Antisense oligonucleotide-sense oligonucleotide coupling molecule and application thereof

Abstract

The invention relates to an antisense oligonucleotide-sense oligonucleotide coupling molecule and application thereof. The antisense oligonucleotide-sense oligonucleotide coupling (ASSOC) molecule comprises the following three parts: an antisense strand (ASO); a sense strand (SO) comprising a nucleotide sequence capable of forming reverse complementation with part or all of the nucleotide sequence of the antisense strand; and a linking strand (L) for linking the antisense strand and the sense strand and not containing a base. By constructing an intramolecular hybridization structure, the selectivity of the antisense oligonucleotide (ASO) to a substrate sequence is stably improved, and the off-target effect of the ASO is effectively reduced. The constructed ASSOC molecule can be applied to development of next-generation high-efficiency and low-toxicity ASO drugs, and has important value in the field of antisense oligonucleotide drugs.

Description

Technical Field

[0001] The invention belongs to the field of antisense oligonucleotide drugs, and specifically relates to an antisense oligonucleotide-sense oligonucleotide coupling molecule and an application thereof. Background Art

[0002] Antisense oligonucleotides (ASOs) are a class of short-chain nucleic acid molecules that can bind to target RNA through complementary base pairing and regulate RNA translation. ASOs can precisely downregulate or upregulate the expression of disease-related proteins using biological mechanisms such as RNase H1-mediated RNA degradation, hybridization-occupied RNA translation inhibition, and exon skipping. These functions allow ASOs to be used for the precise treatment of diseases, and 12 ASO drugs have been approved for clinical use. These ASO drugs have shown good efficacy in the treatment of diseases such as Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and familial chylomicronemia syndrome (FCS). In addition, a large number of studies have shown that ASOs have great application prospects in the treatment of diseases such as infectious diseases, cancer, cardiovascular diseases, and autoimmune diseases (Nat. Rev. Drug Discov. 2021, 20, 427).

[0003] However, ASO drugs also have serious toxic and side effects, which is an important factor limiting the large-scale clinical application of ASO drugs (Nucleic Acid Ther. 2023, 33, 1). Many ASO drugs have been forced to suspend clinical research due to serious liver toxicity problems (such as EZN-4176 for the treatment of prostate cancer and vupanorsen for the treatment of dyslipidemia). Even ASOs that have passed clinical approval can cause liver damage during use (such as mipomersen, inotersen, volanesorsen, etc.). The currently known mechanisms that cause ASO drug hepatotoxicity can be divided into two categories: one is the disorder of cellular proteins caused by the chemical composition of ASO; the other is the disorder of RNA function caused by ASO binding to non-target RNA, that is, off-target effect. For the first type of mechanism, many studies have shown that it can be effectively inhibited by specific chemical modifications (Nat. Biotechnol. 2019, 37, 640; Nucleic Acids Res. 2022, 50, 7224). As for off-target effects, there is currently no very effective method to inhibit them.

[0004] Although ASO drugs are expected to act only on RNA substrates that are completely complementary to their sequences, this is difficult to achieve in practice. In fact, under physiological conditions, ASOs can also bind to non-completely complementary RNAs and affect RNA translation. Tod M. Woolf et al. reported that even with a 9-base mismatch, an ASO with a length of 25 bases can still effectively degrade RNA substrates in vivo (Proc. Natl. Acad. Sci. USA 1992, 89, 7305). This high mismatch tolerance makes it easy for ASO drugs to affect the expression of normal proteins and produce off-target toxicity. This type of ASO off-target toxicity can cause apoptosis and hepatotoxicity (Nucleic Acids Res. 2016, 44, 2093; Mol. Ther. Nucleic Acids 2018, 10, 45). In response to this problem, the Oligonucleotide Safety Working Group recommends computer simulation screening of ASO sequences to select sequences with fewer potential off-target sites (Nat. Biotechnol. 2012, 30, 920). However, this approach is also difficult to avoid off-target toxicity. On the one hand, the human genome is huge, and the types of RNA sequences produced are very diverse; on the other hand, RNase H has a high tolerance for DNA / RNA double-strand mismatches, which makes the number of potential off-target sequences very large. For example, for an ASO with a length of 20 bases, the average number of exon RNAs with less than 5 base mismatches with its sequence exceeds 200 (Genes Cells 2018, 23, 448). Therefore, it is necessary to develop strategies to inhibit ASO off-target toxicity. Tang Xinjing's research group found that cyclic ASOs have lower off-target effects than linear ASOs in model reactions, but their off-target effects at the cellular or in vivo level have not been reported (J. Med. Chem. 2021, 64, 16046). Takao Inoue's team reported that gapmer ASOs with a length of 18 bases have lower off-target effects than gapmer ASOs with a length of 14 bases, suggesting that off-target effects can be reduced by increasing the length of ASOs (Mol. Diagn. Ther. 2022, 26, 117). However, this finding does not form a universal strategy. This is mainly because the increase in the length of ASO will increase the binding force with RNA, and the tolerance for mismatches and possible mismatched substrates will also increase rapidly, which may increase the off-target effect. Therefore, for different ASOs, the optimal sequence and length still need to be screened through a large number of experiments. Recently, Tsuyoshi Yamamoto's team reported that hybridizing a peptide nucleic acid through base complementary pairing on the ASO chain can improve the specificity of ASO (Nat. Commun. 2023, 14, 7972).This study shows that the selectivity of ASO chain binding to RNA can be improved by pre-hybridizing a sense chain on the ASO chain. However, the structure of this intermolecular hybridization is not stable, it relies on the use of special nucleic acid chains with high affinity and cannot guarantee a fixed ratio of sense chain and ASO chain, which makes this strategy very uncertain in practical applications. In short, although the selectivity problem of ASO drugs has long restricted its clinical application, there is still no effective way to solve this problem. Summary of the invention

