RNA interference triggering molecule and application thereof

Abstract

The invention relates to the field of nucleic acid medicines, in particular to an RNA (Ribonucleic Acid) interference triggering molecular structure and clinical application thereof. The RNA interference triggering molecule provided by the invention is a partial double-stranded molecule consisting of an antisense strand and a heterozygous strand, and the heterozygous strand comprises three modules: (1) a shortened sense strand, (2) a DNA strand, and (3) other coupled chemical molecules (including lipids, small molecules, polypeptides, sugar molecules and the like). The length of the sequence of the positive-sense strand is remarkably shortened, the opportunity that the positive-sense strand is competitively combined with RISC can be eliminated, the miss-target effect possibly existing in the positive-sense strand is reduced, meanwhile, due to the fact that the positive-sense strand is shortened and the number of phosphate groups is reduced, the hydrophilicity of small nucleic acid molecules is reduced, and the electronegativity and hydrophilicity of the molecules are reduced; the antisense strand and the positive-sense strand-conjugate are separated from each other when passing through a cell membrane, and the antisense strand independently enters cytoplasm to avoid the ring resistance of endosome / lysosome.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid drugs, and in particular to an RNA interference triggering molecule and its uses. Background Technology

[0002] In its natural state, nucleic acid molecules are polyanionic macromolecules that do not possess the typical physicochemical properties of classic drug molecules or lack "druglikeness". Although the concept of "druglikeness" was initially aimed at the development of small molecule chemical drugs. Because data analysis has found that existing drugs often have some common physicochemical properties. Therefore, by comparing the similarity of the physicochemical properties of a new chemical molecule with those of existing drugs, it is possible to predict the chances of the chemical molecule becoming a candidate drug and whether it is worthwhile to carry out further development[1]. However, historical data shows that this principle is also applicable to guiding the development of RNAi drugs. For example, various chemical modifications that have been widely explored and used are aimed at improving the stability of small nucleic acid molecules and making them more "druglike". Various chemical modifications improve the physicochemical properties of small nucleic acid molecules as drug molecules from various aspects, giving them good in vivo ADME properties, including: making small nucleic acid drug molecules resistant to RNase digestion, improving lipophilicity, improving efficacy, reducing the side effects of innate immunity, reducing off-target effects, etc. In short, the progress of chemical modification of small nucleic acid molecules has made the successful development of RNAi drugs a reality[2,5].

[0003] Alnylam's GalNAc-siRNA chemical conjugation technology platform has achieved great success, enabling breakthroughs in the treatment of liver-related clinical diseases with small interfering RNA drugs. However, there has been no equivalent progress in extending this technology platform to extrahepatic applications. Clearly, only by successfully achieving extrahepatic delivery of small nucleic acid drugs such as siRNA or ASO can the anticipated grand vision—that RNAi drugs become an effective independent drug class—be truly realized.

[0004] Currently approved siRNA therapies target liver diseases. This is not surprising, as the liver is actually a natural accumulation organ for RNAi drugs. Especially after effective chemical modifications largely solved stability issues, the circulating half-life of RNAi drugs has been significantly prolonged. Overcoming liver retention has become a bottleneck for expanding in vivo applications.

[0005] Different strategies have been proposed to develop feasible methods for extrahepatic delivery, with most of the work focusing on finding biocompatible systems similar to GalNAc-ASGPR. However, progress in this area has been less than satisfactory, possibly because the GalNAc-ASGPR system is unique in the human body.

[0006] From the historical experience of siRNA drug development, chemical modification is perfect in promoting the hepatocyte-targeted delivery of small nucleic acid drug molecules. Chemical modification has greatly improved the druglikeness of small nucleic acid molecules in many aspects, significantly improved the stability of small nucleic acid molecules, reduced off-target effects, and reduced immune stimulation, enabling RNAi drugs to break through in the treatment of liver diseases and leading to the commercial success of small nucleic acid drugs [3]. So what are the key issues that chemical modification should address in breaking through extrahepatic delivery?

[0007] The small nucleic acid drugs that have been successfully applied in clinical practice mainly include siRNA, miRNA, and ASO, whose molecular weight (~4000 to ~15000 Da) is much lower than the lower limit of renal filtration (~40000 Da). Most of the disease targets of these approved small nucleic acid drugs are located in the liver. The half-life of fully chemically modified GalNAc-siRNA conjugate molecules in the blood is less than 2 hours (in rats) [4], while the half-life in the liver is close to 25 hours. Similarly, although the blood half-life of LNP-siRNA preparations is less than 1 hour, about 80% of LNPs remain in the liver, which is similar to the distribution ratio of actively targeted GalNAc-siRNA preparations. These results suggest that the passive targeting of small nucleic acid drugs to the liver by nanoparticles such as LNP is similar to the active liver targeting of GalNAc, and most of the drugs will always be distributed in the liver. In addition, both drug preparations are released into the cytoplasm through the endosome / lysosome pathway, and the release rate is low. Although the release rate of LNP drugs is slightly higher than that of GalNAc conjugates, GalNAc conjugates have >80% of their contents entering hepatocytes[5].

[0008] Therefore, the key to effective extrahepatic delivery may lie in how to avoid the retention of small nucleic acid drugs by the liver. There may be two reasons for this: (1) Due to the tissue characteristics of the liver, blood flow slows down significantly when passing through the sinusoids of the liver lobules, and the gaps between the endothelial cells of the sinusoids are wide, which facilitates the diffusion of blood components, especially macromolecules, into the interstitial space, increasing the chance of exchange between blood components and liver tissue cells. Therefore, >60-80% of small nucleic acid drug molecules remain in the liver, becoming the main bottleneck for extrahepatic delivery of small nucleic acid drugs. Although there are various reported extrahepatic delivery technologies, including the use of antibodies, antibody fragments, aptamers, peptides, etc., the efficiency still needs to be improved. (2) Small nucleic acid molecules are polyanionic biomolecules and cannot enter cells on their own. Current delivery methods all use the endocytosis pathway to bring small nucleic acid drug molecules into cells. The endocytosis pathway is a cellular physiological process of biological evolution. Endosomes / lysosomes contain many acidic hydrolases, whose main function is to digest and degrade the swallowed substances. In addition to the rapid degradation of unmodified small nucleic acid molecules in endosomes / lysosomes, only a very small fraction of chemically modified small nucleic acid molecules escape from the entrapment of endosomes / lysosomes.

[0009] Currently, the mainstream methods for delivering small nucleic acid molecules in vivo are divided into two main categories: LNP and GalNAc. Both of these methods suffer from low intracellular release efficiency. Although some literature reports that chemical reagents such as quinine chloride or transmembrane peptides such as melittin are used in vitro to treat cells to disrupt the stability of endosomes / lysosomes and promote the escape efficiency of small nucleic acid molecules, unfortunately, these endosome lysins cause unacceptable cytotoxicity at effective dose concentrations and cannot be used clinically[6].

[0010] Therefore, in order to enable small nucleic acid drugs to be used to treat extrahepatic diseases, the bottleneck of delivery—the large amount of small nucleic acid drugs being retained by the liver—must be overcome, including how to improve the efficiency of intracellular drug release.

[0011] References

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[0013] 2.Chelliah Selvama,*,Daniel Mutisyab,Sandhya Prakashc,KasturiRangannaa,and Ramasamy Thilagavathi,Therapeutic potential of chemicallymodified siRNA:Recent Trends.Chem Biol Drug Des.2017November;90(5):665–678.

