RNA interference trigger molecule, and corresponding nucleic acid interference pharmaceutical preparation and use thereof

Abstract

Provided are an RNA interference trigger molecule, and a corresponding nucleic acid interference pharmaceutical preparation and the use thereof, which belong to the field of nucleic acid drugs. One end of the structure of the provided RNA interference trigger precursor molecule has an asymmetric pharmaceutically active duplex composed of an antisense strand and a significantly shortened sense strand, wherein the antisense strand and the shortened sense strand are of unequal length in the asymmetric pharmaceutically active duplex. The other end of the structure has a double-stranded loop sequence, wherein a single-stranded LCS complementary to a loop single-stranded sequence LS is added, the LS and the LCS bind to each other by means of base complementarity, and LCS is pre-conjugated with a fusogenic molecule that promotes cellular transfection efficiency. The two parts of the structure are linked by a short linker duplex.

Description

An RNA interference trigger molecule and corresponding nucleic acid interference drug preparation and use thereof Technical Field

[0001] The present invention relates to the field of nucleic acid drugs, and in particular to an RNA interference trigger molecule and a corresponding nucleic acid interference drug preparation and uses thereof. Background Art

[0002] More than 40 years ago, Zamecnik and Stephenson first proposed that sequence-specific antisense nucleic acids could be chemically synthesized to inhibit RSV replication[1]. Small nucleic acids have come a long way in becoming a new class of drugs. In their natural state, small nucleic acid molecules do not have "drug-like" properties[2] because they are large polyanionic molecules that are easily digested by ribonucleases and unstable in the body. Small nucleic acids also have multiple limitations, such as activation of nonspecific immune responses, insufficient therapeutic effects, poor PK / PD, instability in the body, and off-target effects, which hinder the clinical application of nucleic acids. However, these various defects have been effectively overcome to a large extent due to the tremendous progress in nucleic acid chemical modification technology[3]. For example, with the progress of research on chemical modification of nucleic acid monomers, the stability of small nucleic acid molecules has been greatly enhanced and off-target effects have been significantly reduced. Now, computer algorithms can be used to minimize the possibility of immune response activation when starting sequence selection design. The ultimate improvement in the drug-likeness of small nucleic acid molecules comes from breakthroughs in the in vivo delivery technology of nucleic acid drugs. Among the many successful nucleic acid drug delivery technologies, liposome nanoparticles and GalNac conjugate technologies are the most prominent [4].

[0003] In order for siRNA to become a new class of drugs with practical clinical application, two bottlenecks that hinder the realization of siRNA's promising future need to be effectively addressed: off-target effects and in vivo delivery. Off-target effects are the nonspecific knockdown of non-target gene expression by siRNA, which is sequence-dependent and occurs on both the antisense and sense strands. Alnylam and other R&D teams have confirmed that nonspecific knockdown of non-target gene expression is one of the key side effects of siRNA drugs[5].

[0004] Based on the mechanism of action, off-target effects can be divided into three categories: microRNA-like off-target effects, innate inflammation response through activation of TLRs system, and saturation of endogenous RNAi machinery due to excessive drug use, thereby hindering the normal function of RNAi [6].

[0005] Some research results on the mechanism of siRNA off-target effects indicate that the chemical structure basis for miRNA-like off-target effects may lie in the presence of a partial sequence in the siRNA sequence that pairs with the 3'-UTR (untranscribed region), thereby acting similarly to miRNA. The activation of nonspecific TLR systems may also be closely related to the length of the siRNA nucleic acid chain and / or the presence of certain special sequences. In short, these off-target effects may be caused by the special sequence information of the nucleic acid [7].

[0006] To address off-target effects, the main measure currently adopted by the industry is to perform a series of chemical modifications on nucleic acid sequences, especially to reduce the binding affinity of the 5'-end seed sequence of the antisense strand to non-target mRNAs through chemical modifications. However, the miRNA-like off-target effects of siRNA are not limited to the antisense strand; the sense strand faces the same problem. In 2007, a Danish research team proposed a solution to address the off-target effects of the sense strand, sisiRNA[8]. The authors claim that this structure not only maintains the knockdown efficacy of the traditional structure with the same sequence, but also eliminates the off-target effects caused by the sense strand. However, the knockdown efficiency of chemically synthesized sisiRNA has not been significantly improved[9].

[0007] Research results suggest that the RISC complex selects the guide strand by detecting differences in the thermal stability of complementary pairs at the two ends of the RNA duplex

[0010] . Both the antisense and sense strands have the potential to become guide strands. Although chemical modification has been used to reduce the thermal stability of the complementary pairing of the antisense strand seed sequence, resulting in a lower complementary binding affinity at the 5' end of the antisense strand than at the 3' end, allowing the antisense strand to be preferentially selected as the guide strand and loaded into RISC, this approach cannot be simultaneously applied to reduce the off-target effects of the sense strand, because the seed sequence of the sense strand is located at the 3' end of the antisense strand. Methods that reduce the thermodynamic stability of the seed region sequence are not suitable for eliminating the off-target effects of the sense strand. As the saying goes, "You can't have your cake and eat it too."

[0008] Although GalNAc has successfully replaced LNP and is widely used in the liver-targeted delivery of small nucleic acid drugs, research data show that the endosomal escape rate of GalNAc-siRNA is extremely low (less than 0.1%), becoming the rate-limiting step in the in vivo delivery of small interfering nucleic acids

[0011] . Therefore, how to increase the intracellular release rate is also one of the major bottlenecks and difficulties in the development of small interfering nucleic acid drugs. Regarding how to improve the efficiency of endosomal escape, various solutions have been reported in the literature, including the use of chemicals such as chloroquine to increase the osmotic pressure inside the endosomal body, thereby increasing the permeability of the endosomal body; or using transmembrane peptides to disrupt the stability of the endosomal membrane. However, these treatments have a common drawback, that is, the toxicity of these methods cannot be used in clinical practice. Even for LNP preparations with higher release efficiency, data show that their endosomal escape rate is 1-2%, and the duration is short.

[0009] Although the GalNAc technology platform is only suitable for liver delivery, this success provides new ideas and concepts for the development of small nucleic acid drugs, indicating that chemical coupling can improve the drug-like properties of small nucleic acid molecules, thereby widely promoting the clinical treatment application of small nucleic acid drugs to extrahepatic disease indications.

[0010] Therefore, no matter how we try to improve the efficacy of RNAi drugs, we must first solve the problem of low in vivo delivery efficiency of small nucleic acid drugs. In order for small nucleic acid drugs to effectively reach the target organs in the body, the main obstacles that need to be overcome by the designed small nucleic acid drug structure, in addition to the precursor must be sufficiently stable, are: (1) how to extend the half-life in the blood circulation, which is the basis for improving the efficiency of in vivo delivery. (2) how to promote the efficiency of drug entry into cells. (3) how to improve the efficiency of intracellular release.

