesized linear single-stranded RNA; Figure 3E shows the results of circularizing and assembling complementary strands of directly synthesized linearly modified single-stranded RNA.

[0142] Example 2: In vitro Hep3B cell transfection assay of circRNA assemblies targeting different sites

[0143] Both linear single-stranded RNA generated by the IVT method and linear single-stranded RNA synthesized directly can be used for the preparation of circular RNA, and the results of in vitro cell validation show no difference.

[0144] Hep3B cells were seeded in 24-well plates at 7.5 × 10⁻⁶ wells. 4 After approximately 24 hours, the medium was changed to 500 μL / well of Opti-MEM medium. The circRNA assemblies CON-1, CON-8, CON-11, and CON-14 targeting different sites in Table 4 were diluted to the corresponding concentrations. 50 μL / well of the transfection complex was added to the cell plate (final compound concentration was 10 nM). After 4-6 hours, 1 mL of complete medium was added. After approximately 24 hours, RNA extraction, reverse transcription, and qPCR were performed.

[0145] The test results are summarized in Figure 4. 10 nM CON-1 showed a 91.0% inhibition rate against PCSK9 in Hep3B cells, CON-8 a 96.6% inhibition rate against ANGPTL3 in Hep3B cells, CON-11 a 95.4% inhibition rate against AGT in Hep3B cells, and CON-14 a 53.1% inhibition rate against FXI in Hep3B cells. The low inhibition rate of CON-14 against FXI may be due to the slightly lower expression level of FXI in Hep3B cells. These results demonstrate that this circRNA assembly structure can target and inhibit the expression of corresponding gene mRNAs based on the target RNA sequence.

[0146] Example 3: In vitro Hep3B cell transfection assay for assembling circRNA assemblies with different complementary strands

[0147] Hep3B cells were transfected with circRNA assemblies CON-1, CON-5, CON-6, and CON-7, which were assembled with different complementary strands, according to the procedure in Example 2, with a final concentration of 10 nM, and mRNA was detected.

[0148] The test results are summarized in Figure 5. CON-5, CON-6, and CON-7, based on CON-1, assembled three sequences with non-target complementary regions, respectively, and their inhibition rates against PCSK9 in Hep3B cells were 92.1%, 90.9%, and 92.4%, respectively. The results show that the inhibitory effect of assembling sequences with different non-target complementary regions on the target is comparable to that of circRNA assemblies that only assemble the target complementary region.

[0149] Example 4: In vitro Hep3B cell transfection assay using multiple concentrations of circRNA assemblies

[0150] The ANG-circRNA assemblies CON-8, CON-9, and CON-10 were transfected into Hep3B cells according to the procedure in Example 2, with final concentrations of 10, 2.5, 0.625, and 0.156 nM, respectively, and mRNA was detected.

[0151] The test results are summarized in Figure 6. Based on CON-8, the complementary strand of CON-9 was linked to L96, and the complementary strand of CON-10 was modified with VP and linked to L96. Both still showed good inhibitory effects on the mRNA expression level of ANGPTL3 in Hep3B cells, and the inhibitory levels were basically the same among different concentrations.

[0152] Example 5: Free-uptake assay of circRNA assemblies in primary mouse liver cells

[0153] Mouse primary liver cells were resuscitated and plated into 12-well plates according to the manufacturer's instructions, with a cell density of 4 × 10⁶ cells / well. 5 Each well contains 1 unit of circRNA. After adhesion, the medium is replaced with 500 μL of maintenance medium. The circRNA assemblies CON-9 and CON-10 are diluted with the maintenance medium and 100 μL of the diluted compound is added (final concentrations of 40, 20, 10, 5, and 2.5 nM, respectively). RNA extraction, reverse transcription, and qPCR are performed after about 48 hours.

[0154] The test results are summarized in Figure 7. Both CON-9 and CON-10 with the L96 vector can be freely taken up into mouse primary liver cells. The highest concentration of 40 nM showed an inhibition rate of >50% against ANGPTL3. CON-10 with VP modification on the complementary strand can enhance the inhibitory effect of circRNA assemblies on the target, with the highest concentration of 40 nM showing an inhibition rate of 74.6% against ANGPTL3.

