Novel double-stranded RNA delivery vector and use

Abstract

Provided is a double-stranded RNA delivery vector, comprising the extracellular protein domain of a specific double-stranded RNA-binding protein SID-1 of Caenorhabditis elegans and a GPCR ligand having strong targeting capability and cell specificity. The vector can specifically bind a double-stranded RNA without being affected by DNAs, is independent of sequences, has a relatively high broad spectrum property and does not need artificial screening of RNA fragments, thereby greatly shortening the time for determining druggability. In addition, the combined GPCR ligand solves the problem of poor targeting capability and the problem of being incomprehensive; furthermore, cell-specific GPCRs all have a very strong endocytosis effect, thus ensuring that the SID-1 extracellular domain bound with the RNA can be endocytosed by cells so as to achieve drug effects.

Description

A novel double-stranded RNA delivery vector and its application

Technical field

[0001] The present invention belongs to the technical field of RNA delivery vectors, and more specifically, relates to a novel double-stranded RNA delivery vector and its application. [Background Technology]

[0002] Nucleic acid drugs have emerged as a rising star in the field of disease treatment in recent years. They hold significant clinical significance for their potential to address specific, elusive protein targets and develop treatments for "undruggable" diseases. Nucleic acid drugs primarily target DNA or RNA at the genetic level. Currently, the most widely used drugs include antisense oligonucleotides (ASOs), siRNA, miRNA, and aptamers, as shown in Figure 1. Antisense oligonucleotides, siRNA, and miRNA are composed of 15-25 nucleotides and are complementary or partially complementary to messenger RNA, potentially downregulating the translation of target proteins. Aptamers can recognize target proteins through their molecular structure and directly interact with them.

[0003] As shown in Table 1, according to incomplete statistics, there are currently 4 siRNA drugs approved by the FDA for marketing: In 2018, the FDA approved the world's first siRNA drug, Patisiran, for the treatment of hereditary transthyretin amyloidosis (hATTR amyloidosis). In 2019, the FDA approved the siRNA drug Givosiran for the treatment of acute hepatic porphyria (AHP) in adults. In 2020, Lumasiran also became the world's first siRNA drug for the treatment of primary hyperoxaluria. In the field of cardiovascular disease, Inclisiran, a siRNA drug targeting PCSK9 developed by Alnylam and Novartis, was approved by the FDA in December 2021 for the treatment of atherosclerotic cardiovascular disease. At present, many siRNA drugs are in clinical phases I-III, and small RNA drugs for various diseases are constantly being launched. Nucleic acid drugs are expected to become a type of precision medicine drug after small molecule drugs and monoclonal antibodies. The current RNA drug delivery technologies are mainly the following four types:

[0004] Table 1

[0005] 1) RNA drug delivery. Currently, nucleic acid drugs also face several challenges: poor in vivo stability and susceptibility to degradation by nucleases in the body; negatively charged nucleic acid drugs make it difficult to cross cellular barriers; and poor in vivo targeting, preventing precise targeting of target cells and tissues. Therefore, the modification and delivery of RNA drugs have become critical issues. Currently, the main delivery technologies for RNA interference drugs include nanoparticle delivery, viral vector technology, targeted delivery based on specific chemical modifications, antibody-based targeted delivery, and novel exosome delivery technology (Figure 2).

[0006] 2) Nanoparticle delivery technology. Lipid nanoparticles (LNPs) are the mainstream in nanoparticle delivery technology. A typical example is the neutral LNP developed by Alinylam, which can be enriched in hepatocytes through apolipoprotein E (ApoE)-mediated endocytosis and is therefore suitable for treating liver-related diseases. As shown in Figure 1, the mRNA drug AZD8601, which aims to increase angiogenesis, uses nanoparticle delivery technology to package VEGF-A mRNA in lipid nanoparticles (LNPs) and be internalized by host cells. The mRNA is then translated into VEGF-A protein, thereby increasing angiogenesis. As shown in Figure 3, other types of nanocarrier technologies have entered the clinical stage, such as peptide nanoparticle (PNP) technology, but nanoparticle delivery technology still requires a lot of exploration and research in terms of drug targeting.