[0011] To improve the intracellular release efficiency of small nucleic acid drugs, Gao, Xiaohu from the University of Washington (in Seatle)

[0012] The group pioneered cholesterol tagging technology to address the problem of low release rates of small interfering nucleic acid drugs (siRNAs) trapped in endosomes. According to the group's proposed model, cholesterol molecules can be attached to the surface of nucleic acid molecules using chimeric nucleic acid molecules. The cholesterol molecule and the chimeric small molecule are chemically conjugated. When the chimeric molecule non-covalently binds to the nucleic acid double strand, the cholesterol molecule is also non-covalently tagged to the surface of the small nucleic acid molecule. Because the cholesterol-chimeric molecule conjugate is not covalently linked to the small nucleic acid molecule, the cholesterol molecule fuses with the cell membrane lipids upon penetrating the cell membrane and is retained there. It then detaches from the small nucleic acid molecule and excretes the nucleic acid directly into the cytoplasm. This delivery method bypasses endocytosis and directly enters the cytoplasm, thus avoiding entrapment by endosomes / lysosomes and degradation by hydrolases. However, the effective chimeric nucleic acid molecules they used were all clastogens, making them unsuitable for clinical application. In summary, how to effectively reduce the off-target effects of small nucleic acid drugs, how to improve the targeted delivery of small nucleic acid drugs in vivo, and the efficiency of drug release in cells are still the key bottleneck technical problems faced by the development and application of small nucleic acid drugs, especially when targeting extrahepatic indications.

[0013] Summary of the Invention

[0014] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to solve the problems of obvious off-target effects and low delivery efficiency of small nucleic acid drugs in the prior art.

[0015] To achieve the above-mentioned purpose and other related purposes, the present invention provides a novel RNA interference trigger (RNAi trigger) precursor molecule, which is similar to the small hairpin RNA (shRNA) or stem-loop structure of pre-miRNA. For the convenience of description, the structure of the novel RNA interference trigger precursor molecule is divided into three parts: the first part: comprising an asymmetric drug-active double strand located at one end of the structure, consisting of an antisense strand and a significantly shortened sense strand, wherein the antisense strand and the shortened sense strand in the asymmetric drug-active double strand are unequal in length, wherein the length of the antisense strand is conventionally 19-27nt, and the length of the shortened sense strand is 8-14nt; the second part: comprising a single-stranded loop sequence LS (Loop Strand) located at the other end of the structure; the third part: comprising a connecting portion double strand, wherein the connecting portion double strand is located between the asymmetric drug-active double strand and the single-stranded loop sequence LS. The present invention names the above-mentioned RNA interference trigger precursor molecule as asymmetric stem-loop siRNA (Asymmetric Stem Loop-siRNA, or ASL-siRNA), and its basic structure is shown in Figure 14.

[0016] Preferably, the double-stranded linker comprises 4-8 base pairs.

[0017] The present invention further improves upon the aforementioned ASL-siRNA structure by utilizing the base-pairing complementarity of the single-stranded loop sequence LS and adding a loop-complementary single strand capable of complementary pairing with the single-stranded sequence LS. This loop-complementary single strand is named LCS (Loop Complementary Strand). The loop-complementary single strand LCS can be pre-formed into a conjugate coupled to a fusogenic molecule. Through base pairing and complementary binding between LCS and LS, the fusogenic molecule passively approaches the main ASL-siRNA structure. The present invention names the composite structure of ASL-siRNA + LCS-conjugate, i.e., the aforementioned nucleic acid interference drug structure, fusogenic-tagged-siRNA (FsiRNA). Its basic structure is shown in Figure 15.

[0018] The present invention also provides a pharmaceutical composition, which contains the aforementioned RNA interference triggering precursor molecule or the aforementioned nucleic acid interference drug precursor, and pharmaceutically acceptable excipients.

[0019] The present invention also provides a method for treating a disease, which comprises administering the aforementioned RNA interference trigger precursor molecule, the aforementioned nucleic acid interference drug precursor, or the aforementioned pharmaceutical composition to a patient.

[0020] As described above, the RNA interference trigger molecule and the corresponding nucleic acid interference pharmaceutical preparation and use thereof of the present invention have the following beneficial effects:

[0021] The present invention provides an overall structure of RNA interference effect designed as an asymmetric "stem-double-stranded loop" structure. The stem-loop structure is a precursor molecular structure that exercises RNAi effects in cells and is a natural substrate of the endonuclease Dicer. It releases mature double-stranded RNA effector molecules such as miRNA or siRNA through the action of the endonuclease Dicer. The present invention has made a series of modifications to the classic stem-loop structure, making the sense chain asymmetric in length, significantly shortening the chain length of the sense chain; allowing the antisense chain to be non-covalently connected to other sequences, and having at least one nucleotide gap near one end of the connecting structure. The stem-loop drug structure designed in this way will release asymmetric double-stranded RNA effector molecules under the action of Dicer. Although the length of the sense chain may vary, it will ensure the release of a complete antisense chain sequence. The antisense strand of the resulting asymmetric siRNA or miRNA partial double-stranded molecule is full length and can be selectively loaded into the RISC complex. However, the sense strand, being less than 15 bases long, cannot be loaded into the RISC complex. This doubles the relative effective concentration of the antisense strand, allowing more of it to enter the RISC complex and exert RNAi effects. This enhances the targeted knockdown efficiency of the antisense strand while effectively avoiding the off-target effects and corresponding toxic side effects that may be caused by the sense strand, achieving the desired precision targeted therapy.

[0022] At the same time, fusogenic tagging technology is used to non-covalently attach fusogenic molecules to the surface of small nucleic acids, thereby promoting their efficient entry into cells and avoiding the need for complex nanodelivery systems. Fusogenic molecules, such as cholesterol, are easily detached from the main nucleic acid sequence and therefore do not affect the efficacy of RNA interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 shows the results of the in vitro preliminary screening of candidate siRNAs of the present invention.

[0024] FIG2 shows the results of the rescreening performed by IC50 determination in the present invention.

[0025] FIG3 shows the results of verifying the structure formation of ASL-siRNA by agarose gel electrophoresis in the present invention.

[0026] FIG4 shows the comparison results of the knockdown efficiency of ASL-siRNA and conventional siRNA structures in the present invention.

[0027] FIG5 shows the results of further enhancing the efficacy of ASL-siRNA structure through chemical modification in the present invention.

[0028] FIG6 shows the results that the ASL-siRNA structure of the present invention is highly tolerant to different chemical modifications.

[0029] FIG. 7 shows the results of verifying the structure formation of FsiRNA by agarose gel electrophoresis in the present invention.

[0030] FIG8 shows the gel electrophoresis results of the present invention, which show that the fluorescent molecules have limited influence on the formation of ASL-siRNA structure.

[0031] FIG9 shows the results of the fluorescent labeling and tracing experiment to verify the transfection efficiency and intracellular distribution of FsiRNA in the present invention.

[0032] FIG. 10 shows the summary of the results of the fluorescent tracing imaging data showing the process of FsiRNA entering cells in the present invention.

[0033] FIG11 shows the monochromatic image of a single light channel of FsiRNA and the locally enlarged result of the superimposed image in the present invention.

[0034] FIG12 shows a single-channel monochrome image and a partially magnified result of an overlaid image of FsiRNA transfected with Lipofectamine in the present invention.

[0035] FIG13 shows the results of the knockdown efficiency verification of the FsiRNA structure self-transfection in the present invention.

[0036] FIG14 is a schematic diagram showing the structure of the nucleic acid molecule ASL-siRNA of the present invention.

[0037] FIG15 is a schematic diagram showing the structure of the nucleic acid drug FsiRNA in the present invention.