[0014] 3.Aaron D.Springer and Steven F.Dowdy,GalNAc-siRNA Conjugates:

[0015] Leading the Way for Delivery of RNAi Therapeutics.NUCLEIC ACIDTHERAPEUTICS,Volume 28,Number 3:109-1182018

[0016] 4.Guohua An,Pharmacokinetics and Pharmacodynamics of GalNAc-Conjugated siRNAs.The Journal ofClinical Pharmacology,2024,64(1)45–57

[0017] 5.Alexandre J.Debacker,1Jon Voutila,1Matthew Catley,1David Blakey,1and Nagy Habib,Delivery of Oligonucleotides to the Liver with GalNAc:FromResearch to Registered Therapeutic Drug,Molecular Therapy 2020,28(8):1579-1771

[0018] 6.Dakota J.Brock1, Helena M.Kondow-McConaghy1, Elizabeth C.Hager1, and Jean Philippe Pellois. Endosomal escape and cytosolic penetration of macromolecules mediated by synthetic delivery agents. BioconjugChem. 2019February 20;30(2):293–304.

[0019] 7.Yosef Landesman*,Nenad Svrzikapa,Armand Cognetta III,Xuemei Zhang,Brian R Bettencourt,Satya Kuchimanchi,Keri Dufault,Sarfraz Shaikh,MapleGioia,Akin Akinc,Renta Hutabarat,Rachel Meyers.In vivo quantification offormulated and chemically modified small interfering RNA by heating-in-Tritonquantitative reverse transcription polymerase chain reaction(HIT qRT-PCR).Silence 2010,1:16 Summary of the Invention

[0020] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an RNA interference triggering molecule and its uses to solve the problems in the prior art.

[0021] To achieve the above and other related objectives, this invention provides an RNA interference triggering molecule composed of an antisense strand and a hybrid strand. The hybrid strand includes a modified sense strand sequence, which is shorter than the antisense strand. The antisense strand and the modified sense strand sequence bind through complementary base pairings.

[0022] Preferably, the RNA interference triggering molecule is a molecule having any of the following structures:

[0023] Molecular 1: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence;

[0024] Molecular 2: Composed of an antisense chain and a hybrid chain, wherein the hybrid chain is a conjugate molecule formed by a sense chain sequence and one or more chemical conjugates;

[0025] Molecular 3: Composed of an antisense strand and a hybrid strand, wherein some nucleotides in the sense strand of the hybrid strand are replaced by lipid-soluble chemical molecules;

[0026] Molecular 4; consists of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence, and some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules;

[0027] Molecular 5: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is a conjugate molecule formed by a sense strand sequence and one or more chemical conjugates, wherein some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules.

[0028] The present invention also provides a pharmaceutical composition comprising the aforementioned RNA interference triggering molecule and pharmaceutically acceptable excipients.

[0029] The present invention also provides the use of the aforementioned RNA interference triggering molecule or the aforementioned pharmaceutical composition in the preparation of disease treatment products.

[0030] The present invention also provides a method for preventing or treating a disease, the method comprising administering to a subject an effective amount of the aforementioned RNA interference triggering molecule or the aforementioned pharmaceutical composition.

[0031] As described above, the RNA interference triggering molecule and its uses of the present invention have the following beneficial effects:

[0032] (1) In this invention, the length of the positive chain is significantly shortened to about 10 nt, which can eliminate the opportunity for the positive chain to compete for binding to RISC, reduce the off-target effect that the positive chain may have, and at the same time, because shortening the positive chain reduces the number of phosphate groups, the hydrophilicity of small nucleic acid molecules is reduced, thus reducing the electronegativity and hydrophilicity of the molecules.

[0033] (2) In this invention, fusogenic molecules similar to TEG-LIPID are coupled with a shortened positive strand, which can improve the hydrophobicity of the entire small nucleic acid molecule, increase its binding ratio with plasma proteins, and promote cell transfection efficiency. This also improves the druglikeness of the entire small nucleic acid molecule. Fusogenic molecules include various lipid molecules, lipid-soluble small molecule chemicals, transmembrane peptide molecules, Fab molecules, Aptamer molecules, etc.

[0034] (3) In this invention, the RNA interference triggering molecule can be directly used as the antisense strand for delivery. Therefore, the fabrication of this structure is simple, freeing it from complex in vivo small nucleic acid drug delivery systems, and it can be effectively applied to clinical drug development.

[0035] (4) The antisense and sense strands-coupled molecules separate from each other when passing through the cell membrane. The antisense strand enters the cytoplasm independently, avoiding the entrapment of endosomes / lysosomes. Attached Figure Description

[0036] Figure 1 The diagram shown is a schematic representation of the structure of the RNA interference triggering molecule in Embodiment 1 of the present invention.

[0037] Figure 2 The results of RNA interference-triggered in vitro gene knockdown in Example 1 of this invention are shown; the dangling sequence positions are divided into two orientations: F2LS and LSF1. LS is a single-stranded circular sequence, which is a dangling sequence unrelated to the target gene.

[0038] Figure 3 The diagram shown is a schematic diagram of the structure of the self-transfected RNA interference triggering molecule (named FsiRNA-2.0) in Example 2 of this invention.

[0039] Figure 4 The image shows the result of agarose gel electrophoresis verification of the structure of the RNA interference triggering molecule FsiRNA-2.0 in Example 2 of this invention after the annealing reaction.

[0040] Figure 5 The image shows the results of in vitro gene knockdown using the structure of the RNA interference trigger molecule FsiRNA-2.0 in Example 2 of this invention.

[0041] Figure 6 The diagram shows the structure of FsiRNA-3.0, the RNA interference triggering molecule in Example 4 of this invention.

[0042] Figure 7 The image shows the result of agarose gel electrophoresis to verify the structure formation of the RNA interference triggering molecule FsiRNA-3.0 after the annealing reaction in Example 4 of this invention.

[0043] Figure 8 The results shown are from the in vitro knockdown of gene expression by the RNA interference triggering molecule FsiRNA-3.0 in Example 4 of this invention.

[0044] Figure 9 The results show the knockdown of gene expression in different in vitro cells using the RNA interference triggering molecule FsiRNA-3.0 in Example 4 of this invention.

[0045] Figure 10 The results of the fluorescence tracing experiment in Example 6 of this invention are shown; wherein, FAM is labeled on the AS chain; Lipo cells are transfected with LF2000; RNA interference triggering molecules are directly added to the culture medium. After incubation for 24 hours, cells are fixed with 4% paraformaldehyde, washed, mounted, and confocal images are acquired.

[0046] Figure 11 The results of the fluorescent labeling tracer experiment in Example 6 of this invention are shown; wherein, FAM labels the AS strand; Cy5 labels the SS strand; and RNA interference triggering molecules are directly added to the culture medium. After incubation for 10 minutes, cells are fixed with 4% paraformaldehyde, washed, mounted, and then confocal images are acquired.

[0047] Figure 12 The results shown are from a dual-color fluorescent labeling tracer experiment in Example 6 of this invention, in which conventional siRNA molecules were transfected into HeLa cells using LF2000.

[0048] Figure 13 The results shown are from a dual-color fluorescent labeling tracer experiment of RNA interference triggering molecules in HeLa cells, as described in Example 6 of this invention.

[0049] Figure 14 The image shows the in vivo knockdown results of the RNA interference triggering molecule FsiRNA-3.0 in Example 7 of this invention, targeting the target gene.

[0050] Figure 15 The diagram shows the structure of FsiRNA-4.0, the RNA interference triggering molecule in Example 8 of this invention.

[0051] Figure 16 The image shows the result of agarose gel electrophoresis verification of the structure formed after the annealing reaction of the RNA interference triggering molecule FsiRNA-4.0 in Example 8 of this invention.

[0052] Figure 17 The results shown are from the in vitro knockdown of gene expression by the RNA interference triggering molecule FsiRNA-4.0 in Example 9 of this invention.

[0053] Figure 18 The diagram shows the structure of FsiRNA-6.0, the RNA interference triggering molecule in Example 10 of this invention.