[0005] One object of the present invention is to provide an antisense oligonucleotide molecule with a novel structural form, namely, an antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule.

[0006] Another object of the present invention is to provide a method for preparing such molecules.

[0007] Another object of the present invention is to provide a pharmaceutical composition, comprising the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule of the present invention and a pharmaceutically acceptable carrier.

[0008] Another object of the present invention is to provide a kit, which comprises the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule of the present invention.

[0009] Another object of the present invention is to provide application of such molecules in the field of antisense nucleic acid drugs.

[0010] In one aspect, the present invention provides an antisense oligonucleotide-sense oligonucleotide coupling molecule in the form of a primary structure, which comprises the following three parts:

[0011] Antisense strand (ASO);

[0012] a sense strand (SO), the sense strand comprising a nucleotide sequence capable of forming a reverse complement to a portion or all of the nucleotide sequence of the antisense strand; and

[0013] The base-free linker (L) used to connect one end of the antisense strand and one end of the sense strand has the following structure:

[0014]

[0015] Wherein, R represents a C2-C30 straight or branched alkylene group, or other divalent linker without a base, X represents oxygen or sulfur, and n represents the number of repeating units, which is an integer ranging from 1 to 20.

[0016] Wherein, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

[0017] On the other hand, the present invention provides an antisense oligonucleotide-sense oligonucleotide coupling molecule in the form of a secondary structure, which comprises the following three parts:

[0018] Antisense strand (ASO);

[0019] a sense strand (SO), the sense strand comprising a nucleotide sequence capable of forming a reverse complement to a portion or all of the nucleotide sequence of the antisense strand; and

[0020] The base-free linker (L) used to connect one end of the antisense strand and one end of the sense strand has a structure as shown below:

[0021]

[0022] Wherein, R represents a C2-C30 straight or branched alkylene group, or other divalent linker without a base, X represents oxygen or sulfur, and n represents the number of repeating units, which is an integer ranging from 1 to 20.

[0023] The sense strand and the antisense strand form a hybridization region through reverse complementary pairing, that is, the bases of the antisense strand are completely or partially covered by the sense strand through base pairing, and

[0024] Wherein, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

[0025] It should be noted that the formation of the secondary structure of the antisense oligonucleotide-sense oligonucleotide coupling molecule of the present application is reversible, and an intramolecular hybridization structure can be formed under specific conditions, such as physiological conditions, PBS buffer, sodium chloride solution, Tris buffer, magnesium chloride solution, etc.; and when the target RNA is present in urea solution or solution, the intramolecular hybridization structure is opened.

[0026] In some embodiments, the other non-basic divalent linker is a -[(CH2) p -O] q -(CH2) p - a divalent linker of the structure shown, wherein p is an integer between 2 and 6, and q is an integer between 1 and 20. In a specific example, p is 2, 3, 4, 5, or 6, and in a specific example, q is an integer between 1 and 15, 1 and 12, 1 and 10, 1 and 5, or 1 and 3.

[0027] In a specific embodiment, R is a C2-C30, C2-C15, C2-C10, C2-C7 or C2-C5 straight chain alkylene group, such as ethylene, propylene, butylene, pentylene, etc.

[0028] In some embodiments, the linker strand L is used to connect the 3' end of the antisense strand to the 5' end of the sense strand, or to connect the 5' end of the antisense strand to the 3' end of the sense strand.

[0029] In some embodiments, in the linker chain L, n is selected from 3, 4, 5, 6, 7, 8, 9 or 10, for example, n is 3.

[0030] In some embodiments, some bases of the sense strand are complementarily paired with some bases of the antisense strand.

[0031] In some embodiments, all bases of the sense strand are fully complementary to a portion of the bases of the antisense strand, or all bases of the sense strand are fully complementary to all bases of the antisense strand.