[0038] FIG16 is a schematic diagram showing the structure of the nucleic acid drug targeting FsiRNA in the present invention. DETAILED DESCRIPTION

[0039] The present invention provides an RNA interference triggering precursor molecule, ASL-siRNA. The structure of the RNA interference triggering precursor molecule comprises: a first portion comprising an asymmetric drug-active double strand located at one end of the structure, consisting of an antisense strand and a significantly shortened sense strand. The antisense strand and the shortened sense strand in the asymmetric drug-active double strand are unequal in length, wherein the antisense strand is conventionally 19-27 nt in length, and the shortened sense strand is 8-14 nt in length; a second portion comprising a single-stranded loop sequence LS located at the other end of the structure; and a third portion comprising a double-stranded linker located between the asymmetric drug-active double strand and the single-stranded loop sequence LS. The RNA interference triggering precursor molecule is a nucleic acid molecule that is converted into a small interfering RNA nucleic acid molecule in vivo.

[0040] In some embodiments, the double-stranded linker comprises 4-8 base pairs. Specifically, the double-stranded linker comprises 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, or 8 base pairs. Preferably, the double-stranded linker comprises 4-6 base pairs.

[0041] In some specific embodiments, in the RNA interference trigger precursor molecule, the end connected to the single-stranded loop sequence LS is a blunt end, the end connected to the asymmetric drug-active double strand is a sticky end, and / or the linker sequence covalently connected to the positive chain has a gap of 1-3nt.

[0042] In some specific embodiments, the significantly shortened sense chain is connected to the double-stranded linker through a covalent chemical bond, the antisense chain is connected to the sense chain through a hydrogen bond, the double-stranded linker is connected to the single-stranded loop sequence LS through a covalent chemical bond, and there is no covalent chemical bond between the antisense chain and the double-stranded linker, and there is a gap of 1-3 nt.

[0043] In some specific embodiments, the significantly shortened sense strand, the double-stranded connector, and the single-stranded loop sequence LS are continuously synthesized into a single nucleic acid chain without interruption, connected by covalent bonds, and the sequence order is: significantly shortened sense strand, followed by connector sequence I (connector sequence I and the sense strand are on the same side of the DNA double-linked structure, also referred to as the "connector sequence on the same side of the sense strand"), followed by the loop single-stranded sequence LS, and then followed by the connector sequence II (connector sequence II and the antisense strand are on the same side of the DNA double-linked structure, also referred to as the "connector sequence on the same side of the antisense strand"). The connector sequence on the same side of the sense strand and the connector sequence on the same side of the antisense strand are reversely complementary, and after annealing, the double-stranded connector is formed by intrachain pairing and complementation. The formation of the double-stranded connector simultaneously causes the unpaired sequence located between them, i.e., the loop single-stranded sequence LS, to bend into a circular single strand.

[0044] In some embodiments, the aforementioned looped single-stranded sequence LS is a single-stranded nucleic acid with the physical and chemical properties to form a double-stranded structure with a complementary single-stranded sequence. Therefore, the present invention utilizes this property of complementary bases to pair and bind with each other. By adding a single-stranded nucleic acid (LCS) that is complementary to the LS nucleic acid single-stranded sequence to the ASL-siRNA, a fusogenic lipid molecule is pre-coupled to the LCS single-stranded sequence to form a conjugate. This allows the fusogenic lipid molecule to passively bind to the ASL-siRNA nucleic acid molecule when the LS and LCS complement each other. The fusogenic molecule facilitates transfection of the nucleic acid molecule across the cell membrane, enhancing the efficiency of nucleic acid delivery into cells.

[0045] The antisense strand and the significantly shortened sense strand are connected to each other through hydrogen bonds of complementary paired bases to form a partial double strand.

[0046] In some embodiments, the significantly shortened sense strand is 8-14 nt in length. Specifically, the sense strand is 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, or 14 nt in length. Preferably, the sense strand is 8-12 nt in length.

[0047] In some embodiments, the antisense strand is 19-27 nt in length. Specifically, the antisense strand is 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt in length. Preferably, the antisense strand is 19-23 nt in length.

[0048] In the present invention, the purpose of the unequal lengths of the antisense strand and the sense strand is to eliminate the possibility of the sense strand being loaded into the RISC complex. This is because nucleic acid chains shorter than 15nt will be difficult to load into the RISC complex, which can eliminate the off-target effects of the sense strand as much as possible. The shortened sense strand contains 8-14nt at the 5' end or 3' end of the strand and is aligned with one end of the antisense strand. That is, the shortened sense strand and the complementary paired antisense strand form a flat end on one side of the formed duplex, and the unpaired sequence at the other end of the antisense strand becomes a single-stranded overhang sequence. The overhang sequence contains chemical modifications to resist nuclease attack.

[0049] In some embodiments, the length of the circular single-stranded sequence LS is 7-12 nt. Specifically, the length of the circular nucleic acid double strand is 7 nt, 8 nt, 9 nt, 10 nt, 11 nt or 12 nt.

[0050] Furthermore, the nucleotide sequence of the loop single-stranded sequence LS can be designed according to the candidate siRNA sequence to avoid forming an intrachain double strand with the sense strand sequence of the candidate siRNA.

[0051] Furthermore, the looped single-stranded sequence LS may contain one or more pseudouridines, 5-methyl bases, or other special nucleotides, or various combinations thereof.

[0052] In the present invention, the aforementioned RNA interference trigger precursor molecule is composed of at least the following two independent oligonucleotide fragments:

[0053] Oligonucleotide fragment 1: a significantly shortened sense strand sequence-a linker sequence on the same side of the sense strand-a loop single-stranded sequence LS-a linker sequence on the same side of the antisense strand;

[0054] Oligonucleotide fragment 2: is the antisense strand sequence, and the length can be 19-27 nt.

[0055] In some specific embodiments, the linker sequence on the same side of the sense strand is longer than the linker sequence on the same side of the antisense strand. Preferably, the linker sequence on the same side of the sense strand is 1-3 nt longer than the linker sequence on the same side of the antisense strand. The portion of the linker sequence on the same side of the sense strand that is longer than the linker sequence on the same side of the antisense strand is the overhang sequence of the double-stranded linker. The overhang sequence can create a gap between the antisense strand and the double-stranded linker. The aforementioned gap can provide space for chemical modification of the 5' end of the antisense strand and can accommodate groups that occupy a larger space. The phosphodiester bonds of the nucleotides in the base complementary pairing portion of the linker sequence on the same side of the sense strand and the linker sequence on the same side of the antisense strand are both thiolated. The aforementioned thiolation modification can guide the Dicer enzyme to act on the unmodified overhang sequence and prevent the Dicer enzyme from acting on the antisense strand molecule.

[0056] In some embodiments, the free 5' end of the antisense strand sequence is chemically modified. Further, the chemical modification is the introduction of a derivatized phosphate group at the free 5' end. Preferably, the modifying group of the chemical modification is vinyl phosphodiester (Vinyl Phosphorous), and the derivatized phosphate group is 5'-vinyl phosphate (5'-phosphoethylene ester). The purpose of the chemical modification is to ensure that the phosphate group is not degraded, and the phosphate group is a structural feature required for the selective loading of the antisense strand fragment into the RISC complex.

[0057] In some embodiments, the free 5' end of the significantly shortened sense strand sequence contains a chemical modification that prevents phosphorylation; preferably, the free 5' end of the significantly shortened sense strand fragment contains a trifluoromethylation modification.