[0007] 3) Targeted delivery via specific chemical modification. Among targeted delivery methods based on specific chemical modification, the most well-known is liver-targeted delivery mediated by N-acetylgalactosamine (GalNAc). GalNAc has a high affinity for asialoglycoprotein (ASGPR), which is primarily expressed on the surface of hepatocytes. Therefore, as shown in Figure 4, siRNA is modified with GalNAc to achieve specific delivery to the liver, allowing siRNA to enter cells and exert its function through ASGPR-mediated endocytosis. GalNAc trimers are a typical modification method.

[0008] 4) Exosome delivery technology. Virtually all cells in the body secrete extracellular vesicles (EVs), a group of vesicles enclosed by a lipid bilayer membrane. They transport payloads such as proteins and nucleic acids to recipient cells, playing a central role in intercellular communication. Exosomes are nanoscale EVs derived from endosomal secretions and can regulate many pathophysiological processes, including immune responses and inflammation, tumor growth, and infection. Healthy individuals and patients with various diseases release exosomes with varying RNA and protein content into the bloodstream, which can be measured as biomarkers. The discovery that exosomes are natural carriers of functional small RNAs and proteins has sparked significant interest in the drug delivery field due to the potential for using these vesicles to therapeutically deliver miRNAs, siRNAs, mRNAs, lncRNAs, peptides, and synthetic drugs. However, systemically delivered exosomes accumulate in the liver, kidneys, and spleen. Displaying glycosylphosphatidylinositol (GPI)-anchored nanobodies on exosomes is a new technology that allows exosomes to display various proteins, including antibodies, reporter proteins, and signaling molecules, such as peptides or antibody fragments that recognize target antigens, to obtain targeted exosomes. Targeting can be achieved by engineering the mother cells using fusion constructs between targeting ligands and EV transmembrane proteins (such as LAMP-2b or PDGFR™ domains) (as shown in Figure 5). Exosomes are composed of natural human proteins and lipids, are non-toxic and non-immunogenic, which is the biggest advantage of exosomes as drug delivery vehicles.

[0009] Currently, nucleic acid drugs face the following problems:

[0010] 1) Poor in vivo stability and easy to be degraded by nucleases in the body;

[0011] 2) Nucleic acid drugs are negatively charged and have difficulty crossing cell barriers;

[0012] 3) Poor in vivo targeting, unable to accurately target target cells and tissues.

[0013] Therefore, the delivery of RNA drugs has become a key technical issue that needs to be solved. The current delivery technologies of RNA drugs mainly include:

[0014] 1) Nanoparticle delivery technology;

[0015] 2) Viral vector technology;

[0016] 3) Targeted delivery based on specific chemical modifications;

[0017] 4) Antibody-based targeted delivery;

[0018] 5) Novel exosome delivery technology

[0019] The common disadvantages of the above technologies are that RNA needs to be modified and screened, which has great restrictions on the RNA sequence and does not have broad spectrum; the delivery vector production process is complicated and the cost is too high, so the drug cannot be used in large quantities; chemical modification has poor targeting and the targeting sites are not comprehensive; the drug efficacy is not high and it is easily captured by the immune system, reducing the drug efficacy.

[0020] [Summary of the invention]

[0021] The present invention utilizes the extracellular protein domain of the double-stranded RNA-binding protein SID-1, unique to Caenorhabditis elegans, in combination with a highly targeted, cell-specific GPCR ligand to construct a double-stranded RNA delivery vector. The vector specifically binds to double-stranded RNA, is unaffected by DNA, and is unaffected by sequence. It exhibits a high broad-spectrum activity, eliminating the need for manual screening of RNA fragments and significantly reducing drug development time. Furthermore, the combined GPCR ligand addresses the issues of poor targeting and incompleteness, and cell-specific GPCRs possess a strong endocytosis effect, ensuring that the RNA-bound SID-1 extracellular domain is internalized by cells to exert its pharmacological effects.

[0022] According to a first aspect of the present invention, a double-stranded RNA delivery vector is provided, comprising an extracellular domain of a SID-1 protein and a GPCR ligand, wherein the GPCR ligand is integrated into the extracellular domain of the SID-1 protein; the amino acid sequence of the extracellular domain of the SID-1 protein is shown in SEQ ID NO: 1.

[0023] Preferably, the GPCR ligand is a hepatocyte GPCR ligand, a muscle cell GPCR ligand, a pancreatic β cell GPCR ligand or a nerve cell GPCR ligand.

[0024] Preferably, the double-stranded RNA delivery vector can be linked to the double-stranded RNA through electrostatic interaction.