[0032] In some embodiments, the connecting strand L connects the antisense strand and the sense strand through a phosphorothioate diester bond or a phosphodiester bond. In some embodiments, the length of the antisense strand can be 13 to 23 nt, and the length of the sense strand depends on the binding energy of the sense strand and the antisense strand. In theory, as long as the binding force between the sense strand and the antisense strand is strong enough, considering the function of the ASO, preferably, the binding force is close to the binding force between the target RNA and the antisense strand. In a specific embodiment, the number of bases of the complementary pairing of the antisense strand and the sense strand is at least 1, for example, the number of bases of the complementary pairing of the antisense strand and the sense strand is any integer in the range of 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. To achieve the purpose of the present invention, the melting temperature of the hybridization of the sense strand and the antisense strand should be equal to or less than the melting temperature of the hybridization of the target RNA strand and the antisense strand, and the difference between the two melting temperatures should be less than 15 ° C.

[0033] In some embodiments, the antisense strand may be a single-stranded DNA molecule; the sense strand may be a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid molecule.

[0034] In the present invention, in the ASSOC molecule, each nucleotide is independently a modified or unmodified nucleotide. The modification of nucleotides is well known in the art, for example, 2'-F, 2'-OMe, 2'-MOE, PMO, GNA, UNA, PNA, 5'-VP, LNA (for example, see Nat. Rev. Drug Discov. 2020, 19, 673; Nucleic Acids Res. 2023, 51, 2529, which are incorporated herein by reference). Preferably, the modification of nucleotides is 2'-OMe, 2'-MOE, LNA. For example, the 5' end of the antisense strand is modified with 5'-VP or GNA / PNA / UNA is used as the sense strand to perform base complementary pairing with the antisense strand.

[0035] In some embodiments, the ASSOC molecules of the invention may comprise at least one modified nucleotide and / or at least one interstrand modification.

[0036] In some embodiments, all nucleotides on the sense strand are modified nucleotides.

[0037] In some embodiments, all of the nucleotides on the antisense strand are modified nucleotides.

[0038] In some embodiments, substantially all of the nucleotides of the sense strand and the antisense strand are modified nucleotides.

[0039] In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0040] In some embodiments, the modified nucleotides include 2'-F, 2'-OMe, 2'-MOE, PMO, GNA, UNA, PNA, 5'-VP, and LNA modified nucleotides.

[0041] In some embodiments, the modified nucleotides include locked nucleic acid modifications.

[0042] In some embodiments, the interchain modification comprises phosphorothioate, PMO, or PNA modification.

[0043] In a particularly preferred embodiment, the ASSOC molecules of the present invention include phosphorothioate interchain modifications. Preferably, all nucleotides and nucleotides and nucleotides and ethylene glycol units in the ASSOC molecules of the present invention include phosphorothioate interchain modifications.

[0044] In some embodiments, the ASSOC molecule of the present invention further comprises a conjugated group connected to the 5' end and / or 3' end of the molecule. The conjugated group can be routinely selected by a person skilled in the art to provide corresponding targeting, imaging, cellular efficiency, in vivo circulation and other functions.

[0045] In some embodiments, the ASSOC molecule is selected from one of the following:

[0046] MyD88 ASSOC-16nt: 5'-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*C*A*G*A*G*C*A*A*G*G*A*A*T*G*T*G-3';

[0047] MyD88 ASSOC-15nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*A*G*A*G*C*A*A*G*G*A*A*T*G*T*G-3’;

[0048] MyD88 ASSOC-14nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*A*G*A*G*C*A*A*G*G*A*A*T*G*T-3’;

[0049] MyD88 ASSOC-13nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*G*A*G*C*A*A*G*G*A*A*T*G*T-3’;

[0050] MyD88 ASSOC-12nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*G*A*G*C*A*A*G*G*A*A*T*G-3’;

[0051] MyD88 ASSOC-11nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*A*G*C*A*A*G*G*A*A*T*G-3’;

[0052] MyD88 ASSOC-10nt:5’-C*A*C*A*T*T*C*C*T*T*G*C*T*C*T*G*E*E*E*A*G*C*A*A*G*G*A*A*T-3’;

[0053] ApoB ASSOC-13nt:5’-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*C*A*A*G*C*T*G*G*C*C*A*T*T-3’;

[0054] ApoB ASSOC-12nt:5’-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*A*A*G*C*T*G*G*C*C*A*T*T-3’;

[0055] ApoB ASSOC-11nt:5’-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*A*A*G*C*T*G*G*C*C*A*T-3’;

[0056] ApoB ASSOC-10nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*A*G*C*T*G*G*C*C*A*T-3';

[0057] ApoB ASSOC-9nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*A*G*C*T*G*G*C*C*A-3';

[0058] ApoB ASSOC-8nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*G*C*T*G*G*C*C*A-3';

[0059] ApoB ASSOC-7nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*G*C*T*G*G*C*C-3';

[0060] ApoB ASSOC-6nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*C*T*G*G*C*C-3';

[0061] ApoB ASSOC-5nt: 5'-A*A*T*G*G*C*C*A*G*C*T*T*G*E*E*E*C*T*G*G*C-3';

[0062] Among them, the bold font represents the locked nucleic acid base, E represents the ethylene glycol linking chain unit, and * represents the thiophosphate backbone.

[0063] In another aspect, the present invention provides a method for preparing the above-mentioned ASSOC molecule, the method comprising:

[0064] The ASSOC molecule is synthesized by nucleic acid automated synthesis in the presence of unmodified or modified nucleic acid monomers and linker monomers of the following structure,

[0065]

[0066] wherein R is as defined above.