[0155] Example 6: In vitro Hep3B cell transfection assay of modified circRNA assemblies

[0156] The modified circRNA assembly CON-19 and PC-siRNA (linear SS chain SEQ ID NO.51: AmsCmsAmUmAmUmUfUmGfAfUfCmAmGmUmCmUmUmUmUmUm_L96; AS chain SEQ ID NO.30: AmsAfsAmAmAmGfAmCmUmG mAmUmCmAfAmAfUmAmUmGmUmsUmsGm) were transfected into Hep3B cells according to the procedure in Example 2, with final concentrations of 10 and 1 nM, respectively, and mRNA was detected.

[0157] The test results are summarized in Figure 8. Modified circRNA CON-19 showed a good inhibitory effect on the mRNA expression level of ANGPTL3 in Hep3B cells, and was comparable to that of PC-siRNA.

[0158] Example 7: Free-uptake assay of modified circRNA assemblies in primary mouse liver cells

[0159] Mouse primary liver cells were resuscitated and plated into 12-well plates according to the manufacturer's instructions, with a cell density of 4 × 10⁶ cells / well. 5 Each well contains 1 cell. After adhesion, the medium is replaced with 500 μL of maintenance medium. The circRNA assembly CON-19 and PC-siRNA are diluted with the maintenance medium (same as in Example 6). 100 μL of the diluted compound is added (final concentration of 20 and 10 nM). RNA extraction, reverse transcription, and qPCR are performed after about 48 h.

[0160] The test results are summarized in Figure 9. The modified circRNA CON-19 can also enter mouse primary liver cells via free uptake, and has a good inhibitory effect on the mRNA expression level of ANGPTL3 in the cells, which is comparable to that of PC-siRNA.

[0161] Example 8: Transfection assay of dual-target circular RNA assemblies in primary mouse liver cells

[0162] The circRNA can carry two different target sequences simultaneously, and the construction method is the same as in Example 1. Freshly isolated primary mouse liver cells were seeded in 6-well plates at 800 μL / well. The circRNA assembly CON-17 targeting PCSK9 and ANGPTL3 was diluted with opti-MEM to the corresponding concentrations. 200 μL / well of the transfection complex was added to the cell plate (final concentrations: 20, 10, 5, 2.5, 1, 0.33, 0.11, 0.04 nM, respectively). RNA extraction, reverse transcription, and qPCR were performed after 24 h.

[0163] The test results are summarized in Figure 10. The results show that the circRNA assembly CON-17, targeting PCSK9 and ANGPTL3, can simultaneously inhibit the mRNA expression of both PCSK9 and ANGPTL3 in primary mouse liver cells. At concentrations above 2.5 nM, the inhibition rate of PCSK9 is >70%, and the inhibition rate of ANGPTL3 is >80%. Even at the lowest concentration of 0.04 nM, the inhibition rate of ANGPTL3 is still >50%. These results indicate that the circRNA assembly can simultaneously inhibit the expression of the mRNA of two genes.

[0164] Example 9: In vivo testing of modified circRNA assemblies in mice

[0165] The inhibitory effect on ANGPTL3 in plasma was evaluated by subcutaneously injecting 6 mpk of PC-siRNA (same as in Example 6) and the same molar amount of CON-19 into female C57BL / 6J mice on day 1. Plasma samples were collected before drug administration (D-2), on day 4, day 7, day 14, day 21, day 28, and day 42.

[0166] The test results are summarized in Figure 11. The results showed that throughout the study period, the change in mouse body weight was similar to that of the PBS group, and CON-19 had no effect on mouse body weight. On day 4 after administration, CON-19 reached its maximum inhibition of ANGPTL3 in mouse plasma, with an inhibition rate of 94.8%. This maximum inhibition was maintained until day 14, with an inhibition rate of 93.7%. Approximately 40% inhibition remained until the end of the study on day 42. The inhibitory level of CON-19 on the target ANGPTL3 was comparable to that of PC-siRNA.