[0054] Figure 19 The image shows the results of agarose gel electrophoresis verification of the structure formed after the annealing reaction of the RNA interference triggering molecule FsiRNA-6.0 in Example 10 of this invention.

[0055] Figure 20The results of agarose gel electrophoresis verification of the structure formation after annealing of RNA interference triggering molecules FsiRNA-6.0 with different nucleic acid chain lengths in Example 10 of the present invention are shown, comparing the effect of nucleic acid chain length on the completeness of structure formation in the FsiRNA-6.0 structure.

[0056] Figure 21 The results of agarose gel electrophoresis verification of the annealed structure formation of RNA interference triggering molecules FsiRNA-6.0 with different nucleic acid chain lengths in Example 10 of the present invention are shown. The influence of nucleic acid chain length on the completeness of structure formation in the FsiRNA-6.0 structure is systematically compared.

[0057] Figure 22 This shows the results of knocking down gene expression in vitro using the structures of the RNA interference triggering molecule FsiRNA-6.0 with different nucleic acid chain lengths in Example 10 of the present invention.

[0058] Figure 23 The diagram shows the in vivo pharmacodynamic verification experiment grouping scheme for the structure of the RNA interference triggering molecule FsiRNA-6.0 in Example 11 of this invention.

[0059] Figure 24 The results shown are the PK study results of the RNA interference triggering molecule FsiRNA-6.0 drug in Example 11 of this invention.

[0060] Figure 25 The results shown are the PD study results of the RNA interference triggering molecule FsiRNA-6.0 drug in Example 11 of this invention. Detailed Implementation

[0061] This invention provides an RNA interference triggering molecule, which is composed of an antisense strand and a hybrid strand. The hybrid strand contains a sense strand sequence of unequal length to the antisense strand. The hybrid strand also contains other molecules covalently coupled to the sense strand sequence; these other molecules are: 1) an unrelated dangling sequence, or 2) one or more lipid-soluble chemical molecules. The hybrid strand binds complementary to the antisense strand through its contained sense strand sequence.

[0062] In some specific embodiments, if the hybrid chain contains an unrelated dangling sequence, the unrelated dangling sequence is complementary to another matching chain, and the matching chain is coupled with one or more other lipophilic chemical molecules to form a conjugate molecule.

[0063] In some specific embodiments, the length of the positive chain sequence is shorter than the length of the negative chain. Specifically, the length of the negative chain is 15-27 nt. More specifically, the length of the negative chain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nt.

[0064] In some specific embodiments, the antisense strand in the RNA interference triggering molecule is complementary to the sense strand sequence in the hybrid strand.

[0065] In some specific embodiments, the hybrid strand in the RNA interference triggering molecule has one or more of the following characteristics:

[0066] 1) The hybrid chain contains unrelated dangling sequences;

[0067] 2) The hybrid chain contains chemical conjugates;

[0068] 3) Some nucleotides in the hybrid chain are replaced by lipid-soluble chemical molecules.

[0069] In some specific embodiments, the RNA interference triggering molecule is a molecule having any of the following structures:

[0070] Molecular 1: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence.

[0071] Molecular 2: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is a fusible molecule formed by a sense strand sequence and one or more chemical conjugates;

[0072] Molecular 3: Composed of an antisense strand and a hybrid strand, wherein some nucleotides in the sense strand of the hybrid strand are replaced by lipid-soluble chemical molecules;

[0073] Molecular 4; consists of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence, and some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules;

[0074] Molecular 5: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is a conjugate molecule formed by a sense strand sequence and one or more chemical conjugates, wherein some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules.

[0075] In some specific embodiments, when the hybrid strand contains an unrelated dangling sequence, the unrelated dangling sequence is a sequence protruding from the 5' end of the antisense strand or a sequence protruding from the 3' end of the antisense strand.

[0076] In some specific embodiments, when the hybrid chain contains unrelated dangling sequences, the connection order of the parts of the hybrid chain is any of the following:

[0077] A) 5' end - 3' end: Justice chain sequence - unrelated dangling sequence;

[0078] B) 5' end - 3' end: Unrelated dangling sequence - justice chain sequence.

[0079] Furthermore, the length of the unrelated dangling sequence is 6-15 nt. Specifically, the length of the unrelated dangling sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nt.

[0080] In some specific embodiments, when the hybrid strand contains an unrelated dangling sequence, the RNA interference triggering molecule also contains a complementary chain conjugate molecule. The complementary chain conjugate molecule is a conjugate molecule covalently coupled to a complementary nucleotide chain and a chemical conjugate, wherein the complementary nucleotide chain pairs with the unrelated dangling sequence in the hybrid strand. The general formula for the complementary chain conjugate molecule is as follows: chemical molecule-complementary nucleotide chain-chemical molecule.

[0081] In this invention, the purpose of unequal lengths of the sense strand sequences in the antisense and hybrid strands is to eliminate the possibility of the sense strand being loaded into the RISC complex. This is because nucleic acid strands shorter than 15 nt are difficult to load into the RISC complex, thus minimizing off-target effects that may be caused by the sense strand. The sense strand sequence in the hybrid strand includes 8-14 nt of its 5' or 3' end and is aligned with one end of the antisense strand. That is, when the hybrid strand consists of a shortened sense strand and an unrelated dangling sequence, the antisense strand forms a blunt-end complementary pair with the shortened sense strand on one side, forming an incomplete double helix, resulting in the unpaired sequence at the other end of the antisense strand becoming a single-stranded dangling sequence. In this case, the nucleic acid molecule structure of this invention contains two dangling sequences: one is the unrelated sequence in the hybrid strand, and the other is the unpaired sequence in the antisense strand. The dangling sequence contains chemical modifications to resist nuclease attack.

[0082] Furthermore, when the hybrid strand contains an unrelated dangling sequence, the nucleic acid sequences in the complementary strand conjugate molecule of the RNA interference triggering molecule pair with the unrelated dangling nucleic acid sequences and are interconnected by hydrogen bonds formed by base complementarity. Simultaneously, since the 5' or 3' end sequence in the antisense strand, after pairing and binding with the shortened complementary sense strand sequence, results in an unpaired sequence forming a dangling sequence at the other end, another complementary strand conjugate molecule can be designed, in which the complementary nucleotide sequence pairs complementaryly with and binds to the dangling sequence of the antisense strand. Therefore, the complete molecular structure at this point will contain two complementary strand conjugate molecules, one pairing and binding with the unrelated dangling nucleic acid sequence, and the other binding complementaryly with the dangling sequence of the antisense strand.

[0083] In some specific embodiments, when some nucleotides in the hybrid chain are replaced by lipid-soluble chemical molecules, the lipid-soluble chemical molecules may be selected from spermine molecules or C6 alkyl chains. Specifically, spermine molecules may replace 2 or 3 nucleotides; C6 alkyl chains may replace 1 nucleotide.

[0084] In some specific embodiments, one or both ends of the heterozygous strand and / or antisense strand of the RNA interference triggering molecule contain various chemical molecules coupled thereto.

[0085] In some specific embodiments, the chemical molecules coupled to the RNA interference triggering molecule are fusion molecules and / or cell-targeting molecules. Specifically, the fusion molecules can be selected from one or more of lipid molecules, peptide molecules, sugar molecules, or small chemical molecules. Specifically, lipid molecules include one or more of aliphatic hydrocarbon molecules of different chain lengths, cholesterol molecules, various phospholipids, amino fatty acid molecules, or lipid-soluble small molecule complexes; the cell-targeting molecules are selected from one or more of peptide molecules, sugar molecules, small chemical molecules, aptamer molecules, or Fab molecules. Specifically, peptide molecules are represented by RGD molecules or EED-endosome escape domain peptides; sugar molecules are represented by GalNac molecules; small chemical molecules are represented by folic acid molecules; fusion molecules are represented by cholesterol; and the Fab molecules of HER-2 are represented by monoclonal antibodies.