[0067] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule of the present invention and a pharmaceutically acceptable carrier.

[0068] In yet another aspect, the present invention provides a kit comprising the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule of the present invention.

[0069] In another aspect, the present invention provides use of the above ASSOC molecule in the preparation of antisense oligonucleotide drugs.

[0070] In another aspect, the present invention provides a method for regulating the expression of a target gene in a cell, the method comprising: contacting the above-mentioned ASSOC molecule with the cell.

[0071] In a specific embodiment, said modulating comprises decreasing the expression of a target gene.

[0072] Beneficial Effects

[0073] The present invention designs a class of antisense oligonucleotide-sense oligonucleotide coupling (Antisenseoligonucleotide-sense oligonucleotide conjugate, ASSOC) molecules, by constructing an intramolecular hybridization structure, thereby stably improving the selectivity of ASO to substrate sequences and effectively reducing its off-target effect. Experiments have shown that this type of molecule can not only retain efficient target RNA degradation capabilities, but also has significantly lower off-target toxicity than traditional ASO molecules. Compared with the strategy of chemically modifying single-stranded ASO reported in the literature, the strategy of the present invention has better effect robustness. Compared with the intermolecular hybridization strategy reported in the literature (for example, using a special nucleic acid chain of peptide nucleic acid, which has a high affinity with the ASO chain), the intramolecular hybridization strategy of the present invention makes the SO chain more capable of binding to the ASO chain, without the need to use a special nucleic acid chain with high affinity, and the required SO chain length is shorter and does not require annealing before use. The present invention can be applied to the development of the next generation of efficient and low-toxic ASO drugs, and is of great value in the field of antisense oligonucleotide drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The schematic diagrams of the structure of the ASSOC molecule of the present application are shown. A: Schematic diagram of the primary structure of the ASSOC molecule; B: Schematic diagram of the secondary structure of the ASSOC molecule.

[0075] Figure 2 Schematic diagram of the synthesis process of ASSOC molecule.

[0076] Figure 3 Schematic diagram of the structure of traditional ASO molecules and ASSOC molecules targeting MyD88 mRNA.

[0077] Figure 4 The figure shows the melting temperature determination results of MyD88 ASSOC molecules with different sense chain lengths.

[0078] Figure 5The results of the degradation of target and non-target RNAs by traditional ASO targeting MyD88 mRNA and the corresponding MyD88 ASSOC-16nt molecule in the model reaction. A: The degradation degree-time curve of the traditional ASO simultaneously degrading the target and non-target RNAs; B: The degradation degree-time curve of the ASSOC simultaneously degrading the target and non-target RNAs.

[0079] Figure 6 This is a graph showing the results of caspase3 / 7 activity detection in 3T3 cells treated with traditional ASO molecules and ASSOC molecules targeting MyD88 mRNA.

[0080] Figure 7 This is a graph showing the results of measuring the MyD88 mRNA expression level in 3T3 cells treated with traditional ASO molecules and ASSOC molecules targeting MyD88 mRNA.

[0081] Figure 8 Schematic diagram of the structure of traditional ASO molecules and ASSOC molecules targeting ApoB mRNA.

[0082] Fig. 9 The figure shows the results of caspase 3 / 7 activity detection in Huh-7 cells treated with traditional ASO molecules and ASSOC molecules targeting ApoB mRNA.

[0083] Fig.10 This is a graph showing the results of measuring the MyD88 mRNA expression level in Huh-7 cells treated with traditional ASO molecules and ASSOC molecules targeting ApoB mRNA. DETAILED DESCRIPTION

[0084] The present invention will be further described below by way of examples and drawings. These examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0085] the term:

[0086] Herein, among the various modified forms of nucleotides, 2'-F, 2'-OME, and 2'-MOE modifications refer to the replacement of the hydroxyl group at the 2' position of the furanose ring of the nucleotide by fluorine (F), methoxy (OMe), or methoxyethoxy (MOE), respectively; GNA modification refers to glycerol nucleotides, which are composed of glycerol (DNA and RNA are pentoses) linked by phosphate bonds; UNA modification refers to open-ring nucleotides, which lack the C2' and C3' chemical bonds of the RNA ribose ring, but can still mimic the unmodified RNA ribose ring RNA when bound to the RNA double-stranded structure; PNA modification refers to peptide nucleic acids, a class of DNA analogs with a polypeptide backbone replacing the sugar-phosphate backbone; 5'-VP modification refers to the modification of the 5' end of the ASSOC molecule with vinyl-phosphate.

[0087] Herein, the term phosphorothioate modification refers to the substitution of a hydroxyl group on the phosphate backbone with a thiol group, and -OMe modification refers to the substitution of a hydrogen of a hydroxyl group on the phosphate backbone with a methyl group.