[0025] Preferably, the connection sites are R172, A173, K179, K181, K193, K198, R293, K294, K299 and K301 sites of the extracellular domain of the SID-1 protein in the double-stranded RNA delivery vector.

[0026] Preferably, the GPCR ligand is integrated into the extracellular domain of the SID-1 protein through glycosylation modification.

[0027] Preferably, the sites where the GPCR ligand is integrated into the extracellular domain of the SID-1 protein are aspartic acid 27, 44, 54, 73, 123, 136 and 165.

[0028] According to another aspect of the present invention, there is provided a use of any one of the double-stranded RNA delivery vectors for preparing a double-stranded RNA vector for drug delivery.

[0029] Preferably, the drug double-stranded RNA is liver cancer gene inhibitory RNA, hormone polypeptide expression type RNA or blood lipid formation inhibitory RNA.

[0030] Preferably, the length of the drug double-stranded RNA is greater than or equal to 25 bp.

[0031] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0032] (1) The present invention utilizes the extracellular protein domain of the double-stranded RNA binding protein SID-1, which is unique to Caenorhabditis elegans, in combination with a GPCR ligand with strong targeting and in vivo cell specificity to form a novel double-stranded RNA delivery vector. The present invention structurally analyzes the overall conformation of the nematode SID-1 and deeply explores the details of the specific binding of the SID-1 extracellular domain to double-stranded RNA. At the same time, the cell-specific GPCR ligand can express the cell's own glycosylation modification method to bind to specific amino acid residues in the SID-1 extracellular domain, thus making the construction method of the novel RNA delivery vector feasible.

[0033] (2) The extracellular domain of the SID-1 protein of the present invention can specifically bind to double-stranded RNA and is not affected by DNA, thereby avoiding the influence of DNA contamination during RNA extraction or preparation.

[0034] (3) The extracellular domain of the SID-1 protein of the present invention can specifically bind to double-stranded RNA of more than 25 bp and is not affected by the sequence. It has a high broad spectrum and does not require manual screening of RNA fragments, greatly reducing the time for drug development.

[0035] (4) The present invention combines the extracellular domain of the SID-1 protein with a cell-specific GPCR ligand to construct a new type of RNA delivery vector for the application of double-stranded RNA delivery of drugs with strong targeting and comprehensiveness, which solves the problems of poor targeting and incompleteness. Almost all tissues and organs of the human body have their own specific GPCRs, and the mechanism of GPCR endocytosis by target cells is relatively clear, which can ensure that the SID-1 extracellular domain bound to RNA is endocytosed by cells to exert a pharmacodynamic effect. Therefore, GPCR ligands of different cell types can be designed to be integrated into the SID-1 extracellular domain, and RNA delivery vectors specific to different cell types can be obtained.

[0036] (5) The extracellular domain of the SID-1 protein of the present invention is combined with a cell-specific GPCR ligand to construct a new type of RNA delivery vector that does not require a bilayer liposome as a medium for transport and can act directly on the human body. In addition, RNA modification can reduce the human immune response and improve drug efficacy, achieving simple preparation, low cost, and wide-scale use.

Brief Description of the Drawings

[0037] Figure 1 shows the therapeutic mechanism of nucleic acid drugs.

[0038] Figure 2 shows different types of RNA delivery methods and disease treatment types.

[0039] Figure 3 shows different types of RNA nanoparticle carriers.

[0040] Figure 4 shows the liver-targeted delivery technology mediated by N-acetylgalactosamine (GalNAc) modification.

[0041] Figure 5 shows the cell exosome technology.

[0042] Figure 6: A. Molecular sieve peak diagram of SID-1 protein; B. SDS-PAGE results of SID-1 protein molecular sieve at different peak positions; C. Schematic diagram of the incubation of SID-1 protein and 70bp double-stranded RNA at a molar ratio of 1:1.

[0043] Figure 7: A. Electron microscopy density map of SID-1 / double-stranded RNA, the yellow part represents double-stranded RNA; B. Structural model corresponding to the map in Figure A.

[0044] Figure 8: A. Surface potential of SID-1 / dsRNA, with blue representing positive charge, red representing negative charge, and white representing hydrophobicity; B. Structural model corresponding to Figure A, with positively charged amino acid side chains shown in green as stick figures.