[0086] Furthermore, the coupling chemical molecule can directly link the nucleic acid chain or link the nucleic acid chain through a linker molecule. Specifically, the linker molecule can be selected from one or more of alkyl carbon chains, tetraethylene glycol (TEG), or dibenzocyclooctene (DBCO).

[0087] In some specific embodiments, one or both ends of the hybrid strand in the RNA interference triggering molecule contain free carboxyl groups, which are connected to the hybrid strand via alkyl carbon chains; and / or, one or both ends of the antisense strand contain free carboxyl groups, which are connected to the antisense strand via alkyl carbon chains.

[0088] In some specific embodiments, the free 5' end of the antisense strand in the RNA interference triggering molecule is chemically modified. Further, the chemical modification involves introducing a phosphate group derivative at the free 5' end. Preferably, the modifying group is vinyl phosphate, and the derivatized phosphate group is 5'-ethylene phosphate. The purpose of this chemical modification is to ensure that the phosphate group is not degraded, and the phosphate group is a structural feature necessary for the selective loading of the antisense strand fragment into the RISC complex.

[0089] In some specific embodiments, the free 5' end of the positive strand sequence in the heterozygous strand of the RNA interference triggering molecule contains a chemical modification that prevents phosphorylation; preferably, the free 5' end of the positive strand sequence contains a trifluoromethylation modification.

[0090] In some specific embodiments, the 2' carbon of the pentose sugar in the RNA interference trigger molecule is chemically modified. Specifically, the chemical modification includes one or more of fluorine modification, oxymethyl modification, or other chemical modifications.

[0091] In some specific embodiments, the phosphodiester bond in the RNA interference triggering molecule is modified with a thiophosphate.

[0092] In some specific embodiments, the RNA interference triggering molecule can be obtained by annealing an antisense strand and a hybrid strand. In this invention, the aforementioned antisense strand is the active ingredient of a nucleic acid drug. Specifically, the antisense strand can be the active strand of siRNA, or the active strand of microRNA, or the active strand of ASO, or the active strand of Antagomir.

[0093] The present invention also provides a pharmaceutical composition comprising the aforementioned RNA interference triggering molecule and pharmaceutically acceptable excipients. The pharmaceutical composition or conjugate is administered via a systemic or local route, selected from inner ear administration, ophthalmic administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration. The dosage form of the pharmaceutical composition or conjugate is one or more selected from injections, tablets, capsules, aerosols, eye drops, or nasal drops.

[0094] The excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When used in an aqueous solution for injection, the excipients are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50).

[0095] In the pharmaceutical composition provided by this invention, the aforementioned RNA interference triggering molecule is a single active ingredient, but it can also be combined with one or more other active ingredients useful for disease treatment to form a combined formulation. The active ingredients are various other drugs used for disease treatment.

[0096] The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the aforementioned nucleic acid molecule or the active ingredient of the aforementioned nucleic acid drug depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as the active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0097] The present invention also provides the use of the aforementioned RNA interference triggering molecule in the preparation of disease prevention or treatment products.

[0098] In some specific embodiments, the disease prevention or treatment product is a liver disease prevention or treatment product and / or a non-liver disease prevention or treatment product.

[0099] The present invention also provides a method for preventing or treating a disease, the method comprising administering an effective amount of the aforementioned RNA interference triggering molecule or the aforementioned pharmaceutical composition to a subject.

[0100] In some specific embodiments, the route of administration can vary and includes, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, tracheal, intraperitoneal, intraarterial, intravesical, intraocular, intratumoral, intraocular, intratumoral, inhalation, infusion, lavage, and oral administration and formulation. Treatment regimens may also vary and are generally dependent on the type of disease, the site of disease, the progression of disease, and the patient's health condition and age.

[0101] In some specific embodiments, the object can be a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, or humans. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines such as domestic cats, canines such as dogs, foxes, wolves, and bird species.

[0102] Some terms in this invention are defined as follows:

[0103] Fusogenic molecules: chemical molecules that facilitate the fusion of biological macromolecules with the cell membrane and help them enter the cell.

[0104] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0105] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0106] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0107] Example 1: In vitro verification of knockdown activity of long suspension structure.

[0108] Based on previous experimental data, the dangling sequence at the 3' end of a conventional siRNA structure can be much longer than 2 nt. Therefore, this invention designs... Figure 1 The long suspension structure was used to test the design.

[0109] Brief description of the experimental procedure: Based on the sequence design, two single-stranded components were ordered from a CRO company. First, the single strands were annealed at a certain molar concentration ratio to obtain a double-stranded structure. Then, the product was transfected into cells in vitro and incubated for 24 hours. RNA was extracted and reverse transcribed to obtain cDNA. The cDNA was used as a template for qRT-PCR to quantitatively detect the degree of knockdown of the target gene mRNA.

[0110] In vitro knockdown results as follows Figure 2 As shown. As expected, this long dangling structure (with a dangling sequence LS reaching 9 nt) still maintains the knockdown activity of the original sequence, and the significantly extended dangling sequence was shown to have no significant effect on the knockdown activity.

[0111] Depend on Figure 2 It can be seen that the F2LS orientation structure has a slight advantage in activity compared to the LSF1 orientation, although this may not be significant. However, the results show that the dangling sequence can be attached to either end of the shortened SS single strand, and the resulting long dangling sequence structure still has knockdown activity comparable to the original parent sequence.

[0112] Example 2: In vitro detection of FsiRNA-2.0 structural formation.

[0113] The experimental results of Example 1 above show that the long dangling structure still retains the knockdown activity of the original sequence, suggesting that the long dangling structure may still be a substrate for Dicer. The purpose of designing long dangling sequence structures is to utilize this unpaired sequence to bind with other structures. For example, to create a [nucleic acid-lipid] complementary chain conjugate, in which the nucleic acid sequence is... Figure 1 The long, dangling nucleic acid sequences in the structure pair up and complement each other, thereby non-covalently attaching the coupled lipid molecules to the small nucleic acid molecules, forming a structure like... Figure 3 The structure shown is named FsiRNA-2.0. The characteristics of the FsiRNA-2.0 structure prepared in this way include: increased hydrophobicity of the molecule, decreased hydrophilicity of the small nucleic acid molecule, and the attached fusion molecule can promote the transfection of the small nucleic acid molecule across the cell membrane.

[0114] Obtained from CRO company Figure 3 After identifying each single-chain component as shown in the molecular structure, the single chains are first annealed at a specific molar concentration ratio. Then, the annealed samples are separated and verified by electrophoresis on an agarose gel. The specific procedure is as follows.

[0115] Annealing reaction settings:

[0116] (1)AS+F2LS

[0117] (2) AS+F2LS+LCS-C12(FsiRNA)

[0118] (3) AS+F2LS+LCS-C18(FsiRNA)

[0119] PCR instrument annealing reaction temperature setting:

[0120] step temperature time 1 50℃ 1min 2 The temperature drops by 1°C per minute, until it reaches 25°C. Approximately 25 minutes 3 4℃ continued

[0121] Agarose gel electrophoresis:

[0122] The suitability of the annealing reaction conditions and the formation of the structure were verified using 3% agarose gel electrophoresis. The sample loading for the agarose gel electrophoresis lanes is as follows:

[0123] Group 1: (1) F2LS single chain

[0124] (2) AS single chain

[0125] (3)AS+F2LS

[0126] (4)AS+F2LS+C 12 -LCS-C 12

[0127] (5)AS+F2LS+C 18 -LCS

[0128] Group 2: (6) LS-F1 single chain

[0129] (7)MW standard AS

[0130] (8) AS single chain

[0131] (9)LSF1+AS

[0132] (10)LSF1+AS+C 12 -LCS-C12

[0133] (11)LSF1+AS+C 18 -LCS

[0134] The above results are as follows Figure 4 As shown, the results indicate that the position of the dangling sequence affects the efficiency of structural integrity formation. Comparing the same LS sequence, the LS dangling at the 3' end of the SS chain has a higher efficiency in forming a complete structure by combining with the [LCS sequence-fusion molecule] complementary chain conjugate. Among them, [C 12 -LCS-C 12] ] or [C 18 [-LCS] is an LS complementary chain conjugate.