[0088] Herein, the "complementarity" of an oligonucleotide means that at least one of the oligonucleotide nucleic acid or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof can form hydrogen bonds with each other when the nucleobase sequences of the oligonucleotide and the other nucleic acid are arranged in opposite directions. Complementary nucleobases mean nucleobases that can form hydrogen bonds with each other according to the principle of classical base complementary pairing. That is, adenine (A) and thymine (T); adenine (A) and uracil (U); cytosine (C) and guanine (G); and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleobase complementarity at each nucleoside. In some embodiments, some mismatches can be tolerated. "Complete complementarity" or "100% complementarity" of an oligonucleotide means that an oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of an oligonucleotide.

[0089] Theoretical analysis

[0090] 1. The introduction of the positive chain is conducive to improving the selectivity of hybridization between ASO and target RNA:

[0091] The reaction equations for the hybridization reaction between the ASO chain (abbreviated as A) and the target RNA chain (abbreviated as B) and the non-target RNA chain (abbreviated as C) can be written as:

[0092]

[0093]

[0094] The equilibrium constant of the two reactions is:

[0095]

[0096]

[0097] Assume that the initial concentration of chain A is a, the initial concentration of chain B is b, the initial concentration of chain C is c, the equilibrium concentration of chain AB is x, and the equilibrium concentration of chain AC is y.

[0098] The selectivity of the reaction can be quantified by the ratio of the concentrations of the AB and AC duplexes at equilibrium:

[0099]

[0100] For nucleic acid double-strand hybridization reactions, the reaction equilibrium constant K is related to the standard Gibbs free energy change ΔG of the reaction. oThere is the following relationship (Nat.Chem.2012,4,208):

[0101] ΔG o =-RT ln K-(Δn)RT ln m

[0102] Where R is the ideal gas constant, T is the temperature, Δn is the difference between the number of species after the reaction and the number of species before the reaction (for example, for AB hybridization reaction, Δn = -1), and m is the initial concentration value of the reactant with a lower initial concentration. From this, we can get:

[0103]

[0104] For AB hybridization reaction:

[0105]

[0106] Where m AB Indicates the initial concentration value of the reactant with lower initial concentration in the AB hybridization reaction. represents the standard Gibbs free energy change of the AB reaction;

[0107] For AC hybridization reaction:

[0108]

[0109] Where m AC Indicates the initial concentration value of the reactant with lower initial concentration in AC hybridization reaction, represents the standard Gibbs free energy change of AC reaction;

[0110] So the selectivity of the reaction is:

[0111]

[0112] When the concentration of each chain is fixed, is a constant. Due to the huge number of genomic sequences, the initial concentrations of target RNA and ASO can be considered to be much smaller than non-target RNA, and the binding force between non-target RNA and ASO is relatively weak. We can assume that c>>y, cy≈c. For traditional ASO, It is generally a large negative value (i.e., the ASO should have a strong binding force with the target RNA). When the ASO concentration is high, x≈b, bx≈0, and Q≈0, indicating that the ASO has no selectivity and the off-target effect is obvious.

[0113] If it can effectively improve So that x→0, then:

[0114]

[0115] When the concentration of each chain is fixed, is a constant greater than 0, so Q tends to a constant greater than 0 at this time.

[0116] Furthermore, since the B chain is the target chain, the binding ability of the A chain and the B chain is greater than the binding ability of the A chain and the C chain, that is:

[0117]

[0118] therefore:

[0119]

[0120] so:

[0121]

[0122] So at this time Q tends to be greater than Obviously, the selectivity of ASO chain is higher at this time.

[0123] The following proves that the introduction of justice chain can effectively improve Values:

[0124] The hybridization reaction formula of the sense strand (abbreviated as D) and the antisense strand (A) is:

[0125]

[0126] The Gibbs free energy change for the reaction is:

[0127]

[0128] The hybridization reaction formula of the target strand (B) and the antisense strand is:

[0129]

[0130] The Gibbs free energy change for the reaction is:

[0131]

[0132] The hybridization reaction formula of the target chain (B) and the AD double chain is:

[0133]

[0134] The Gibbs free energy change for the reaction is:

[0135]

[0136] Substituting the Gibbs free energy change of the AD reaction into the equation:

[0137]

[0138] because so That is, the introduction of the justice chain makes Increase.

[0139] In summary, the introduction of the positive chain is conducive to improving the selectivity of hybridization between ASO and target RNA, thereby reducing the off-target effect of ASO.

[0140] 2. The introduction of the positive chain can improve the robustness of the ASO chain selectivity:

[0141] because:

[0142]

[0143] Significantly For traditional ASO, Δn = -1, so The larger the Q, the smaller the Q. The introduction of the positive chain makes the corresponding reaction equation become:

[0144]

[0145]

[0146] This makes the Δn of the reaction equal to 0:

[0147]

[0148] at this time No longer affected , thereby eliminating the effect of changes in the relative concentration between the ASO chain and the target RNA chain on the ASO selectivity.