[0058] In some embodiments, the 2' carbon of the pentose in the RNA interference triggering precursor molecule is chemically modified, specifically, the chemical modification includes one or more of fluorine modification, oxymethyl modification, or other chemical modifications.

[0059] In some embodiments, the phosphodiester bond in the RNA interference triggering precursor molecule contains a phosphorothioate modification.

[0060] In some specific embodiments, the RNA interference triggering precursor molecule may comprise a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 19 and SEQ ID No. 20.

[0061] In some embodiments, the RNA interference trigger precursor molecule can be obtained by annealing oligonucleotide fragment 1 and oligonucleotide fragment 2. In the present invention, the antisense strand sequence is the active ingredient of the nucleic acid drug. Specifically, the antisense strand sequence can be the antisense strand of siRNA, or the active strand of microRNA, or the active strand of ASO, or the active strand of Antagomir.

[0062] The present invention also provides a nucleic acid interference drug preparation FsiRNA that can be effectively self-delivered, wherein the nucleic acid interference drug preparation comprises the aforementioned RNA interference triggering precursor molecule and a loop-complementary single-stranded LCS, wherein the loop-complementary single-stranded LCS comprises a nucleic acid fragment that is complementary to the base pairing of the single-stranded loop sequence LS in the aforementioned RNA interference triggering precursor molecule, that is, the loop-complementary single-stranded LCS pairs with the single-stranded loop sequence LS to form a double-stranded loop.

[0063] In some embodiments, the nucleic acid interference pharmaceutical preparation FsiRNA comprises at least the following three independent oligonucleotide fragments:

[0064] Oligonucleotide fragment 1: sense strand sequence-double-stranded linker sequence on the same side of the sense strand-loop single-stranded sequence LS-double-stranded linker sequence on the same side of the antisense strand;

[0065] Oligonucleotide fragment 2: antisense strand sequence, length can be 19-27 nt;

[0066] Oligonucleotide fragment 3: loop complementary single-stranded sequence LCS, with a length of 7-12nt.

[0067] In some embodiments, the ring-complementary single-chain LCS further comprises a fusogenic molecule. The fusogenic molecule can be selected from one or more of a lipid molecule, a polypeptide molecule, a sugar molecule, or a small chemical molecule. Specifically, the lipid molecule includes one or more of aliphatic hydrocarbon molecules of varying chain lengths, cholesterol molecules, various phospholipids, amino fatty molecules, or lipid-soluble small molecule complexes. Preferably, the fusogenic molecule is a cholesterol molecule.

[0068] In some embodiments, the loop-complementary single-chain LCS also contains a cell-targeting molecule, enabling targeted delivery to organs and tissues. Figure 16 shows its basic structure. The cell-targeting molecule is selected from one or more of a polypeptide molecule, a sugar molecule, a small chemical molecule, an aptamer molecule, or a Fab molecule. Specifically, polypeptide molecules are represented by RGD molecules or EED-endosomal escape domain polypeptides; sugar molecules are represented by GalNac molecules; small chemical molecules are represented by folic acid molecules; membrane-melting molecules are represented by cholesterol; and HER-2 Fab molecules are represented by monoclonal antibodies.

[0069] In the present invention, the cholesterol and other membrane-melting molecules in the nucleic acid interference drug formulation FsiRNA are not covalently conjugated to the sense or antisense strands of the aforementioned RNA interference trigger precursor molecule, allowing the active portion of the aforementioned RNA interference trigger precursor molecule to remain intact. In traditional methodologies, cholesterol or other lipid molecules are covalently attached to one end of the sense strand. However, in the present invention's membrane-melting attachment technology platform, the physicochemical interaction between the membrane-melting chemical and the primary active sequence is not a traditional covalent conjugation, but rather mediated by weak non-covalent forces such as hydrogen bonds between the complementary base pairing of the two single strands. This unique structural feature enables FsiRNA to be transfected and released into the cytoplasm with higher efficiency. The present invention demonstrates that the intracellular distribution pattern of FsiRNA is diffuse, which is significantly different from the pattern of liposome transfection, a classic example of transfection via the endosomal-lysosomal pathway.

[0070] Secondly, the ring-complementary single-chain LCS can be further derived and customized to meet the specific requirements of selective delivery to certain tissues or organs. For example, PEG molecules can be added to the ring-complementary chain to increase circulation duration; or specific ligands can be added to the ring-complementary chain to achieve tissue or organ targeting, and so on.

[0071] The present invention also provides a pharmaceutical composition comprising the aforementioned RNA interference triggering precursor molecule ASL-siRNA or the aforementioned nucleic acid interference pharmaceutical preparation FsiRNA, and pharmaceutically acceptable excipients. The pharmaceutical composition or conjugate is administered via a systemic or local route selected from auris interna, ophthalmic administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, topical contact, intraperitoneal administration, and intralesional administration. The pharmaceutical composition or conjugate is administered in the form of one or more of an injection, tablet, capsule, aerosol, eye drops, or nasal drops.

[0072] The adjuvant includes various excipients and diluents. These adjuvants are not necessary active ingredients and do not have excessive toxicity after administration. The adjuvant includes sterile water or normal saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or adhesives. The adjuvant also includes other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. When the adjuvant is used for the aqueous solution of injection, it is selected from normal saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannose or sugar alcohol isotonic solution. The aqueous solution of the injection includes a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and / or nonionic surfactant (Tween 80 or HCO-50).

[0073] In the pharmaceutical composition provided by the present invention, the aforementioned RNA interference triggering precursor molecule or the aforementioned nucleic acid pharmaceutical preparation is a single active ingredient, and can also be combined with one or more other active ingredients useful for disease treatment to form a combined preparation. The active ingredients are various other drugs used for disease treatment.

[0074] 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 active ingredient of the aforementioned nucleic acid molecule or 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.

[0075] The present invention also provides the use of the aforementioned RNA interference triggering precursor molecule ASL-siRNA or the aforementioned nucleic acid pharmaceutical preparation FsiRNA in preparing a disease treatment product.

[0076] The present invention also provides a method for treating a disease, which comprises administering the aforementioned RNA interference triggering precursor molecule ASL-siRNA, the aforementioned nucleic acid pharmaceutical preparation FsiRNA, or the aforementioned pharmaceutical composition to a patient.

[0077] Some of the terms in this invention are defined as follows:

[0078] Fusogenic molecules: Chemical molecules that facilitate fusion with cell membranes and assist biomacromolecules in entering cells.

[0079] Asymmetric: refers to the unequal lengths of the sense and antisense strands of a double-stranded small nucleic acid molecule.

[0080] Asymmetric stem-loop: refers to a shRNA structure similar to pre-miRNA, in which the length of the sense strand is shorter than the conventional length of the antisense strand.

[0081] Loop sequence: refers to the single-stranded loop sequence in the shRNA structure.

[0082] Loop complementary strand sequence LCS (Loop complementary strand): refers to a single-stranded sequence that is complementary to the base pairing of the single-stranded loop sequence LS.

[0083] sisiRNA is the abbreviation for small internally segmented interfering RNA.

[0084] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the technical basis of the present invention.

[0085] Before further describing the 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0086] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0087] Example 1 Construction of a novel RNA interference triggering precursor molecule

[0088] Experimental procedure: First, order the components according to the structural design

[0089] 1. Candidate siRNA Screening Process: Through years of experience, the screening process for candidate siRNA sequences has become largely standardized. For ease of description, the screening process for candidate siRNA sequences targeting TGFβ1 (ASL-siRNA) is used as an example.