[0045] Figure 9: A. Double-stranded RNA bound to the ECD of SID-1. ECDI, ECDII, and double-stranded RNA are indicated in blue, pink, and yellow, respectively; solid squares represent the regions shown in panels B to D. Panels B to D show detailed interactions between double-stranded RNA and the ECD in regions 1 (B), 2 (C), and 3 (D); interacting residues such as R135, K136, and R137, as well as the phosphodiester and ribose side chains, are depicted as stick figures. The density of hypothetical cations in region 2 is represented by a red grid with a 12σ intensity, and magenta spheres represent hypothetical positive ions.

[0046] FIG10 shows the interaction between SID-1 and nucleic acid detected by microscale thermophoresis (MST).

[0047] Figure 11 is a schematic diagram of the seven glycosylation sites in the extracellular domain of SID-1.

[0048] FIG12 is a diagram showing the effect of SID-1 targeted delivery of YFP fluorescent protein inhibitory RNA.

[0049] FIG13 is a negative control diagram showing the results of the SID-1 extracellular domain without ligand integration. [Specific implementation method]

[0050] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0051] Example 1: Preparation method of the novel RNA delivery vector of the present invention

[0052] 1) Clone and construct the SID-1 protein extracellular domain gene plasmid to be expressed;

[0053] 2) Using HEK-293 cells to express the extracellular domain of SID-1 protein;

[0054] 3) Purifying the extracellular domain of the SID-1 protein using protein affinity purification technology; the amino acid sequence of the extracellular domain of the SID-1 protein is shown in SEQ ID NO: 1;

[0055] 4) In vitro binding of the extracellular domain of SID-1 protein to double-stranded RNA and detection of its specificity;

[0056] 5) Using amino acid glycosylation modification to bind the extracellular domain of SID-1 protein to GPCR ligands;

[0057] 6) Validate the efficacy of novel RNA delivery methods using S2 cells or muscle cells from animal models;

[0058] The following are the specific steps:

[0059] 1) Cloning of the sid-1 gene

[0060] Based on the SID-1 coding sequence NM_071971.6 given in the Uniprot database, the following primers were designed:

[0061] First, the sid-1 Forward 1 and sid-1 Reverse 1 primers were used to retrieve the sid-1 gene from the cDNA library according to the touch-down method.

[0062] Table error! There is no text of the specified style in the document. Touch down RCR reaction system

[0063] After adding the above sample reagents, use the PCR instrument to perform the following reaction program:

[0064] Table 3 Touch down RCR reaction procedure

[0065] After the reaction is complete, use the product from the first round as a template and perform a second round of PCR with primers sid-1 Forward 2 and sid-1 Reverse 2 (see Table 4). This aligns the sid-1 gene with the corresponding vector homology arms and is then recovered using the gel extraction kit. The pEG2 vector is also linearized using the corresponding primers for PCR.

[0066] Table 4 PCR reaction system

[0067] Add the sid-1 gene and pEG2 vector linearized in 3 × 50 μL each according to the above system, and perform the following reaction using a PCR instrument:

[0068] Table 5 PCR reaction procedure

[0069] After the reaction, the sample was added to DNA loading buffer and subjected to agarose gel electrophoresis. The gel block corresponding to the molecular weight of the target band, sid-1, was then excised and recovered using the gel recovery kit. Following the PCR reaction, the pEG2 linearized vector was incubated overnight in a 37°C water bath with 1% Dpn I and recovered the next day. After recovery, the plasmid concentration was determined, and homologous recombination reactions were performed.

[0070] Table 6 Homologous recombination reaction system

[0071] After the above reaction, the homologous recombination products were transformed into DH5α competent cells using standard chemical transformation methods and revived. They were then plated onto LB plates containing ampicillin resistance medium and incubated inverted at 37°C overnight. The following day, colony PCR was performed. If a band of the corresponding molecular weight was identified, it was identified as a positive clone, picked, cultured overnight, and sent for testing. Successful sequencing yielded the pEG2-IL2SP-twinstrep-GFP-HRV3C-sid1 plasmid.

[0072] Example 2: Expression and purification of the extracellular domain of SID-1 protein

[0073] The DH5α Escherichia coli containing the previously constructed pEG2-IL2SP-twinstrep-GFP-HRV3C-sid1 plasmid was revived and extracted according to the standard method of the plasmid extraction kit, and then the concentration was measured.