[0149] Example

[0150] Materials and reagents

[0151] All reagents used were used directly without special treatment. Solvents were purchased from Sinopharm Group (analytical grade) or Inotech Technology Co., Ltd. Reaction raw materials were purchased from Aladdin, Inotech and other chemical reagent companies with a purity greater than 95%. Nucleotide monomers and reagents required for ASSOC molecule synthesis were purchased from Dynaxin Biotech, model RNA substrates were purchased from Hongxun Biotech Co., Ltd., cells were sourced from the Westlake University Cell Bank, caspase 3 / 7 kits were purchased from Bioengineering, and mRNA extraction and qPCR experimental kits were purchased from TakaRa.

[0152] Example 1

[0153] This example illustrates that the automated synthesis of the ASSOC molecules in this application can be achieved using a nucleic acid synthesizer.

[0154] A synthesis process of ASSOC molecules targeting MyD88 mRNA, the main technical points are as follows (synthesis route diagram see Figure 2 ):

[0155] a) Prepare monomer solution: four DNA monomers A, T, C, and G are dissolved in acetonitrile to a final concentration of 50 g / L; four locked nucleic acid monomers LA, LT, LC, and LG are dissolved in acetonitrile: dichloromethane = 1:1 solution to a final concentration of 80 g / L; ethylene glycol linker monomer (DMT-EG-CEP) is dissolved in acetonitrile to a final concentration of 50 g / L. DMT-EG-CEP was synthesized by the method of reference (Chemistry Select 2021, 6, 10648), and its structure is as follows:

[0156]

[0157] b) Synthesized using a nucleic acid automatic synthesizer (Qingke Bio Single-stranded Nucleic Acid Synthesizer-192P), 200 nmol The universal CPG synthesis column is a solid phase carrier, synthesized Figure 3 and 8 The ASSOC molecules and conventional ASO molecules shown in the figure. The sequences of the molecules are shown in Tables 1 and 2 below, where the normal fonts are DNA bases, the bold fonts are locked nucleic acid bases, E is the ethylene glycol linker unit, and * indicates the thiophosphate backbone. The parameters recommended by the manufacturer were used for each step of the reaction.

[0158] c) Cleavage and deprotection: The synthesis column was transferred to an aminolysis apparatus, concentrated aqueous ammonia was added, and the reaction was carried out at 95° C. for 2 hours.

[0159] d) Use 0.2mL deionized water to dissolve the product, and then add 20μL 3M sodium chloride solution and 500μL ethanol to precipitate the product. After storing at -20℃ overnight, collect the product precipitate by centrifugation. Then redissolve the product precipitate in 500μL deionized water and transfer it to an ultrafiltration tube, and perform ultrafiltration purification more than 3 times using deionized water as solvent. Collect the product solution after ultrafiltration to obtain the product stock solution for subsequent experiments. Identify the product by mass spectrometry (theoretical value: 10952.4, measured value: 10953.0) and non-denaturing polyacrylamide gel electrophoresis.

[0160] Table 1

[0161]

[0162]

[0163] Table 2

[0164]

[0165]

[0166] Example 2

[0167] Determination of melting temperature of ASSOC molecule:

[0168] MyD88 ASSOC molecules with different sense chain lengths were prepared (structures such as Figure 3 The test solution is shown in the figure. The final solution composition is: 1×PBS, nucleic acid molecule concentration is 2μM, 1×EvaGreen. Add 20μL of the test solution to each PCR tube and transfer to the PCR instrument (Bio-Rad CFX Connect). Annealing program: 98.0℃ for 2 minutes, cool down to 25.0℃ at 0.05℃ / s, and 25.0℃ for 1 minute; melting program: 25℃ for 1 minute, heat up to 98.0℃ at 0.05℃ / s, and measure fluorescence every 10 seconds. Finally, the instrument reads the melting temperature of the sample according to the fluorescence curve, and the result is as follows: Figure 4 shown.

[0169] The blue dotted line indicates the melting temperature of the double chain formed by the traditional ASO and the 16nt sense chain (60.0℃), which is much lower than the melting temperature of the 16nt ASSOC (76.0℃), indicating that the intramolecular hybridization structure can significantly improve the affinity of the sense chain and the antisense chain. The melting temperature of the 10nt ASSOC (59.5℃) is close to 60.0℃, indicating that to achieve similar sense chain-antisense chain affinity, the ASSOC structure requires a shorter sense chain length. The red line indicates the melting temperature of the double chain formed by the traditional ASO and the target RNA chain (70.0℃). Therefore, by adjusting the length of the sense chain in the ASSOC, the intramolecular affinity can be effectively adjusted to be close to or even greater than the affinity with the target RNA.

[0170] Example 3

[0171] The selectivity of ASSOC molecules is more robust as demonstrated by model reactions:

[0172] Prepare the model RNA solution: dissolve the target RNA model molecule (R0, sequence: 5'-Cy3-CAGAGCAAGGAAUGUG-BHQ2-3', SEQ ID No.: 14) with a Cy3 fluorescent label and a quencher group BHQ2 at both ends and the non-target RNA model molecule (R2, sequence: 5'-Cy5-CAGAGC-AGGAAU-UG-BHQ2-3', the long horizontal line indicates the base deletion, SEQ ID No.: 15) with a Cy5 fluorescent label and a quencher group BHQ2 at both ends in 1× RNase buffer, so that the final concentration of the two RNA molecules is 2 μM.