[0090] 1.1 First, use the company's independent siRNA candidate sequence screening software ScoreSequence to design candidate sequences.

[0091] The screening criteria for candidate sequences (antisense strand) include: (1) sequence length of 19-25 nt; (2) G:C content of 30%-60%; (3) the 5' end of the sequence starts with A or U; (4) the A:U ratio of 2-8 nt from the 5' end is greater than 50%; (5) no more than 4 consecutive Gs; (6) no more than 5 consecutive GC / CGs; (7) no more than 3 pairs of paired sequences within the chain; etc.

[0092] Download the target gene cDNA sequence from the NCBI Nucleotide database: Homo sapiens transforming growth factor beta 1 (TGFB1), mRNA. NCBI Reference Sequence: NM_000660.7. Copy the downloaded sequence into a computer program and press Enter to run the program. A typical result is shown in Table 1.

[0093] Table 1. Candidate in silico siRNA sequences for TGFβ1 target genes

[0094] 1.2 Screening of siRNAs with conventional structures in an in vitro cell experimental system.

[0095] The routine screening procedure has become the standard method. The whole procedure is divided into the following steps:

[0096] (1) First, place an order with the reagent supplier and chemically synthesize the candidate sequences in the table.

[0097] (2) Based on the results of previously published literature, select a working cell line for the identified target gene.

[0098] (3) Screening is done using cells in a rapid growth phase. First, plate the cells overnight (usually in a 24-well culture plate, about 20-22 hours), and the next day, transfect the cells with the candidate siRNA using positive liposomes (such as Lipofectamine-2000 from Invitrogen).

[0099] (4) Incubate the transfected cells for 24-72 hours.

[0100] (5) Extract RNA from transfected cells using a commercial kit (such as Omega).

[0101] (6) Reverse transcribe the extracted RNA into cDNA using a commercial kit (such as AG).

[0102] (7) Using cDNA as a template, designed primers, and a commercial kit (such as AG) to perform qRT-PCR to quantitatively analyze the content of target gene mRNA in cells and detect the efficiency of candidate siRNA in knocking down target gene mRNA expression.

[0103] (8) The knockdown percentage of the screened siRNA was calculated using the comparative ΔΔCt method and untransfected cells as the comparison baseline. Typical results are shown in Figure 1.

[0104] (9) Then, sequences with high knockdown efficiency (e.g., T1, T3, T5, T9) were selected for review and further validation. To make the data more reliable, IC50 determination was used for review and validation. Based on the predicted data, the sample was serially diluted, for example, starting from 100 nM, and a 4-5-fold serial dilution was performed, with 5-6 points. Typical results are shown in Figure 2.

[0105] 1.3 Next, based on the selected sequence, the corresponding ASL-siRNA fragment sequence is ordered according to the structural design of the present invention.

[0106] Here we take TGFβ1 siRNA sequence T5 as an example.

[0107] Order the following RNA sequences:

[0108] TGFβ1(ASL#3)-F2: 5'CACAGCAUA(psA)A(psG)(psC)(psU)CCUGACCCAAGCU3' (SEQ ID No. 19);

[0109] TGFβ1(ASL#3)-AS: AAGGAGAACUCGGGAAUCAGGtt (SEQ ID No. 20).

[0110] 1.4 After receiving the two ordered nucleic acid sequences, they are annealed in vitro to obtain the asymmetric stem-loop siRNA duplex (Asymmetric Stem Loop siRNA, ASL-siRNA) of the present invention.

[0111] The specific procedure is as follows: First, order the individual RNA single strands from a supplier. Then, dissolve the RNA fragments and mix them at equimolar concentrations. Annealing is performed in a PCR machine to obtain the ASL-siRNA structure. The reagent formula for the annealing reaction is shown in Table 2.

[0112] Table 2. Annealing reaction reagent formula

[0113] Annealing reaction was performed in a PCR instrument. Annealing reaction program settings are shown in Table 3.

[0114] Table 3. Annealing reaction program

[0115] 1.5 Structure verification.

[0116] Successful annealing was verified using 3% agarose gel electrophoresis. The results are shown in Figure 3: M represents a molecular weight marker; F2 represents the 3'-end 10 nt of the sense strand plus the linker sequence and the LS sequence; AS represents the antisense strand; and ASL represents the ASL-siRNA structure after annealing F2+AS. The band in the ASL electrophoresis lane exhibits a significantly larger molecular weight than the nucleic acid fragments in the other lanes, confirming successful annealing and the formation of the ASL-siRNA structure.

[0117] Example 2 Novel RNA interference triggering precursor molecules have higher target gene knockdown efficiency

[0118] 2.1 The validation data obtained in the in vitro cell system demonstrated that the ASL-siRNA structure has a high inhibitory efficiency on the target gene mRNA.

[0119] Take TGF-β1 as the target gene. The construction method of the asymmetric stem-loop-siRNA (ASL-siTGFβ1) is the same as that described in Example 1. The method used to detect the knockdown efficiency is the same as that described in 1.2, and the detection method used is also in vitro cell transfection and corresponding qRT-PCR. To demonstrate the advantages of the structure of the present invention, a comparison was made with the classic siRNA double-stranded structure. The results showed that the efficiency of ASL-siRNA in knocking down the target gene was significantly higher than that of the traditional structure siRNA. The specific detection results are shown in Figure 4.

[0120] 2.2 The comparison results of IC50 further confirmed the advantages of the ASL-siRNA structure, as shown in Figure 5.

[0121] Knockdown efficiency was assessed using the same in vitro cell transfection and qRT-PCR methods as described in 1.2. Figure 5 shows typical assay results, comparing the ASL construct with a conventional siRNA. The results demonstrate that the ASL-siRNA construct significantly outperforms the conventional siRNA construct in knocking down the target gene TGF-β1.

[0122] Example 3 Chemical modification of novel RNA interference triggering precursor molecules

[0123] 3.1 Chemical modification further enhances the knockdown activity of ASL-siRNA. To increase the stability of siRNA molecules in vivo, chemical modification is usually applied to the 2'-position of the pentose of nucleotides. Published data have clearly shown that chemical modification not only enhances the stability of siRNA molecules, but also appropriate chemical modification can improve the activity of siRNA in knocking down target genes. In this invention, a series of chemical modifications were also carried out on ASL-siRNA, and the structures before and after chemical modification were compared to detect whether the knockdown efficiency changed. The method used was the same in vitro cell transfection and corresponding qRT-PCR as described in 1.2. Taking the screened T5 sequence as an example, the knockdown efficiency was in the order of classical structure siRNA < ASL < chemically modified ASL. The results in Figure 5 show that chemical modification can significantly further improve the knockdown efficacy of ASL-siRNA. After fitting calculation by the professional data analysis software Graphpad prism, it can be seen that the IC 50 of the chemically modified ASL structure reached 0.52 nM, which was significantly lower than that of the classical siRNA (IC50 = 4.10 nM) structure.