[0074] The specific method is as follows:

[0075] a) HEK293T cell preparation: One day before transfection, seed HEK293T cells at a concentration of 1×105 cells / mL in a 6-well cell culture plate. Add 2 mL of complete medium (DMEM + 10% FBS) and culture overnight in an incubator at 5% CO2 and 37°C. Proceed with the subsequent transfection experiment when the cell adherence density reaches approximately 60%.

[0076] b) Preparation of transfection materials: Coat the cells with PEI 25000 and sid-1 plasmid at a mass ratio of 3:1. Mix 3 μg of plasmid and 9 μg of PEI 25000 in 250 μL of DMEM, mix well, and incubate at 37°C for 30 minutes.

[0077] c) Transfection: Remove complete medium from the 6-well plate and wash once with PBS. Then add 500 μL of the incubated PEI and sid-1 plasmid mixture and mix thoroughly in an "8" pattern. Place the plate in a cell culture incubator and let it stand for 5 hours. After that, replace the plate with complete medium. After 48 hours, observe and photograph the cell fluorescence using a fluorescence microscope. Collect and freeze the cells.

[0078] Then, 2 mL of HEK293T cells were collected and membrane lysed, and GFP green fluorescence was photographed by SDS-PAGE and processed by high-performance liquid chromatography. The specific steps are as follows:

[0079] a) Resuspension: Remove 2 mL of frozen HEK293T cells from a -80°C freezer and resuspend in 150 μL of buffer A (20 mM HEPES, 150 mM NaCl, 2 mM β-mercaptoethanol, pH 7.5, and protease inhibitor cocktail including 0.8 μM pepstatin, 2 μM leupeptin, and 2 μM aprotinin).

[0080] b) Dissolution of membranes: Place 150 μL of the resuspended solution into a 2 mL EP tube and rotate the membrane at 4°C for 1.5 hours. Then, centrifuge at 45,000 rpm for 20 minutes and collect the supernatant.

[0081] c) Gel running: 20 μL of supernatant was added to loading buffer and mixed evenly, and electrophoresed on a 10% polyacrylamide SDS-PAGE gel, and GFP fluorescence was captured using a protein imager.

[0082] d) HPLC: Pre-equilibrate a Superose 6 Increase 10 / 300GL column with buffer B (20 mM HEPES, 150 mM NaCl, 2 mM β-mercaptoethanol). 100 μL of the supernatant after centrifugation of the membrane solution was analyzed by high-performance liquid chromatography (HPLC) for GFP signal detection (excitation wavelength 480 nm, emission wavelength 517 nm).

[0083] Following the above protocol, 200 mL of HEK293F cells were induced for expression, snap-frozen in liquid nitrogen, and stored at -80°C. The frozen cells were removed and treated with buffer A (20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol (β-ME, which has a certain reducing property and reduces cysteine, thereby reducing protein aggregation). The cells were homogenized using a homogenizer and then centrifuged at 36,900 rpm for 40 minutes. The pellet was collected and resuspended in buffer B (buffer A supplemented with 1% DDM (n-Dodecyl-β-D-Maltopyranoside) and 0.2% CHS (cholesteryl hemisuccinate, a cholesterol analog that helps stabilize membrane proteins). The membranes were solubilized at 4°C for 2 hours. Then ultracentrifuge and take the supernatant to hang on the gravity column of pre-equilibrated streptactin medium, and perform affinity chromatography binding through the twinStrepII tag carried on the N-terminus of the SID-1 protein. After the column hanging is completed, use buffer C [buffer A plus 0.03% (w / v) GDN (Glyco-diosgenin), 1mM ATP and 5mM MgCl2 (to help remove the heat shock protein bound to the target protein)] to wash for about 10 column volumes. Then, using buffer C as the base liquid, prepare 5mM desthiobiotin for protein elution. After elution, the solution is concentrated to 1mL through a 100kDa concentrator tube. Use buffer A to pre-equilibrate (buffer A plus 0.007% (w / v) GDN (Glyco-diosgenin) Superose6 Increase 10 / 300GL) molecular sieve column, and then inject the concentrated SID-1 protein into Size exclusion chromatography purification was performed. Size exclusion chromatography produced two peaks: the first 9 mL or so was a protein polymer peak, and the second was the main protein peak, with a peak tip of 14.29 mL. Protein samples near the peak tip were taken for SDS-PAGE. As shown in Figure 2-12B, the SID-1 protein bands were between 100 kDa and 130 kDa, with minimal degradation and a distinct main band, making them suitable for frozen sample preparation and electron microscopy data collection. Therefore, 1 mL of the peak tip was concentrated to 30 uL at a concentration of 6.8 mg / mL for subsequent frozen sample preparation and incubation with double-stranded RNA at a molar ratio of 1:1 (as shown in Figure 6).