Claims

1. A double-stranded circular siRNA, which is assembled by one circular sense strand (circSS strand) and one or more antisense strands (AS strand), The circular sense strand (circSS strand) comprises: a circularization region, one or more antisense strand (AS strand) complementary regions (target regions), and one or more spacer regions (linker regions) ; The one or more antisense strands (AS strands) are independently bound to the antisense strand (AS strand) complementary region of the circular sense strand (SS strand) in a complementary pairing manner, and can independently generate the function of interfering RNA. 2.The double-stranded circular siRNA of claim 1, wherein the circular sense strand (circSS strand) comprises the following structures from 5′ end to 3′ end: (1) a circularization initiator: -GGGA-non-complementary region-X 1 X 2 X 3 X 4-; (2) a lock motif; (3) a first antisense strand complementary region; (4) a spacer region between the antisense strand complementary regions; (5) a second antisense strand complementary region; (6) a spacer region between the antisense strand complementary region and the key motif; (7) a key motif; the 5′ end and the 3′ end of the sense strand (circSS strand) are connected to form a ring; Optionally, a sequence of -(gap between antisense strand complementary regions - nth antisense strand complementary region) is inserted after the 3' end of the second antisense strand (AS strand) complementary region, where n = 3-5. n-2 - any antisense strand (AS strand) complementary region is the same, or different. 3.The double-stranded circular siRNA of any one of claims 1 to 2, wherein The circularized region of the circular sense strand (circSS strand) has the structure shown below: wherein -GGGA- is a non-complementary region -X1X2X3X4- 4- is a circularization promoter, said GGGA- is fully or partially complementary to said -X1X2X3X4- such that the circularization promoter forms a stem-loop structure; said lock motif is fully complementary to the key motif to form a paired structure of at least 3 base pairs; preferably, the GGGA- is completely complementary or at least 3 complementary pairings with the -X 1 X 2 X 3 X 4-, more preferably, there is only a mismatch between G and X 3. 4.The double-stranded circular siRNA of claim 3, wherein the non-complementary region of the circularization initiator has a length of 3-15 nt; the sequence of the non-complementary region is not complementary to GGGA-, -X 1 X 2 X 3 X 4-, and the target RNA. 5.The double-stranded circular siRNA of claim 3 or 4, wherein the proportion of bases C and A in the non-complementary region of the circularization initiator is 66%-100%. 6.The double-stranded circular siRNA of any one of claims 3 to 5, wherein the non-complementary region in the circularization initiator is a sequence of -CCAAC- from 5′ end to 3′ end. 7.The double-stranded circular siRNA of any one of claims 3 to 6, wherein the circularization initiator is a sequence of SEQ ID NO. 40 GGGACCAACUCUC from 5′ end to 3′ end. 8.The double-stranded circular siRNA of any one of claims 2 to 7, wherein the lock motif and the key motif are complementary to form a paired structure of 3-15 bp, preferably a paired structure of 3-9 bp. 9.The double-stranded circular siRNA of any one of claims 2 to 8, wherein the lock motif is 1-5 repeated UAG units from 5′ end to 3′ end, and the key motif which is completely complementary to the lock motif is 1-5 repeated CUA units from 5′ end to 3′ end; preferably, the lock motif is a sequence of SEQ ID NO. 41 UAGUAG from 5′ end to 3′ end, and the key motif is a sequence of SEQ ID NO. 42 CUACUA from 5′ end to 3′ end.

10. The double-stranded circular siRNA of any one of claims 2 to 9, wherein, The sequence of the linker region between the complementary regions of the antisense strand is from 5' to 3' the sequence of SEQ ID NO. 43 UUACCGGUUUGAAGGC or SEQ ID NO. 44 UUACCGGU.

11. The double-stranded circular siRNA of any one of claims 2 to 10, wherein, The sequence of the linker region between the complementary regions of the antisense strand and the key motif is the sequence of SEQ ID NO. 45 UUGACAUCUUACGUGG or SEQ ID NO. 46 UUGACAUC.

12. The double-stranded circular siRNA of any one of claims 1 to 11, wherein the length of the circular sense strand (circSS strand) is 28 nt or more, preferably 50 nt or more; the length of the antisense strand (AS strand) is 19-28 nt.

13. The double-stranded circular siRNA of any one of claims 1 to 12, wherein the circular sense strand (circSS strand) or the antisense strand (AS strand) is further conjugated with a delivery molecule.

14. The double-stranded circular siRNA of claim 13, wherein in the antisense strand (AS strand) conjugated with a delivery molecule, the delivery molecule is conjugated at the 5' or 3' end of the antisense strand (AS strand), which is partially or fully complementary to the sequence of the complementary region (target region) of the antisense strand (AS strand); or in the circular sense strand (circSS strand) conjugated with a delivery molecule, the delivery molecule is conjugated at the 5' or 3' end of the complementary strand of the circular sense strand (circSS strand); the complementary strand of the circular sense strand (circSS strand) is partially or fully complementary to the non-target region of the circSS strand, and the complementary strand of the circular sense strand (circSS strand) is selected from modified or unmodified RNA or single-stranded DNA, preferably with a length of 5-30 nt, preferably 19 nt or 21 nt; or in the circular sense strand (circSS strand) conjugated with a delivery molecule, the delivery molecule is conjugated on the circular sense strand (circSS strand) at a conjugation connection position which is the non-target region of the circular sense strand (circSS strand).