[0124] 3.2 ASL-siRNA has strong tolerance to different chemical modifications, that is to say, the chemical modification that enhances the knockdown activity of ASL-siRNA is not single. Although chemical modification can improve stability and enhance the knockdown effect, the optimal chemical modification format often varies due to unique sequences and often requires corresponding screening and modification due to different sequences. Here, taking the screened T5 as an example, the T5 effector molecule structures were respectively constructed as: conventional siRNA structure and ASL-siRNA structure. And two chemical modification formats were respectively adopted for the two structures: one is the ESC modification format of Alnylam, and the other is the carboxylate modification format. The experimental method for detecting the knockdown efficiency was the same as described in 1.2, and the same in vitro cell transfection and qRT-PCR were used to determine the influence of different chemical modification formats on the knockdown efficacy of candidate sequences. ASL-JL and ASL-ALN are both ASL structures based on the T5 sequence.

[0125] ASL-JL: The carboxylate modification format was adopted. The specific sequence is as follows:

[0126] ASL-JL-F2: mCmAmCmAmGmCmAmUmAC(Aps)G(Cps)UCCUGACCCAA(Gps)C(Ups)

[0127] ASL-JL-AS:

[0128] p-(mUps)(fAps)mUmGmCfUmGfUmGfUmGfUmAmCmUmCmU(mGps)(mCps)uu

[0129] ASL-ALN: A modified version of inclisiran developed by Alnylam was used as a positive control.

[0130] ASL-ALN-F2:

[0131] Cf-Am-Cm-Am-Gm-Cm-Am-Um-Am-C-Aps-G-Cps-UCCUGACCCAA-Gps-C-Ups

[0132] ASL-ALN-AS:

[0133] Umps-Afps-Um-Gm-Cm-Uf-Gm-Uf-Gf-Um-Gm-Um-Am-Cf-Um-Cf-Um-Gm-Cmps-dtps-dt

[0134] Capital letters represent bases, and lowercase letters represent chemical modifications. m- represents 2'-O-Methylation; f- represents 2'-Fluorine; ps- represents

[0135] phosphorothioate; d- stands for deoxyribose.

[0136] IC 50 The data were fitted and calculated using the professional data analysis software Graphpad Prism, and the results are shown in Figure 6. These results demonstrate that the ASL-siRNA structure of the present invention not only significantly enhances the inhibitory efficiency of target gene mRNA but also exhibits high tolerance to various chemical modification formats, suggesting that chemical modifications to the ASL-siRNA structure may be more extensive, thereby enhancing sequence stability and enabling more efficient, stable, and long-lasting efficacy upon in vivo administration.

[0137] Example 4: Confirmation of the structure of membrane-attached nucleic acid interference drug preparation FsiRNA

[0138] The membrane-attached nucleic acid drug FsiRNA is a drug precursor and a key component of the present invention. The FsiRNA structure consists of three components: the AS strand, the F2 segment, and the LCS conjugate. The 8-14 nt SS strand contained in the F2 segment can complementarily bind to the AS strand. The LS sequence in the F2 segment can pair with the LCS sequence. The molecular weight changes of the three component sequences upon annealing can be monitored by gel electrophoresis to infer the correct structure.

[0139] Gel electrophoresis is a commonly used method for detecting the molecular weight of biomolecules. The molecular structure of the FsiRNA small nucleic acid drug of the present invention is composed of the above three parts and needs to be formed by in vitro annealing through complementary base pairing. The formation of the detected molecular structure is inferred by the change in electrophoretic mobility shift before and after sample annealing. For example, the FsiRNA structure is composed of the ASL main structure sequence and the LCS sequence through complementary pairing of the LS loop sequence. Other molecules coupled to the LCS (such as lipid molecules) are therefore passively close to the ASL main structure sequence, thereby changing the molecular size and mobility of the ASL. In other words, the complete structure of FsiRNA is composed of the complementary pairing of nucleic acid bases between the component fragments. Therefore, the size of the molecule can reflect the degree of structural integration. That is, when each component is integrated into the main structure, its own electrophoretic band will disappear. At the same time, as the molecular weight increases, the electrophoretic band of the final drug structure - FsiRNA - will shift upward accordingly.

[0140] Electrophoresis experiment group settings:

[0141] (1) Marker: Commercial standard molecular weight;

[0142] (2) ASL-F2 single strand: ASL-siRNA sense strand 8-14 nt + double-stranded linker fragment I 5 nt + LS sequence 9 nt + double-stranded linker fragment II 4 nt;

[0143] (3) AS single strand: ASL-siRNA antisense strand 21 nt;

[0144] (4) ASL(F2+AS): Asymmetric stem-loop-siRNA structure (F2+AS)

[0145] (5) FsiRNA#1: ASL+LCS-lipid 1 (C12-LCS-C12: The two free ends of LCS are each coupled to a carbon 12 aliphatic hydrocarbon)

[0146] (6) FsiRNA#2: ASL+LCS-lipid 2 (C18-LCS: the 5' free end of LCS is coupled to a C18 aliphatic hydrocarbon)

[0147] Brief description of the experimental method: The above groups were annealed and renatured separately on a PCR instrument. Samples were then electrophoresed on a 3% agarose gel (containing ethidium bromide). After approximately 30 minutes of electrophoresis, the nucleic acid-bound ethidium bromide was excited under UV light to produce fluorescence, allowing the electrophoretic migration of nucleic acid molecules to be observed. The rate of electrophoretic migration is related to molecular size and hydrophobicity.

[0148] The experimental results are shown in Figure 7. According to the structural design, F2 is a 28-nt single-stranded protein, AS is a 21-nt single-stranded protein, ASL is an asymmetric duplex of F2 and AS, FsiRNA#1 is ASL+C12-LCS-C12, and FsiRNA#2 is ASL+C18-LCS. The electrophoretic mobilities of the individual molecules differ significantly, suggesting differences in their structural formation.

[0149] Example 5 Comparison of intracellular distribution characteristics of FsiRNA self-transfection and liposome transfection

[0150] Fluorescently labeled small nucleic acids are commonly used in tracing studies. Fluorescence microscopy allows for direct observation of the kinetics of cellular entry and intracellular distribution of labeled small nucleic acids, thereby understanding the impact of chemical modification and molecular structure on the entry pathway and release efficiency of small nucleic acid molecules. One of the objectives of FsiRNA structural design is to enhance the efficiency of polyanionic small interfering RNA molecules across cell membranes by introducing fusogenic molecules into the drug structure, and to encourage siRNA molecules to avoid the endosome pathway, thereby enhancing the release efficiency of siRNA molecules after entry into cells.

[0151] 5.1 Gel electrophoresis images show that the fluorescent molecules have limited effect on the formation of ASL-siRNA structure and do not hinder the formation of the complete structure as expected.

[0152] Briefly describe the experimental procedure: First, order nucleic acid fragments with the 5' end of F2 fluorescently labeled with FAM. One set of FAM-labeled F2 is incubated with the AS chain to anneal, forming an ASL-siRNA structure. Another set of FAM-labeled F2 is incubated with the AS chain and a Cy5-labeled C12-LCS-C12 conjugate to anneal, forming an FsiRNA structure. The samples are then subjected to 3% agarose gel electrophoresis for structural verification to determine whether the introduction of the fluorescent dye affects the expected FsiRNA structure.