[0084] After incubation with double-stranded RNA, in-depth electron microscopic observation revealed that the loop between β7 and β8 in the SID-1 extracellular region is directly involved in the binding of double-stranded RNA. At the same time, the loop between β16 and β17 undergoes a conformational change, which facilitates the binding of double-stranded RNA (Figure 7). Observations revealed that the SID-1 extracellular region displays three strongly positively charged surface regions, resembling a "triangular claw" that firmly grasps the negatively charged double-stranded RNA (Figure 8). Further analysis revealed that in the extracellular region, the loop between the β7 and β8 sheets contains three consecutive positively charged residues, R135, K136, and R137, whose side chains extend into a large groove of the dsRNA, possibly interacting with the phosphate groups of the dsRNA backbone. The protruding loop just below is wedged into the minor groove of dsRNA, becoming a solid platform to support dsRNA binding. At the same time, K43, K4, H166 and R164 in the extracellular region and K193, K198, R293, K294, K299 and K301 insert their positively charged side chains into the minor and major grooves of dsRNA (Figure 9).

[0085] Example 3: Double-stranded RNA binding in vitro

[0086] The interaction between SID-1 and dsRNA was detected by microscale thermophoresis (MST) using a Monolith NT.115 Blue / Red (NanoTemper Technologies) instrument, using the GFP fragment of the SID-1-GFP fusion protein as a site-specific fluorescent marker. Preliminary experiments indicated that a 4.5 nM concentration of purified SID-1 and SID-1 mutants was an appropriate working concentration for this assay. dsRNA (500 bp, 300 bp, 70 bp) and DNA (500 bp) (stock concentration ~2 μM) were diluted in a buffer containing 20 mM HEPES, 150 mM NaCl, 0.007% GDN (w / v), and 2 mM β-ME, pH 7.5, with concentrations ranging from ~50 pM to 1.0 μM. Then, 10 μL of each dsRNA or DNA solution at various concentrations was mixed with 10 μL of SID-1 protein. The mixed sample was incubated at room temperature for 20 minutes, centrifuged at 16000g for 15-30 minutes at 4°C, and loaded into a standard glass capillary (Nanotemper MO-K022). The thermophoresis experiment was performed at 25°C using a Medium MST power supply and an 80% light-emitting diode (LED) power supply. After completing the "capillary scan", the MST curve recorded by each capillary was displayed, and the dissociation constant (Kd) value of each reaction was calculated using MO.Affinity Analysis Software (V2.2.4) (NanoTemper Technologies). For regression analysis, the baseline-corrected normalized fluorescence ΔFnorm expressed as 1 / 1000 (=‰) corresponds to the concentration of dsRNA or DNA. The binding affinity is the average value with standard deviation obtained from at least three independent binding experiments.

[0087] To determine the structural determinants of SID-1's dsRNA recognition, we first used microscale thermophoresis (MST) to test the interaction of synthetic YFP double-stranded RNAs of varying lengths with purified SID-1 protein. Dose-response curves revealed that dsRNA bound to SID-1 in a length-dependent manner, whereas no dsDNA binding was detected (Figure 10), consistent with previous reports that SID-1 specifically recognizes dsRNA rather than dsDNA. MST results revealed that the affinity of 500 bp double-stranded RNA for SID-1 (kd = 1.1 ± 0.3 nM) was approximately 10-fold higher than that of 70 bp double-stranded RNA (kd = 11.2 ± 2.2 nM), likely due to the ability of a longer double-stranded RNA molecule to bind more SID-1 molecules than a shorter one. Furthermore, the fitted Hill coefficient was greater than 1, suggesting a possible synergistic interaction between the double-stranded RNA and SID-1 protein. Our in vitro SID-1 binding results for nucleic acids are consistent with previous double-stranded RNA uptake experiments conducted in S2 cells and the gel retardation binding results of the purified extracellular ECD fraction of SID-1 and double-stranded RNA. SID-1 protein can indeed interact with double-stranded RNA, and the affinity is higher for long-chain double-stranded RNA; there is no obvious interaction with double-stranded DNA.