[0135] Example 3: In vitro verification of FsiRNA-2.0 knockdown of target gene activity.

[0136] The in vitro activity detection method used in this invention to assess the knockdown efficiency of siRNA-mediated target gene expression is the now-standard qRT-PCR method. Methodological summary: Cells were transfected using LF-2000 (Lipofectamine 2000, Invitrogen). After 24 hours of incubation, total RNA was extracted using a commercial RNA extraction kit. cDNA was then obtained through reverse transcription using a commercial kit. Subsequently, RT-qPCR was performed using a commercial SYBR Green kit to detect changes in intracellular target mRNA levels. Finally, the knockdown rate of the target gene was calculated using software. The experimental groups are as follows:

[0137] (1) Blank control;

[0138] (2) Negative control

[0139] (3) Candidate siRNAs with a 19-21nt structure were transfected with LF2000.

[0140] (4) 3' Suspension Structure (F2LS) + F1-Lipid, Self-rotating dyeing

[0141] (5) 3' suspension structure (F2LS) + LCS-TEG cholesterol, self-transfection

[0142] (6) 3' suspension structure (F2LS) + F1-Lipid + LCS-TEG cholesterol, self-transfection

[0143] The above experimental results are as follows Figure 5 As shown. Figure 5 This indicates that FsiRNA-2.0 molecules can effectively knock down target genes. (1) Compared with the screened original 19-21nt sequences, RNA molecules with dangling sequences have the same knockdown efficiency, indicating that the substrate molecules of the RNA endonuclease Dicer may include the above-mentioned long dangling structures. (2) The dangling sequence can be located at the 3' end of the sense strand and protrude from the 5' end of the antisense strand, or it can be located at the 5' end of the sense strand and protrude from the 3' end of the antisense strand. (3) The protruding dangling sequence is an unpaired sticky sequence that can pair and bind with a single strand with a complementary sequence. This also means that, through chemical coupling, lipid-soluble fusion membrane molecules can be pre-linked with nucleic acid single-strand LCS to form complementary strand conjugate molecules. When the nucleic acid single strand in the complementary strand conjugate molecule anneals and pairs with the dangling sequence (LS), the fusion membrane molecule is also passively and non-covalently attached to the small RNA molecule with the dangling sequence. Unlike covalent conjugation, this non-covalent attachment is a weak interaction and is reversible. (4) Figure 3The structure shown can effectively promote the transfection of small nucleic acid molecules across the cell membrane, and the self-transfection efficiency is close to that of LF2000, indicating that long dangling sequences can be another effective triggering structure for exercising RNA interference effects in cells. (5) This structure can effectively self-transfect cells, thereby simplifying the delivery of small nucleic acid molecules.

[0144] Example 4: Agarose gel electrophoresis to verify the formation of FsiRNA-3.0 structure.

[0145] Although Figure 3 The in vitro knockdown efficiency of the FsiRNA-2.0 molecule with the shown structure is satisfactory, but it may have potential risks because its total nucleic acid chain length is close to 30 nt, which is close to the theoretical limit and may induce an innate immune inflammatory response in vivo, leading to unnecessary toxic side effects. To avoid the above-mentioned potential side effects, the structure is further simplified in this invention, designed as follows: Figure 6 The FsiRNA-3.0 molecule with the structure shown.

[0146] The fabrication procedure is the same as described above. The individual single-stranded components of the FsiRNA-3.0 structure were obtained from a CRO company, such as... Figure 6 As shown. Annealing reaction was performed in vitro: the conventional 21nt AS chain, or the SS chain shortened to 9nt, or the [9nt SS chain-TEG-cholesterol] conjugate, were mixed at equimolar concentrations and annealed on a PCR instrument.

[0147] Annealing reaction settings:

[0148] (1)AS+SS-9nt

[0149] (2)AS+full lengthSS

[0150] (3) AS+[9n tSS chain-TEG-cholesterol]

[0151] Electrophoresis lane group:

[0152] Group 1:

[0153] (1) MW standard

[0154] (2) AS single chain

[0155] (3) 9nt SS single chain

[0156] (4) [9nt SS chain-TEG-cholesterol] single chain

[0157] (5) Full-length SS single chain

[0158] Group 2:

[0159] (6) AS+9nt SS chain

[0160] (7) AS+[9nt SS chain-TEG-cholesterol]

[0161] (8)AS+Full Length SS

[0162] (9) AS+ [Full-length SS-TEG-cholesterol]

[0163] The above results are as follows Figure 7 As shown in the gel electrophoresis, both AS and SS with a standard chain length of 21nt exhibit clear bands. Therefore, if the annealing reaction is successful, the corresponding single-chain bands of the components involved in the annealing reaction will disappear and merge into new bands with increased molecular weight, and their migration positions will correspondingly shift upwards. The components annealed with the AS chain include full-length SS, [SS-TEG-cholesterol], 9nt SS chains, [9nt SS chains-TEG-cholesterol], etc. The migration rate of cholesterol conjugates is not correlated with molecular weight due to factors such as the hydrophobicity and steric hindrance of lipid molecules. The completeness of the annealing binding with the AS chain can be initially judged by whether the original independent band staining disappears and the degree of attenuation. Figure 7 The results showed that the individual component single strands could form the expected FsiRNA-3.0 structure after in vitro annealing.

[0164] Example 5: Verification of FsiRNA-3.0 knockdown of target gene activity.

[0165] The same experimental methodology as described in Example 3 was employed. Briefly, cells were transfected using the LF-2000 (Lipofectamine 2000, Invitrogen) cell transfection method. After 24 hours of incubation, total RNA was extracted from the cells using a commercial RNA extraction kit. cDNA was then obtained through reverse transcription using a commercial kit. Subsequently, RT-qPCR was performed using a commercial SYBR Green kit to detect changes in the intracellular target mRNA content. Finally, the proportion of target genes knocked down was calculated using software.

[0166] Experimental groups: (3 concentrations per sample)

[0167] 1) Blank control;

[0168] 2) Negative control, transfected with LF2000;

[0169] 3) Positive control (19-21nt), transfected with LF2000;

[0170] 4) F1+AS, self-transfer;

[0171] 5) FsiRNA-3.0 ([F1-TEG-cholesterol+AS]), self-transfected;

[0172] The experimental results are as follows Figure 8 As shown. The results showed that: (1) the candidate siRNA transfected by LF2000 reacted normally; (2) the results of self-transfection of F1+AS nucleic acid structures indicated that the low activity of small nucleic acid molecules knocked down by themselves may be due to difficulty in transfecting into cells; (3) as Figure 6 The FsiRNA-3.0 molecular structure shown exhibits significant self-transfection capability at a concentration of 50 nM in vitro, effectively and significantly knocking down target gene mRNA compared to naked siRNA.

[0173] like Figure 6 The self-transfection capability of the molecule with the FsiRNA-3.0 structure shown was replicated in Jurkat cells. Figure 9 The results showed that FsiRNA-3.0 exhibited significant knockdown activity in both Jurkat and Hela cell lines, indicating that this structure is universal.

[0174] Example 6: Study on FsiRNA membrane penetration mechanism - fluorescence tracer detection.

[0175] The results of the above embodiments show that, in an in vitro cell experimental system, the FsiRNA structure can effectively self-deliver into cultured cells to knock down target gene expression. Next, a series of fluorescence tracing experiments were conducted to explore the mechanism of action of FsiRNA transmembrane delivery.