15. The double-stranded circular siRNA of any one of claims 1 to 14, wherein the delivery molecule is selected from a lipid, a monosaccharide, a polypeptide, an antibody, an antibody fragment, or a molecule that facilitates penetration through a cell membrane; preferably the delivery molecule is selected from an ASGPR ligand, a C 16 or a derivative thereof.

16. The double-stranded circular siRNA of claim 15, wherein The ASGPR ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; preferably one or more of LICA-1, LICA-2, LICA-3, or L96; The structural formula of LICA-1 is: The structural formula of LICA-2 is: The structural formula of LICA-3 is: The structural formula of L96 is: The C 16 or a derivative thereof is selected from 2'-oxy-hexadecyl.

17. The double-stranded circular siRNA of any one of claims 1 to 16, wherein a phosphate mimic is comprised at the 5'-end of the nucleotide sequence of the antisense strand, selected from the group consisting of 5'-phosphorothioate (5'-PS), 5'-phosphorodithioate (5'-PS2), 5'-vinylphosphonate (5'-VP), 5'-methylphosphonate (5'-MePhos), or 5'-deoxy-5'-C-malonate; preferably 5'-vinylphosphonate (5'-VP).

18. The double-stranded circular siRNA of any one of claims 1 to 17, wherein the one or more antisense strands (AS strands) are modified or unmodified, the circular sense strand (circSS strand) is modified or unmodified; the modification is selected from the group consisting of internucleoside linkage modification, nucleobase modification, sugar modification, and combinations thereof.

19. The double-stranded circular siRNA of claim 18, the modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-methoxyalkyl, 2'-0-alkyl, 2'-0-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-0-N-methylacetamido (2-0-NMA), 2'-0-dimethylaminoethoxyethyl (2'-0-DMAEOE), 2'-0-aminopropyl (2'-0-AP), 2'-ara-F, L-nucleoside modification, and combinations thereof.

20. The double-stranded circular siRNA of any one of claims 1 to 19, wherein each antisense strand (AS strand) interfering target gene is a related gene; preferably each independently selected from one or more of PCSK9, AGT, FXI, or ANGPTL3; more preferably at least two of PCSK9, AGT, FXI, and ANGPTL3.

21. The double-stranded circular siRNA of claim 20, wherein the antisense strand (AS strand) sequence of PCSK9 is selected from the group consisting of the RNA sequence set forth in SEQ ID NO. 26, 27, 28, 29, or 47; the antisense strand (AS strand) sequence of AGT is selected from the group consisting of the RNA sequence set forth in SEQ ID NO. 33, 34, 35, or 48; the antisense strand (AS strand) sequence of FXI is selected from the group consisting of the RNA sequence set forth in SEQ ID NO. 36 or 49; the antisense strand (AS strand) sequence of ANGPTL3 is selected from the group consisting of the RNA sequence set forth in SEQ ID NO. 30, 31, 32, or 50; optionally, the nucleotides in the antisense strand are independently selected from modified or unmodified, the modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-methoxyalkyl, 2'-0-alkyl, 2'-0-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-0-N-methylacetamido (2-0-NMA), 2'-0-dimethylaminoethoxyethyl (2'-0-DMAEOE), 2'-0-aminopropyl (2'-0-AP), 2'-ara-F, L-nucleoside modification, and combinations thereof.

22. The double-stranded circular siRNA of any one of claims 1 to 21, wherein the circular sense strand (circSS strand) is selected from any one of the sequences set forth in SEQ ID NO. 20-25.

23. The double-stranded circular siRNA of any one of claims 1 to 22, wherein the antisense strand is selected from any one of the sequences set forth in SEQ ID NO. 26-36, 47-50. ​ ​ ​ ​ ​ 24. The double-stranded circular siRNA of claim 1 or 2, wherein the assembly of the circular sense strand (circSS strand) and the antisense strand (AS strand) is selected from any one of the assemblies CON-1 to CON-19 shown in Table 4.