[0153] Electrophoresis grouping:

[0154] (1)Marker: Commercial standard molecular weight

[0155] (2) AS single strand: antisense strand fragment

[0156] (3) FAM-F2 single chain: FAM fluorescently labeled F2 fragment

[0157] (4) Cy5-C12-LCS-C12 single chain: Cy5 fluorescently labeled C12-LCS-C12

[0158] (5) ASL-siRNA: Asymmetric stem-loop-siRNA structure (FAM-F2+AS)

[0159] (6)FsiRNA: ASL-siRNA+Cy5-C12-LCS-C12

[0160] Judging from the results in FIG8 , the ASL-siRNA and FsiRNA structures were formed as expected after annealing of the constituent fragments.

[0161] 5.2 Fluorescence tracing experiments were performed in vitro on HeLa cells. ASL-siRNA constructs were transfected using Lipofectamine 2000; FsiRNA was added directly to serum-free culture medium without transfection reagent. Samples were collected 24 hours after transfection and washed three times with PBS for 3 minutes each. The samples were then fixed with 4% paraformaldehyde and coverslipped with Hoechst mounting fluid. Confocal microscopy was used to record the intensity of fluorescence entering the cells and their distribution within the cells.

[0162] Experimental groups:

[0163] 1. Blank control group to eliminate the interference of autofluorescence.

[0164] 2.ASL-siRNA+Lipo: Transfect ASL-siRNA using Lipofectamine 2000.

[0165] 3. FsiRNA: self-transfection group, without any transfection reagent.

[0166] The results in Figure 9 show that ASL-siRNA molecules transfected with Lipofectamine-2000 reagent are distributed in blocks and concentrated around the cell nucleus, which is a typical positive control for endosome distribution. FsiRNA, on the other hand, does not require the assistance of transfection reagent to enter the cell by itself. Its fluorescence in the cell is small and diffusely distributed in the cytoplasm, which is significantly different from the intracellular distribution pattern of Lipofectamine-2000 transfection.

[0167] Example 6 Distribution characteristics of each component of FsiRNA in cells

[0168] The structural design of FsiRNA incorporates fusogenic molecules into the drug structure, attaching them to the ASL-siRNA small nucleic acid core through weak interactions caused by complementary base pairing. This design aims to enhance transfection efficiency by allowing fusogenic molecules, such as cholesterol, to dissociate from the small nucleic acid core during cell membrane penetration, thereby increasing its efficiency.

[0169] Experimental Design: To verify that membrane-integrating molecules (e.g., cholesterol) conjugated to the LCS sequence dissociate from the ASL-siRNA main structure during passage through the cell membrane, the FsiRNA component fragments were individually labeled with different fluorescent molecules: FAM was used at the 5' end of the F2 fragment, and Cy5 was used to label the C12-LCS-C12 conjugate. If FAM-F2 and AS were incubated and annealed together, the resulting ASL-siRNA construct would only emit green fluorescence. However, if FAM-F2, AS, and Cy5-C12-LCS-C12 were incubated and annealed together, the resulting FsiRNA construct would emit both green and red fluorescence. If Cy5-C12-LCS-C12 dissociated from the ASL-siRNA main structure, the green fluorescence would separate from the red fluorescence and remain within the cell.

[0170] 6.1 Perform transfection experiments on Jurkat cells in vitro and observe the distribution and relative positions of the two fluorescent molecules within the cells.

[0171] The experimental group design is as follows:

[0172] 1. Blank control group to eliminate the interference of autofluorescence.

[0173] 2. Negative control group: FAM-labeled ASL-siRNA was directly added into serum-free culture medium to observe the transfection of naked ASL-siRNA.

[0174] 3. FsiRNA self-transfection group: Fluorescently labeled FsiRNA was directly added into serum-free culture medium, which should have both FAM and Cy5 fluorescence.

[0175] 4. FsiRNA transfection group using Lipofectamine-2000: Transfection with liposome reagent should have both FAM and Cy5 fluorescence.

[0176] Transfection samples were taken at predetermined time points and prepared into slides. The distribution of fluorescently labeled molecules within the cells was observed by layered scanning using a confocal microscope. Figure 10 is a summary of the fluorescence distribution results after transfection. Liposome transfection generally enters the cell through endocytosis and is trapped in the endosome / lysosome. As shown in Figures 10 to 12, the FsiRNA transfected into the cell by Lipofectamine was trapped in the endosome / lysosome system, and the overlapping fluorescence appeared in blocks and distributed around the cell nucleus. The two fluorescences of the FsiRNA that entered the cell through self-transfection were clearly separated, diffusely distributed in the cytoplasm, and had a lower fluorescence intensity.

[0177] Example 7 FsiRNA self-transfects HeLa cells and knocks down target gene expression

[0178] Fluorescence tracing experiments demonstrated that FsiRNAs can efficiently self-transfect cells in vitro without the need for any transfection reagents. However, the ultimate criterion for evaluating this novel construct is whether these FsiRNAs that autonomously enter cells possess knockdown activity. Next, the FsiRNAs were tested for their ability to inhibit target gene mRNA expression using conventional in vitro cell transfection and qRT-PCR assays as described in 1.2.

[0179] FsiRNA self-transfection was performed in serum-free medium: After culturing HeLa cells overnight, the medium was discarded and replaced with fresh serum-free medium. Subsequently, FsiRNA samples of varying concentrations were added directly to the serum-free medium and incubated for 4 hours. Complete medium containing 10% FBS was then added. After a further 24 hours of incubation, total RNA from the cells was extracted using a commercial kit. qRT-PCR reactions were then performed using designed primers to detect changes in the intracellular levels of specific target mRNAs, allowing comparison of the efficiency of knockdown of target gene mRNA expression by different candidate constructs.

[0180] The experimental groups included:

[0181] 1. Untreated blank group: baseline calibration control.

[0182] 2. Naked ASL-siRNA group (without LCS-conjugate): No transfection reagent was used. This served as the self-transfection negative control group.

[0183] 3. PC group of ASL-siRNA (w / o LCS-conjugate) was transfected with Lipo2000 and served as a positive control.

[0184] 4. FsiRNA-ASL self-transfection, low-dose group;

[0185] 5. FsiRNA-ASL self-transfection, medium dose group;

[0186] 6. FsiRNA-ASL self-transfection, high-dose group;

[0187] ASL is a naked nucleic acid control, which refers to the ASL-siRNA construct without the addition of LCS-lipid conjugate. ASL+Lipo is a positive control for the naked nucleic acid construct of ASL-siRNA transfected with Lipofectamine-2000.

[0188] Figure 13 shows representative results. These results demonstrate that the FsiRNA of the present invention can effectively self-deliver into cells without the need for transfection reagents. This fact is clearly demonstrated by the fluorescence tracing experimental data presented above. FsiRNA molecules maintain potent knockdown activity after entering cells, effectively inhibiting target gene mRNA expression.