[0176] Experimental design and procedure brief description:

[0177] First, this invention performed conventional fluorescence tracing experiments, labeling nucleic acid molecules with the monochromatic green fluorescent molecule FAM. In vitro annealing yielded 1) conventionally structured FAM-siRNA molecules and 2) structurally modified FAM-FsiRNA molecules.

[0178] The test cells were cultured on chamber slides and incubated overnight. FAM-siRNA molecules were transfected using LF2000, while FsiRNA molecules were directly added to the culture medium for self-transfection. Incubation continued for 24 hours, followed by washing with PBS, fixing with 4% paraformaldehyde, mounting with coverslips, and observation and photography using a confocal microscope. Representative data are presented in […]. Figure 10 .

[0179] Next, this invention designed a dual-fluorescence tracing experiment to further study the transmembrane and intracellular transport pathways of FsiRNA.

[0180] Experimental design: FsiRNA-3.0 structure as follows Figure 6 As shown, the SS chain-lipid conjugate was labeled with Cy5, and the AS chain was labeled with FAM. The labeled components were annealed under standard conditions to obtain the designed molecular structures. Test cells were cultured on chamber slides, and the reagent containing the annealed FsiRNA-3.0 structure was directly added to the cell culture medium for self-transfection. Cell samples were then collected at predetermined time points, washed with PBS, fixed with 4% paraformaldehyde, mounted, and observed under a confocal microscope.

[0181] Experimental Groups:

[0182] 1) Blank control, observe cell background;

[0183] 2) LF2000 transfection with FAM-labeled 19-21nt siRNA served as a positive control for endocytosis;

[0184] 3) F1+AS (containing FAM-labeled SS) self-transfected as a negative control;

[0185] 4) FsiRNA-3.0 self-transfection experimental group; containing FAM marker AS

[0186] 5) FsiRNA-3.0 self-transfection experimental group; containing FAM marker SS and Cy5 marker AS.

[0187] Representative experimental results are shown in Figure 11-13 .

[0188] The above experimental results are as follows Figure 10-13 The following is an analysis of the results. Figure 10 This indicates that comparing LF2000 transfection with FsiRNA transfection (comparing groups 2 and 4), it can be clearly observed that the LF2000 transfection pathway is a typical endocytosis pathway. The FAM-labeled green fluorescent small nucleic acids (19-21 nt structure) are distributed in large, bulky clusters within the cell, suggesting that the small nucleic acids delivered by liposomes are trapped in late endosomes and lysosomes, exhibiting a distribution characteristic of late endosomes and lysosomes. However, as... Figure 6 The FsiRNA molecules with the structure shown exhibit a fine and diffuse distribution pattern within the cell, with FAM-labeled green fluorescent small nucleic acid molecules, suggesting that FsiRNA molecules may enter the cytoplasm directly through different pathways or non-endocytosis.

[0189] Figure 11 Indicates: such as Figure 6The F1-cholesterol conjugate in the FsiRNA molecule with the shown structure, during its passage through the cell membrane, follows the principle of "like dissolves like," where the lipid molecules interact hydrophobically with the lipid bilayer of the cell membrane. This causes the F1 sequence and the AS chain to separate during the passage, and the AS may enter the cytoplasm first to exert its RNA interference effect. The distribution of the two labeled fluorescence patterns indicates that... Figure 6 In the molecule with the structure shown, the AS portion of the small nucleic acid molecule (FAM-labeled green fluorescence) separates from the [F1-cholesterol] conjugate portion (Cy5-labeled red fluorescence) as it passes through the cell membrane. The [F1-cholesterol] conjugate portion remains in the cell membrane, delaying its entry into the cell.

[0190] Compare Figure 12-13 As a result, Figure 6 The FsiRNA molecule with the structure shown exhibits high cell transfection efficiency; a large amount of fluorescence can be observed in the cytoplasm 5 minutes after transfection, reaching its peak at 24 hours. Figure 13 Meanwhile, LF2000 transfection resulted in large clumps at 5 minutes, making it impossible to determine whether the cytoplasm had been reached. The clumps persisted after 24 hours, indicating lysosomal entrapment and a low release rate. Figure 12 The above results indicate that the molecular distribution of FsiRNA structures within cells differs from that of conventional siRNA molecules transfected with LF2000. It is generally believed that siRNA molecules transfected with LF2000 enter cells with a typical endosome / lysosome distribution, appearing as large, bulky masses. However, as... Figure 6 The FsiRNA molecules with the structure shown are distributed in a fine, diffuse manner within the cell. Furthermore, judging from the amount of fluorescence entering the cell, the transfection efficiency of the FsiRNA molecules is at least comparable to that of LF2000.

[0191] Example 7: In vivo efficacy validation of FsiRNA-3.0.

[0192] The purpose of designing this novel FsiRNA structure in this invention is to improve the efficiency of extrahepatic delivery of small nucleic acid molecules in vivo. After obtaining in vitro functional validation, this embodiment selected the target gene IL-4R for in vivo proof-of-concept (POC) validation experiments.

[0193] In vivo experiments were conducted in normal Balb / C mice. All animal experimental plans were approved by the CRO's IACUC (The Institutional Animal Care and Use Committee).

[0194] In vivo validation experiment #1:

[0195] (1) The experimental drug structure was FsiRNA-3.0, such as Figure 6 As shown.

[0196] AS:5'-VP-mU*fA*mUmAmGfAmCfUmAfUmGfAmAmUmUmCmUmGmC*AG;

[0197] SS-cholesterol conjugate: 5'-mG*mC*mAmGmAmAfUmUfC-TEG-cholesterol;

[0198] (2) The dosage is 3 MPa;

[0199] (3) The route of administration is subcutaneous injection;

[0200] (4) Balb / c mice, 6-8 weeks old, were used, with 8 mice per group at each time point;

[0201] (5) The time points for collecting tissue samples are: 2 days, 7 days, 14 days, and 21 days;

[0202] Experimental Groups:

[0203] 1) PBS control group;

[0204] 2) 3mpk - 2 days

[0205] 3) 3mpk - 7 days

[0206] 4) 3mpk - 14 days

[0207] 5) 3mpk - 21 days

[0208] Tissue samples were collected using standard methodologies. The right lobe of the liver was taken and cut into more than 5 pieces. The spleen, kidneys, and lungs were collected intact.

[0209] Target gene knockdown results as follows Figure 14 The results showed that the FsiRNA-3.0 structure exhibits high efficiency and long-lasting effect in vivo. A single subcutaneous administration, such as... Figure 6 The molecule with the structure shown significantly knocked down the expression of target gene mRNA in the liver of mice, with an inhibition efficiency of over 80% after 48 hours. During the experimental observation period (21 days), the drug maintained an effective knockdown efficiency of >50% in the liver and kidneys.

[0210] However, the results suggest that the liver retention rate remains high. Therefore, there is room for further structural modifications.

[0211] Example 8: FsiRNA-4.0.

[0212] To further enhance the hydrophobicity of small nucleic acid molecules, this embodiment replaces nucleotides in the SS chain with lipid-soluble chemical molecules, such as replacing 2-3 nucleotides with one spermine molecule or one nucleotide with a C6 alkyl chain. This type of structure is named FsiRNA-4.0. A representative structure of this design is shown below. Figure 15 As shown.

[0213] Brief description of the production process: Received from the CRO company Figure 15 After processing each single-chain component of the structure shown, an annealing reaction was performed on a PCR instrument to obtain the designed structure.