25. A method for preparing the double-stranded circular siRNA of any one of claims 1-24, comprising the following steps Step 1. sequentially connecting a circularization initiator, a lock motif, one or more antisense strand (AS strand) complementary regions, one or more linker regions, and a key motif to obtain a DNA template strand with a circularization component attached; Step 2: in vitro transcribing the DNA template strand to obtain a linear RNA strand for circularization; Step 3: converting the 5' end triphosphate of the RNA sequence obtained in Step 2 to monophosphate to obtain a precursor strand of the circular sense strand (circSS strand); Step 4: placing the circular RNA precursor strand in a buffer solution to anneal to form a precursor strand of the circular sense strand (circSS strand) in a circular intermediate state; Step 5: treating the RNA precursor strand in the circular intermediate state in Step 4 with an RNA ligase to connect the interface of the 5' end and the 3' end to obtain a covalently closed circular sense strand (circSS strand); Step 6: purifying the circular sense strand (circSS strand) obtained in Step 5; Step 7: preparing the one or more antisense strands (AS strands) using solid-phase synthesis; Step 8: self-assembling the circular sense strand (circSS strand) and the one or more antisense strands (AS strands) into a double-stranded circular siRNA in a reaction system.

26. A method for preparing the double-stranded circular siRNA of any one of claims 1-24, comprising the following steps replacing Steps 1 to 3 of claim 25 with solid-phase synthesis to obtain a linear RNA strand for circularization; Step 4: placing the circular RNA precursor strand in a buffer solution to anneal to form a precursor strand of the circular sense strand (circSS strand) in a circular intermediate state; Step 5: treating the RNA precursor strand in the circular intermediate state in Step 4 with an RNA ligase to connect the interface of the 5' end and the 3' end to obtain a covalently closed circular sense strand (circSS strand); Step 6: purifying the circular sense strand (circSS strand) obtained in Step 5; Step 7: preparing the one or more antisense strands (AS strands) using solid-phase synthesis; Step 8: self-assembling the circular sense strand (circSS strand) and the one or more antisense strands (AS strands) into a double-stranded circular siRNA in a reaction system.

27. The production method according to any one of claims 25 to 26, characterized by, The RNA ligase is selected from T4 RNA ligase 1 (T4 RNA ligase 1) or T4 RNA ligase 2 (T4 RNA Ligase 2).

28. A pharmaceutical composition comprising the double-stranded circular siRNA of any one of claims 1-24 and a pharmaceutically acceptable excipient.

29. The double-stranded circular siRNA of any one of claims 1-24 for use in treating, preventing a disease, or for use in the manufacture of a medicament for treating, preventing a disease.

30. The use of claim 29, wherein the disease is a disease associated with the PCSK9, AGT, FXI or ANGPTL3 gene; preferably the disease is treated by down-regulating the PCSK9, AGT, FXI or ANGPTL3 gene or its expression.

31. The use of claim 29 or 30, wherein the disease is a cardiovascular disease, preferably dyslipidemia, hypertension, or venous thrombotic disease.

32. The use of claim 30 or 31, wherein the double-stranded circular siRNA has comparable gene silencing efficiency and improved stability compared to linear siRNA.

33. A circular sense strand (circSS strand) comprising the following structure from 5' end to 3' end: 8) a cyclization initiator: -GGGA-non-complementary region-X1X2X3X4-; 9) a lock motif; 10) a first antisense strand complementary region; 11) a spacer between the antisense strand complementary regions; 12) a second antisense strand complementary region; 13) a spacer between the antisense strand complementary region and the key motif; 14) a key motif; the 5' end and the 3' end of the sense strand (circSS strand) are connected to form a loop; Optionally, an insertion after the 3' end of the second antisense strand (AS strand) complementary region (spacer region between antisense strand complementary regions - nth antisense strand complementary region) n-2 sequence, n = 3-5; the antisense strand (AS strand) complementary regions are identical, or different; each of the elements has the definition as recited in any one of claims 3-16, 18 or 22.

34. The circular sense strand (circSS strand) of claim 33, having a sequence as recited in any one of SEQ ID NO. 20-25.

35. The circular sense strand (circSS strand) of claim 33, which is obtained by cyclization of a linear RNA strand of any one of SEQ ID NO. 14-19.

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