[0173] Model reaction: Take 80 μL of model RNA solution in a 96-well plate and add 2.5×10 -17 U RNase, incubate at 37°C in a microplate reader (BioTek SYNERGY H1) for 10 min, then add traditional ASO or ASSOC-16nt solution (final ASO or ASSOC molecule concentration is 2 μM), continue to incubate at 37°C in the microplate reader and measure the fluorescence values ​​of Cy3 and Cy5 in the solution every 1 min.

[0174] The results are as follows Figure 5 As shown, it can be seen that for traditional ASO ( Figure 5 A) Although the degradation degree of R0 and R2 is different at the beginning of the reaction, both are quickly and completely degraded as time goes by. The selectivity in the early stage of the reaction mainly comes from the higher R0 concentration, and the ASO chain can bind more to the R0 chain. As the reaction proceeds, the R0 concentration decreases and the relative concentration of ASO increases (i.e., the theoretical analysis shows that the R0 concentration is 1.34, which is 1.77). In the case of increasing, the reaction selectivity decreases, resulting in the degradation of R2, and the final reaction selectivity is 0. Figure 5 In B), not only can R0 be selectively degraded at the beginning of the reaction, but this selectivity tends to be stable over time, indicating that the selectivity of ASSOC is less affected by concentration and has significantly better robustness.

[0175] Example 4

[0176] 3T3 cells were seeded into 96-well plates at a seeding density of 10,000 cells per well, with 100 μL of DMEM medium per well, and cultured at 37°C in a 5% CO2 environment. The next day, each ASSOC molecule and traditional ASO molecule (structured as Figure 3The Lipofectamine 2000 complex solution (as shown) was added to the cell culture medium of each well, and the final concentration of the oligonucleotide chain was 25nM. Each group of three parallel wells. The cells were then cultured for one day at 37°C and 5% CO2. The caspase 3 / 7 kit (Shanghai Biotechnology Co., Ltd.) was then used to detect the caspase 3 / 7 activity of the cells, and the untreated cells were used as blank controls for calculation. The results are shown in the attached figure. Figure 6 shown.

[0177] from Figure 6 It can be seen that for the MyD88 mRNA target, the ASSOC molecule has lower off-target toxicity than traditional ASO molecules.

[0178] Example 5

[0179] 3T3 cells were seeded into 6-well plates at a seeding density of 150,000 cells per well, with 2 mL of DMEM medium per well, and cultured at 37°C in a 5% CO2 environment. The next day, ASSOC-16nt molecules and traditional ASO molecules (structured as Figure 3 The Lipofectamine 2000 complex solution (as shown) was added to the cell culture medium of each well, and the final concentration of the oligonucleotide chain was 25nM, with 3 parallel wells in each group. The cells were then cultured for one day at 37°C and 5% CO2. The TaKaRa kit was then used to extract RNA from each well of cells, prepare cDNA, and detect the MyD88 mRNA expression of the cells by qPCR. The GAPDH mRNA expression was used for normalization, and the relative expression of MyD88 mRNA in untreated cells was calculated as 100%. The measurement results are shown in Figure 7 shown.

[0180] from Figure 7 It can be seen that for the MyD88 mRNA target, the ASSOC molecule has a target mRNA knockdown efficacy similar to that of the traditional ASO molecule.

[0181] Example 6

[0182] Huh-7 cells were seeded into 96-well plates at a seeding density of 10,000 cells per well, with 100 μL of DMEM medium per well, and cultured at 37°C in a 5% CO2 environment. The next day, each ASSOC molecule and traditional ASO molecule (structured as Figure 8The Lipofectamine2000 complex solution (as shown) was added to the cell culture medium of each well, and the final concentration of the oligonucleotide chain was 10nM, and each group was parallel to 5 wells. The cells were then cultured for one day at 37°C and 5% CO2. The culture medium of all cells was then replaced with fresh DMEM culture medium, and the cells were cultured for two days at 37°C and 5% CO2. Finally, the caspase 3 / 7 kit (Shanghai Biotechnology) was used to detect the caspase 3 / 7 activity of the cells, and the untreated cells were used as blank controls for calculation. The results are shown in Fig. 9 shown.

[0183] from Fig. 9 It can be seen that for the ApoB mRNA target, the ASSOC molecule has lower off-target toxicity than traditional ASO molecules.

[0184] Example 7

[0185] Huh-7 cells were seeded into 12-well plates at a seeding density of 150,000 cells per well, with 1 mL of DMEM medium per well, and cultured at 37°C in a 5% CO2 environment. The next day, ASSOC-6nt molecules and traditional ASO molecules (structured as Figure 8 The Lipofectamine 2000 complex solution (as shown) was added to the cell culture medium of each well, and the final concentration of the oligonucleotide chain was 10nM, with 3 parallel wells in each group. The cells were then cultured for one day at 37°C and 5% CO2. The TaKaRa kit was then used to extract RNA from each well of cells, prepare cDNA, and detect the ApoB mRNA expression of the cells by qPCR. The GAPDH mRNA expression was used for normalization, and the relative expression of ApoB mRNA in untreated cells was calculated as 100%. The measurement results are shown in Fig.10 shown.