[0214] Annealing component combination:

[0215] (1) AS+ heterozygous chain #1

[0216] (2) AS+ heterozygous chain #2

[0217] (3) AS+ heterochain #3

[0218] (4) AS+ heterochain #4

[0219] PCR instrument annealing reaction temperature setting:

[0220]

[0221]

[0222] The annealed samples were then verified by agarose gel electrophoresis. The lane groups for 3% agarose gel electrophoresis are as follows:

[0223] MW Standard

[0224] (1) AS single chain

[0225] (2) SS single chain

[0226] (3)AS+SS

[0227] (4) Hybrid chain #1 single chain

[0228] (5) AS+ heterochain #1

[0229] (6) Hybrid chain #2 single chain

[0230] (7)AS+ heterochain #2

[0231] (8) Hybrid chain #3 single chain

[0232] (9) AS+ heterochain #3

[0233] (10) Hybrid chain #4 single chain

[0234] (11)AS+ heterochain #4

[0235] The above results are as follows Figure 16 As shown in the image. According to the gel mapping analysis, except for the incomplete formation of structure #2, the other structures were formed relatively completely. The migration rate of structure #4, which contains cholesterol, is slower, which may be due to the strong hydrophobicity of cholesterol molecules, or it may be due to the interaction between cholesterol molecules to form a larger structure.

[0236] Example 9: In vitro detection of knockdown activity of various FsiRNA-4.0 structures.

[0237] Using the same detection method as in Example 3 above, for Figure 16 The various structures of FsiRNA-4.0 shown were used to detect the target gene knockdown bioactivity. In brief: Cells were plated overnight, and the following day, reagents were prepared according to the experimental design for cell transfection. After 24 hours of incubation, total RNA was extracted from the transfected cells using a commercial kit. The RNA was then reverse transcribed into cDNA. Finally, using the cDNA as a template, qRT-PCR was performed with target gene-specific primers to observe the degree to which the target gene was inhibited by the transfected siRNA.

[0238] Experimental Groups:

[0239] (1) Blank control

[0240] (2) NC control

[0241] (3) Positive control, 19-21nt candidate sequence, LF2000 transfection

[0242] (4)(AS+heterosynthetic chain #1) self-transfection

[0243] (5)(AS+heterosynthetic chain #2) self-transfection

[0244] (6)(AS+heterosynthetic chain #3) self-transfection

[0245] (7)(AS+heterosynthetic chain #4) self-transfection

[0246] The results are as follows Figure 17 As shown in the figure. The results show that replacing nucleotides in the SS chain with hydrophobic molecules can promote the self-transfection of small nucleic acid molecules into cells without affecting the biological function of FsiRNA-4.0 knockdown of target gene expression.

[0247] Further theoretical analysis suggests that in the four tested structures, the spermine molecule may interact electrostatically with the phosphate group of the ascorbic acid (AS) through its polyamino groups. This interaction not only maintains the stability of the asymmetric double-stranded structure but also neutralizes some of the molecule's electronegativity, thus improving transfection efficiency. This data also indicates that various modifications and substitutions can be made to the SS strand of siRNA, which can be utilized as a natural carrier molecule.

[0248] Example 10: In vitro optimization of FsiRNA-6.0.

[0249] In vitro, small nucleic acid molecules demonstrate significant efficacy in knocking down target gene mRNA expression, making them an effective method for studying gene regulation. However, in vivo application often yields unsatisfactory results, particularly in extrahepatic applications, due to various factors affecting the activity, metabolism, and expression (ADME) of small nucleic acid molecules. To achieve efficacy levels approaching those observed in in vitro studies and screenings, and especially to ensure successful extrahepatic delivery, it is essential to extend the circulation time or half-life of small nucleic acid molecules. Only with a satisfactory extension of circulation time can the extrahepatic delivery of small nucleic acid drugs be successful.

[0250] To achieve effective extrahepatic delivery, it is necessary to increase the in vivo circulating half-life of drug molecules and increase the amount of drug passing through the liver. This embodiment is based on the mechanism of fatty acid molecule circulation in vivo, and utilizes the FsiRNA-3.0 molecular structure (…). Figure 6 Based on this, the present invention adds a free carboxyl group to one end of the SS chain to obtain... Figure 18 The molecule with the structure shown is named FsiRNA-6.0. In the figure, COOH represents the terminal free carboxyl group, Cn represents different carbon chain lengths (including aliphatic hydrocarbon chains and PEG chains), and Fusogenic molecules represent various chemicals that can promote transfection into cells (including various lipid molecules, peptide molecules, and small molecule drugs).

[0251] Annealing reaction: Received from CRO Figure 18 The single-stranded components of the structure shown, FsiRNA-6.0, were annealed using a PCR instrument.

[0252] Annealing reaction temperature program settings:

[0253] step temperature time 1 50℃ 1min 2 The temperature drops by 1°C per minute, until it reaches 25°C. Approximately 25 minutes 3 4℃ continued

[0254] Annealing combination settings:

[0255] (1) AS + 5'COOH(COOH-C n -9ntSS-TEG-Cholesterol)

[0256] (2)AS+F1(5' end 9nt-SS)

[0257] Verification by 3% agarose gel electrophoresis:

[0258] MW Standard

[0259] 1: AS single chain

[0260] 2: F1 (5' end 9nt-SS) single strand

[0261] 3: AS+F1 (5' end 9nt-SS)

[0262] 4:5'COOH(COOH-C n -9ntSS-TEG-Cholesterol) single chain

[0263] 5: AS + 5'COOH

[0264] The above results are as follows Figure 19 As shown. The results indicate that the formation of the FsiRNA-6.0 structure with a carboxyl group at the end is not ideal. It is speculated that this may be related to the balance between hydrophobic interactions and base complementarity, and that the length of the nucleic acid chain may need to be increased. This reasoning is confirmed by new data. Figure 20 The gel images showed that when the F1 nucleic acid chain was extended from 9 nt to 14 nt, the efficiency of FsiRNA-6.0 structure formation was significantly improved, approaching complete.

[0265] To further verify the above Figure 19 and Figure 20 As a result, this invention systematically studied the effect of nucleic acid chain length on the structure formation efficiency of FsiRNA-6.0. Single-stranded nucleic acid chains with lengths of 6nt, 9nt, 12nt, and 15nt were ordered from CRO and annealed, and then subjected to gel electrophoresis with AS-annealed samples. Figure 21 The gel images clearly show that the completeness of FsiRNA-6.0 structure formation is directly proportional to the length of the nucleic acid chain. The 6-nt chain length resulted in a low proportion of the expected structure, and at 9 nt, a small portion of free AS failed to form the expected structure. In contrast, the 12-nt and 15-nt chains almost completely formed the expected structure.

[0266] Next, the biological functions of annealed samples of different lengths were compared. Using the same qRT-PCR detection method as in Example 3, the target gene knockdown activity of each FsiRNA-6.0 structure was detected to observe the degree of influence of nucleic acid chain length on target gene knockdown. Results are summarized in... Figure 22 The results showed that: 1) The knockdown effect was lowest with 15nt transfection, whether by self-transfection or transfection with LF2000; 2) The knockdown efficiency of self-transfection was highest to lowest as follows: 12nt > 9nt > 6nt > 15nt; 3) Although the efficiency of 6nt in forming the FsiRNA-6.0 structure was low, the knockdown efficiency achieved by transfection with LF2000 was better than that of 12nt, indicating that the AS single strand may directly exert the effect of knocking down the target gene; 4) 15nt could effectively form the FsiRNA-6.0 structure, but the knockdown efficiency was the worst, suggesting that either the release efficiency of this structure is low, or this structure hinders RISC loading.

[0267] Example 11: In vivo PD / PK study of FsiRNA-6.0.

[0268] The experimental protocol for FsiRNA-6.0 PK / PD can be found here. Figure 23 The experimental details are as follows:

[0269] 1. Mouse age in weeks: 7-8W Ensure that each mouse weighs approximately 25g.

[0270] 2. Administration method: subcutaneous injection (SC);

[0271] 3. Dosage: 6 mg / kg.