[0189] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

[0190] The references used in this application are as follows:

[0191] [1]MARY L.STEPHENSON AND PAUL C.ZAMECNIK,Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide.Proc.Natl.Acad.Sci.USA Vol.75,No.1,pp.285-288,January 1978

[0192] [2]Clement Agoni&Fisayo A.Olotu1&Pritika Ramharack&Mahmoud E.Soliman.Druggability and drug-likeness concepts in drug design:are biomodelling and predictive tools having their say? Journal of Molecular Modeling(2020)26:120

[0193] [3]Aaron D.Springer and Steven F.Dowdy.GalNAc-siRNA Conjugates:Leading the Way for Delivery of RNAi Therapeutics.NUCLEIC ACID THERAPEUTICS,28(3):109,2018

[0194] [4]Bo Hu,Liping Zhong,Yuhua Weng,Ling Peng,Yuanyu Huang,Yongxiang Zhao and Xing-Jie Liang.Therapeutic siRNA:state of the art.Signal Transduction and Targeted Therapy(2020)5:101

[0195] [5]Rafal Bartoszewski and Aleksander F.Sikorski:Editorial focus:understanding off-target effects as the key to successful RNAi therapy.Cellular&Molecular Biology Letters(2019)24:69

[0196] [6]Julia Neumeier and Gunter Meister.siRNA Specificity:RNAi Mechanisms and Strategies to Reduce Off-Target Effects.Frontiers in Plant Science,(2021)11:1.

[0197] [7]Aimee L.Jackson and Peter S.Linsley,Recognizing and avoiding siRNA offtarget effects for target identification and therapeutic application.NATURE Reviews Drug Discovery,9:57,2010

[0198] [8]Jesper B.Bramsen,Maria B.Laursen,Christian K.Damgaard,et al.Improved silencing properties using small internally segmented interfering RNAs.Nucleic Acids Research,2007,35(17):5887.

[0199] [9]O.R.F.Mook,Jeroen Vreijling,Suzy L.Wengel,et al.In vivo efficacy and off-target effects of locked nucleic acid(LNA)and unlocked nucleic acid(UNA)modified siRNA and small internally segmented interfering RNA (sisiRNA)in mice bearing human tumor xenografts.Artificial DNA:PNA &XNA.1:1,36-44;

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[0010] Kumiko Ui-Tei.Optimal choice of functional and off-target effect-reduced siRNAs for RNAi therapeutics.Frontiers in Genetics,2013,4(107):1.

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[0011] Christopher R.Brown, Swati Gupta,et al:Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates.Nucleic Acids Research,2020,Vol.48,No.21 11827–11844.

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[0012] Wanyi Tai,Xiaohu Gao:Noncovalent tagging of siRNA with steroids for transmembrane delivery.Biomaterials,DOI:10.1016 / j.biomaterials.2018.02.007

Claims

1. An RNA interference triggering precursor molecule, characterized in that The structure of the RNA interference trigger precursor molecule includes: a first part: comprising an asymmetric drug-active double strand located at one end of the structure, consisting of an antisense strand and a sense strand, wherein the antisense strand and the sense strand in the asymmetric drug-active double strand are of unequal length, wherein the antisense strand is longer than the sense strand, and the sense strand is 8-14 nt in length; a second part: comprising a single-stranded loop sequence LS located at the other end of the structure; a third part: comprising a connecting portion double strand, wherein the connecting portion double strand is located between the asymmetric drug-active double strand and the single-stranded loop sequence LS.

2. The RNA interference triggering precursor molecule according to claim 1, characterized in that The double-stranded linker contains 4-8 base pairs, and / or the end connected to the single-stranded loop sequence LS is a blunt end, and the end connected to the asymmetric drug-active double-stranded sequence is a sticky end, and / or the linker sequence covalently connected to the positive strand has a gap of 1-3 nt.

3. The RNA interference triggering precursor molecule according to claim 1 or 2, characterized in that The sense strand is connected to the double-stranded linker by a covalent chemical bond, the antisense strand is connected to the sense strand by a hydrogen bond, the double-stranded linker is connected to the single-stranded loop sequence LS by a covalent chemical bond, and there is no covalent chemical bond between the antisense strand and the double-stranded linker, and there is a gap of 1-3 nt.

4. The RNA interference triggering precursor molecule according to any one of claims 1 to 3, characterized in that The length of the antisense strand is 19-27 nt; and / or the length of the single-stranded loop sequence LS is 7-12 nt.

5. The RNA interference triggering precursor molecule according to claim 1, characterized in that The RNA interference triggering precursor molecule is composed of at least the following two independent oligonucleotide fragments: Oligonucleotide fragment 1: positive chain sequence-connector sequence I-single-stranded loop sequence LS-connector sequence II; Oligonucleotide fragment 2: antisense strand fragment, 19-27 nt in length.

6. The RNA interference triggering precursor molecule according to claim 5, characterized in that The linker sequence on the same side of the sense strand is longer than the linker sequence on the same side of the antisense strand; preferably, the linker sequence on the same side of the sense strand is 1-3 nt longer than the linker sequence on the same side of the antisense strand.

7. The RNA interference triggering precursor molecule according to any one of claims 1 to 6, characterized in that The RNA interference triggering precursor molecule has one or more of the following chemical modifications: 1) The free 5' end of the antisense strand sequence is chemically modified; preferably, the modifying group of the chemical modification is vinyl phosphine; 2) The free 5' end of the sense strand sequence contains a chemical modification that prevents phosphorylation; preferably, the free 5' end of the sense strand sequence contains a trifluoromethyl modification; 3) There is a chemical modification on the 2' carbon of the pentose in the RNA interference triggering precursor molecule; preferably, the chemical modification includes one or more of fluorine modification and oxymethyl modification; 4) The phosphodiester bond in the RNA interference triggering precursor molecule contains a phosphorothioate modification.

8. A nucleic acid interference pharmaceutical preparation, characterized in that: The nucleic acid interference pharmaceutical preparation comprises an RNA interference triggering precursor molecule and a loop-complementary single-stranded LCS as described in any one of claims 1-7; the loop-complementary single-stranded LCS comprises a nucleic acid fragment that is complementary to the base pairing of the circular nucleic acid single-stranded LS sequence in the RNA interference triggering precursor molecule as described in any one of claims 1-7.

9. The nucleic acid interference pharmaceutical preparation according to claim 8, characterized in that The nucleic acid interference pharmaceutical preparation comprises at least the following three independent oligonucleotide fragments: Oligonucleotide fragment 1: positive chain sequence-connector sequence I-loop single-stranded sequence LS-connector sequence II; Oligonucleotide fragment 2: antisense strand sequence, 19-27 nt in length; Oligonucleotide fragment 3: loop complementary single-stranded sequence LCS, with a length of 7-12nt.

10. The nucleic acid interference pharmaceutical preparation according to claim 8, characterized in that The loop-complementary single-stranded LCS contains one or more of a membrane-fusing molecule and a cell-targeting molecule.

11. The nucleic acid interference pharmaceutical preparation according to claim 10, characterized in that The membrane-melting molecule is selected from one or more of lipid molecules, polypeptide molecules, sugar molecules or small chemical molecules; or / and, the cell-targeting molecule is selected from one or more of polypeptide molecules, sugar molecules, small chemical molecules, aptamer molecules or Fab molecules.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the RNA interference triggering precursor molecule according to any one of claims 1 to 7 or the nucleic acid interference pharmaceutical preparation according to any one of claims 8 to 11, and pharmaceutically acceptable excipients.

13. Use of the RNA interference triggering precursor molecule according to any one of claims 1 to 7, the nucleic acid interference pharmaceutical preparation according to any one of claims 8 to 11, or the pharmaceutical composition according to claim 12 in the preparation of a disease treatment product.

14. A method for treating a disease, characterized in that: The treatment method comprises administering to the patient the RNA interference triggering precursor molecule according to any one of claims 1 to 7, the nucleic acid interference pharmaceutical preparation according to any one of claims 8 to 11, or the pharmaceutical composition according to claim 12.

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