[0186] from Fig.10 It can be seen that for the ApoB mRNA target, the ASSOC molecule has a target mRNA knockdown efficacy similar to that of the traditional ASO molecule.

[0187] The sequence numbers in the sequence table are as follows:

[0188] name SEQ ID No. MyD88 conventional ASO / antisense chain 1 Sense strand of MyD88ASSOC-16nt 2 MyD88ASSOC-15nt sense strand 3 Sense strand of MyD88ASSOC-14nt 4 Sense strand of MyD88ASSOC-13nt 5 MyD88ASSOC-12nt sense strand 6 MyD88ASSOC-11nt sense strand 7 MyD88ASSOC-10nt positive strand 8 ApoB conventional ASO / antisense strand 9 ApoB ASSOC-13nt positive strand 10 ApoB ASSOC-12nt positive strand 11 ApoB ASSOC-11nt positive strand 12 ApoB ASSOC-10nt positive strand 13 R0 14 R2 15

Claims

1. An antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule in the form of a primary structure, comprising the following three parts: Antisense strand (ASO); a sense strand (SO), the sense strand comprising a nucleotide sequence capable of forming a reverse complement to a portion or all of the nucleotide sequence of the antisense strand; and The base-free linker (L) used to connect one end of the antisense strand and one end of the sense strand has the following structure: in, R represents a C2-C30 straight or branched alkylene group, or other divalent linker without a base, X represents oxygen or sulfur, and n represents the number of repeating units, which is an integer ranging from 1 to 20. Wherein, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

2. An antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule in the form of a secondary structure, comprising the following three parts: Antisense strand (ASO); A sense strand (SO), comprising a nucleotide sequence that is capable of forming a reverse complement to a portion or all of the nucleotide sequence of the antisense strand; The base-free linker (L) used to connect one end of the antisense strand and one end of the sense strand has a structure as shown below: in, R represents a C2-C30 straight or branched alkylene group, or other divalent linker without a base, X represents oxygen or sulfur, and n represents the number of repeating units, which is an integer ranging from 1 to 20. The sense strand and the antisense strand form a hybridization region through reverse complementary pairing, that is, the bases of the antisense strand are completely or partially covered by the sense strand through base pairing, and Wherein, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

3. The ASSOC molecule according to claim 1 or 2, in, R is a C2-C30 straight chain alkylene or -[(CH2) p -O] q -(CH2) p - a divalent linker of the structure shown, wherein p is an integer between 2 and 6, q is an integer between 1 and 20, for example, R is ethylene, propylene, butylene or pentylene; and / or n is selected from 3, 4, 5, 6, 7, 8, 9 or 10.

4. The ASSOC molecule according to claim 1 or 2, in, The connecting strand L is used to connect the 3' end of the antisense strand to the 5' end of the sense strand, or to connect the 5' end of the antisense strand to the 3' end of the sense strand.

5. The ASSOC molecule according to claim 1 or 2, in, Some bases of the sense strand complementarily pair with some bases of the antisense strand; or All bases in the sense strand are fully complementary to some bases in the antisense strand, or All bases in the sense strand are fully complementary to all bases in the antisense strand.

6. The ASSOC molecule according to claim 1 or 2, in, The antisense strand is a single-stranded DNA molecule; the sense strand is a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid molecule.

7. The ASSOC molecule according to claim 1 or 2, in, The nucleotide modification in the sense strand or the antisense strand is selected from 2'-F, 2'-OMe, 2'-MOE, PMO, GNA, UNA, PNA, 5'-VP and LNA, and / or The ASSOC molecule includes phosphorothioate interchain modifications between all nucleotides and between nucleotides and connecting chains, and / or The ASSOC molecule further comprises a conjugated group connected to the 5' end and / or the 3' end of the molecule to provide corresponding targeting, imaging, cell entry efficiency, and in vivo circulation functions.

8. The ASSOC molecule according to claim 1 or 2, wherein: The ASSOC molecule is selected from one of the following: Among them, the bold font represents the locked nucleic acid base, E represents the ethylene glycol linking chain unit, and * represents the thiophosphate backbone.

9. The method for preparing the ASSOC molecule according to claim 1, comprising: The ASSOC molecule is synthesized by nucleic acid automated synthesis in the presence of unmodified or modified nucleic acid monomers and linker monomers of the following structure, Wherein R is defined as in claim 1.

10. A pharmaceutical composition comprising the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule according to claim 1 or 2, and a pharmaceutically acceptable carrier.

11. A kit comprising the antisense oligonucleotide-sense oligonucleotide conjugate (ASSOC) molecule according to claim 1 or 2.

12. Use of the ASSOC molecule according to any one of claims 1 to 8 in the preparation of antisense oligonucleotide drugs.

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