[0272] 4. Dosage time and frequency: Once every 0.0 hours;

[0273] 5. See groupings. Figure 23 ;

[0274] 6. The number of mice in each group was 4. A total of 32 mice were included. Grouping was performed 24 hours before drug administration, and this was recorded as -D1.

[0275] 7. Serum and whole blood cell collection:

[0276] (1) At a set time point, a portion of the blood was collected to prepare serum. The required amount of serum was about 30 μl (about 100 uL of whole blood), which was used to detect the siRNA content.

[0277] (2) The remaining whole blood was anticoagulated and used to separate blood leukocytes for RNA extraction.

[0278] 8. The remaining tissues were collected according to time points: 1) liver, 2) spleen, 3) kidney, and 4) mesenteric lymph nodes.

[0279] After collection, the tissues were rapidly frozen and stored in liquid nitrogen (or dry ice-ethanol).

[0280] 9. Mouse observation: The weight of mice was measured before taking tissue samples at -D1, 0.5h, 2h, 6h, 24h, D5, D12, and D18.

[0281] 10. Experimental endpoint: Euthanasia.

[0282] 11. Drug structure: COOH-C 16 -mIL#2-F1(12nt)-TEG-Cholesterol.

[0283] For the HIT-qRT-PCR method for detecting the AS single-strand content of siRNA in PK studies, please refer to reference [7]. The target gene knockdown method is the same as described in Example 3. The results are summarized in Figure 24 , 25 . Figure 24 PK data showed that although most drugs were still mainly distributed in primary organs such as the liver, spleen, and kidneys, the blood half-life of FsiRNA-6.0 clearly exceeded 24 hours. The highest concentrations of FsiRNA-6.0 were found in the liver and spleen, followed by the kidneys. The amount distributed in the kidneys remained relatively stable, and even slightly increased.

[0284] Combination Figure 25 The PD data showed that, during the observation period, the knockdown efficiency of the drug on target gene expression in the kidneys was relatively stable and superior to that in the liver. This data suggests that a large amount of FsiRNA-6.0 in the liver may only reside in the hepatic sinusoids and not enter the hepatic parenchymal cells. Alternatively, the structure of the FsiRNA-6.0 drug may have a relatively higher affinity for kidney tissue. Figure 25 The data also showed that FsiRNA-6.0 exhibited the best knockdown activity in mesenteric lymph nodes. This indicates that the drug structure possesses differentiated tissue affinity, and the prolonged blood circulation time may be the basis for these effects.

[0285] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An RNA interference triggering molecule named FsiRNA, characterized in that, The RNA interference triggering molecule consists of an antisense strand and a hybrid strand, wherein the hybrid strand contains a sense strand sequence that is shorter than the antisense strand.

2. The RNA interference triggering molecule according to claim 1, characterized in that, The length of the antisense chain is 15-27 nt.

3. The RNA interference triggering molecule according to claim 1, characterized in that, In the RNA interference triggering molecule, the antisense strand and the sense strand in the hybrid strand are complementary base pairs.

4. The RNA interference triggering molecule according to claim 1, characterized in that, The hybrid strand in the RNA interference triggering molecule has one or more of the following characteristics: 1) The hybrid chain contains unrelated dangling sequences; 2) The hybrid chain contains chemical conjugates; 3) Some nucleotides in the hybrid chain are replaced by lipid-soluble chemical molecules.

5. The RNA interference triggering molecule according to claim 4, characterized in that, The RNA interference triggering molecule is a molecule having any of the following structures: Molecular 1: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence; preferably, the unrelated dangling sequence is RNA, or DNA, or a mixture of RNA and DNA; Molecular 2: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is a sense strand sequence coupled with one or more chemicals to form a conjugate molecule; preferably, the chemical is a fusion molecule and / or a cell-targeting molecule; more preferably, the fusion molecule is selected from one or more of lipid molecules, peptide molecules, sugar molecules, or small chemical molecules; or, the cell-targeting molecule is selected from one or more of peptide molecules, sugar molecules, small chemical molecules, aptamer molecules, or Fab molecules; Molecular 3: Composed of an antisense strand and a hybrid strand, wherein some nucleotides in the sense strand of the hybrid strand are replaced by lipid-soluble chemical molecules; Molecular 4; consists of an antisense strand and a hybrid strand, wherein the hybrid strand is formed by a sense strand sequence and a 6-15 nt unrelated dangling sequence, and some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules; Molecular 5: Composed of an antisense strand and a hybrid strand, wherein the hybrid strand is a conjugate molecule formed by the covalent coupling of a sense strand sequence and one or more chemicals, wherein some nucleotides in the sense strand sequence of the hybrid strand are replaced by lipid-soluble chemical molecules.

6. The RNA interference triggering molecule according to claim 4, characterized in that, When the hybrid strand contains an unrelated dangling sequence, the unrelated dangling sequence is either a sequence protruding from the 5' end of the antisense strand or a sequence protruding from the 3' end of the antisense strand, and the connection order of the hybrid strand is any of the following: A) 5' end - 3' end: Justice chain sequence - unrelated dangling sequence; B) 5' end - 3' end: Unrelated dangling sequence - justice chain sequence.

7. The RNA interference triggering molecule according to claim 4, characterized in that, When the hybrid chain contains an unrelated dangling sequence, the RNA interference triggering molecule also contains a complementary chain conjugate molecule, which is a conjugate formed by covalently coupling a complementary nucleotide chain with a chemical, wherein the nucleotide chain sequence is complementary to and paired with the unrelated dangling sequence; preferably, the general formula of the complementary chain conjugate molecule is as follows: chemical-complementary nucleotide chain-chemical.

8. The RNA interference triggering molecule according to claim 4, characterized in that, When a portion of the nucleotides in the hybrid chain is replaced by a lipid-soluble chemical molecule, the lipid-soluble chemical molecule is selected from spermine molecules or C6 alkyl chains.

9. The RNA interference triggering molecule according to claim 1, characterized in that, The RNA interference triggering molecule contains coupled chemicals at one or both ends of the heterozygous strand and / or antisense strand.

10. The RNA interference triggering molecule according to claim 7 or 9, characterized in that, The chemical coupled to the RNA interference triggering molecule is a membrane fusion molecule and / or a cell-targeting molecule; preferably, the membrane fusion molecule is selected from one or more of lipid molecules, peptide molecules, sugar molecules or small chemical molecules; or, the cell-targeting molecule is selected from one or more of peptide molecules, sugar molecules, small chemical molecules, aptamer molecules or Fab molecules.

11. The RNA interference triggering molecule according to claim 1, characterized in that, The RNA interference triggering molecule contains a free carboxyl group at one or both ends of the hybrid strand, the free carboxyl group being connected to the hybrid strand via an alkyl carbon chain; and / or, the antisense strand contains a free carboxyl group at one or both ends, the free carboxyl group being connected to the antisense strand via an alkyl carbon chain.

12. The RNA interference triggering molecule according to claim 1, characterized in that, The RNA interference triggering molecule has one or more of the following characteristics: 1) The free 5' end of the antisense chain is chemically modified; preferably, the chemical modification is the introduction of a phosphate group derivative into the free 5' end; 2) The free 5' end of the positive strand sequence in the hybrid strand contains a chemical modification that prevents phosphorylation; 3) The pentose in the RNA interference triggering molecule has a chemical modification at the 2' carbon position; 4) The phosphodiester bond in the RNA interference triggering molecule is modified with thiophosphate.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the RNA interference triggering molecule as described in any one of claims 1-11, and pharmaceutically acceptable excipients.

14. Use of the RNA interference triggering molecule as described in any one of claims 1-12 or the pharmaceutical composition as described in claim 13 in the preparation of a disease prevention or treatment product.

15. The use according to claim 14, characterized in that, The disease prevention or treatment products are liver disease prevention or treatment products and / or non-liver disease prevention or treatment products.

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