[0088] The results are shown in Figure 10: A. Agarose gel electrophoresis of double-stranded RNAs of different lengths (70 bp, 300 bp, and 500 bp) and 500 bp double-stranded DNA. B. In vitro MST assay of SID-1 protein binding to double-stranded RNAs and DNAs of different lengths. One-way ANOVA analysis shows the mean ± SEM, with three independent biological replicates.

[0089] Example 4: Glycosylation of specific amino acids in the extracellular domain of SID-1 protein to integrate GPCR ligands

[0090] When the SID-1 extracellular domain is expressed in HEK cells, multiple amino acid residues are glycosylated, as shown in Figure 11. This provides seven possible integration sites for glycosylated GPCR ligands: aspartic acid residues 27, 44, 54, 73, 123, 136, and 165. During cell culture, when a glycosylated GPCR ligand, such as myogenin, is added, the specific amino acid glycosylation during SID-1 expression will utilize the sugar groups of the integrated ligand, allowing purification of the SID-1 extracellular domain containing the integrated GPCR ligand.

[0091] Example 5: Validation of a new method for RNA delivery to specific cells using animal models

[0092] The muscle cells of the model animal Caenorhabditis elegans were used as the specific delivery destination. The fluorescent protein YFP was specifically expressed in the muscle cells of the nematode. The effectiveness of RNA delivery was measured by observing and calculating the fluorescence brightness of the muscle cells. A random double-stranded RNA with the fluorescent protein YFP gene was constructed and bound to the extracellular domain of the SID-1 protein. At the same time, the domain was combined with a GPCR ligand targeted to the muscle cells of the nematode. After the delivery vector was constructed, it was injected into the oral cells of the nematode using microinjection at a volume of 1 μg / μL. The fluorescence intensity was observed and counted after 10 hours. The control group of the experiment was microinjected with a mixture without the SID-1 protein domain. The experimental results are shown in Figure 12. The experimental group expressed a functional SID-1 extracellular structure and combined it with the muscle cell targeting ligand myogenin, which significantly inhibited the expression of the fluorescent protein. Figure 13 is a negative control, expressing the SID-1 extracellular region without the linked ligand, and there was no fluorescence inhibition signal. This shows that the new RNA delivery vector and method of the present invention are very effective.

[0093] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A double-stranded RNA delivery vector, characterized in that: The double-stranded RNA delivery vector comprises an extracellular domain of a SID-1 protein and a GPCR ligand, wherein the GPCR ligand is integrated into the extracellular domain of the SID-1 protein; the amino acid sequence of the extracellular domain of the SID-1 protein is shown in SEQ ID NO:

1.

2. The double-stranded RNA delivery vector according to claim 1, characterized in that The GPCR ligand is a hepatocyte GPCR ligand, a muscle cell GPCR ligand, a pancreatic β cell GPCR ligand or a nerve cell GPCR ligand.

3. The double-stranded RNA delivery vector according to claim 1 or 2, characterized in that The double-stranded RNA delivery vector can be linked to the double-stranded RNA through electrostatic interaction.

4. The double-stranded RNA delivery vector according to claim 3, characterized in that The connection sites are R172, A173, K179, K181, K193, K198, R293, K294, K299 and K301 sites of the extracellular domain of the SID-1 protein in the double-stranded RNA delivery vector.

5. The double-stranded RNA delivery vector according to claim 1, characterized in that The GPCR ligand is integrated into the extracellular domain of the SID-1 protein through glycosylation modification.

6. The double-stranded RNA delivery vector according to claim 5, characterized in that The sites where the GPCR ligand is integrated into the extracellular domain of the SID-1 protein are aspartic acid 27, 44, 54, 73, 123, 136 and 165.

7. Use of the double-stranded RNA delivery vector according to any one of claims 1 to 6 for preparing a double-stranded RNA vector for drug delivery.

8. The use according to claim 7, characterized in that The drug double-stranded RNA is liver cancer gene inhibitory RNA, hormone polypeptide expression type RNA or blood lipid formation inhibitory RNA.

9. The use according to claim 7 or 8, characterized in that The length of the drug double-stranded RNA is greater than or equal to 25 bp.

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