SiRNA taking circHECTD1 as target spot and delivery system, construction method and application thereof
By designing siRNA targeting circHECTD1 and using a lipid nanoparticle delivery system, effective inhibition of the expression of circHECTD1 gene in the brain is achieved, solving the shortcomings of ischemic stroke treatment in the prior art, and significantly alleviating brain damage and neuroinflammatory disease Response, providing new drug methods for treating stroke.
Patent Information
- Application Number
- CN202411599219.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-11
- Publication Date
- 2024-12-20
AI Technical Summary
The existing treatment methods for ischemic stroke are limited, especially the targeted treatment technology for circHECTD1 targets has not been developed, resulting in a narrow treatment time window and poor effect, and new treatment methods are urgently needed.
A siRNA was designed to target circHECTD1 and delivered to the brain through a lipid nanoparticle-based delivery system, administered via nasal route to inhibit gene expression of circHECTD1, combined with mannose modification to improve blood-brain barrier penetration Permeability.
It significantly inhibits the expression of circHECTD1 gene in the brain of mice, relieves brain damage, reduces neuroinflammatory response, and improves the distribution efficiency of siRNA drugs in the brain, and has potential drug application value for the treatment of stroke.
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Figure CN119162185A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a siRNA targeting circHECTD1 and a delivery system, a construction method and an application thereof. Background Art
[0002] Stroke is a neurological disease characterized by high morbidity, high mortality, and high disability rate. It is the main cause of permanent disability in adults worldwide, seriously affecting the quality of life of patients and placing a heavy burden on patients' families and the social economy. Ischemic stroke accounts for 85% of all strokes, with brain damage caused by occlusion of brain blood vessels as the main pathological feature. Currently, clinical treatments for ischemic stroke include drug thrombolysis and mechanical thrombectomy. However, the treatment time window is narrow (only within 4-6 hours after onset), and only a very small number of patients can benefit. Mechanical thrombectomy is the most direct and effective measure, but due to its high requirements for the operating environment, patients often cannot receive timely treatment. Therefore, there is an urgent need to develop new therapeutic drugs that can benefit more patients.
[0003] circRNA is a non-coding RNA produced by reverse splicing of precursor RNA. In recent years, many studies have shown that circRNA can act as a "miRNA sponge" to bind to miRNA, making the latter unable to bind to the target mRNA and blocking its RNA interference effect, thereby regulating the occurrence and development of various diseases. Studies have reported that a variety of circRNAs are involved in the pathological process of acute ischemic stroke. In the brain tissue of tMCAO rats, the expression levels of 283 circRNAs changed, suggesting that circRNAs are related to acute ischemic stroke and may play an important role in the disease process. In the plasma of AIS patients, 3 circRNAs were significantly upregulated, and the combination of the three had good diagnostic ability for AIS. In the peripheral blood mononuclear cells of AIS patients, 13 circRNAs were involved in the complex physiological and pathological processes after ischemia, such as neuroinflammatory and immune responses. The above characteristics suggest that circRNA has the potential to be used as a drug target and biomarker for acute ischemic stroke. However, therapeutic technologies and drugs targeting pathogenic circRNAs have not yet been developed for stroke treatment. Therefore, it is of potential clinical significance to develop precise treatment methods and drugs for stroke using circRNA as a target. Studies have shown that circHECTD1 is significantly overexpressed in biological samples of patients with ischemic stroke, but no drugs targeting circHECTD1 have been developed so far. Summary of the invention
[0004] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:
[0005] A siRNA for inhibiting circHECTD1 gene expression, the siRNA comprising a sense chain and an antisense chain, the sense chain nucleotide sequence comprising a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 1; the nucleotide sequence of SEQ ID NO: 1 is: 5'-GAGCUCCACAAUGGUCAACTT-3'; the antisense chain nucleotide sequence comprising a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 2 is: 5'-GUUGACCAUUGUGGAGCUCTT-3'.
[0006] Furthermore, the modification includes substitution of deoxyribonucleotides at the 3'-end overhang of the sense strand and / or the antisense strand; or addition of deoxyribonucleic acid dTdT or addition of ribonucleotide UU at the 3'-end overhang of the sense strand and / or the antisense strand.
[0007] Furthermore, the modification also includes fluorine substitution modification, methoxy modification or thio backbone modification of the nucleotide.
[0008] A delivery system based on lipid nanoparticles for delivering siRNA, the delivery system comprising the siRNA according to any one of claims 1 to 3 and a lipid component, wherein the nitrogen-phosphorus ratio of the lipid component to the siRNA is (3-10):1; the lipid component comprises DOTAP, charged lipids, DSPC, cholesterol, PEG lipids and mannose-modified PEG lipids, and the molar ratio of the ionizable lipids, DSPC, cholesterol and PEG lipids is (40-65):(5-15):(30-50):(1-5):(0.5-1).
[0009] Furthermore, the ionizable lipid is at least one of DLin-MC3-DMA, NT1-O14B, and SM-102.
[0010] A method for constructing a brain-targeted delivery system based on lipid nanoparticles carrying siRNA, the method comprising the following steps:
[0011] S1. Prepare siRNA sodium citrate solution using sodium citrate buffer;
[0012] S2, taking ionizable lipids, DSPC, cholesterol, PEG lipids and mannose-modified PEGylated lipids, and preparing a lipid mixed solution;
[0013] S3, injecting the lipid mixed solution into the siRNA sodium citrate buffer to obtain an initial LNP solution loaded with siRNA;
[0014] S4. Ultrafiltration is performed to remove ethanol in the LNP initial solution to obtain the lipid nanoparticle-based siRNA delivery system.
[0015] Furthermore, the buffer in step S1 is a sodium citrate buffer, the concentration of the sodium citrate is 25 mM, and the pH of the configured siRNA buffer is 5.0.
[0016] Furthermore, the total lipid concentration in the lipid mixture prepared in step S2 is 8-10 mg / mL.
[0017] Furthermore, the buffer in step S3 is sodium citrate buffer, the volume ratio of the lipid mixture to the buffer is 1:3, and the pH value after adjustment in step S4 is about 7.2 to 7.5.
[0018] The above-mentioned lipid nanoparticle-based siRNA delivery system is used to prepare a pharmaceutical composition for down-regulating circular RNA gene expression.
[0019] Beneficial effects of the present invention:
[0020] 1. The present invention administered siRNA directed against the expression of the circHECTD1 target gene to mice three times via the nasal route, significantly inhibiting the gene expression of circHECTD1 in the peri-infarct area of mice after stroke. Therefore, the delivery system for delivering circHECTD1 provided by the present invention can be used as a circHECTD1 inhibitor, and can be prepared into a drug with one or more pharmaceutically acceptable excipients, and used to treat cardiovascular diseases, neurodegenerative diseases, cancers, autoimmune diseases and other indications related to circHECTD1 gene expression.
[0021] 2. In the mouse ischemic stroke model, the mice were given the drug three times via the nasal route. Within one week after the end of the drug administration, the brain, heart, liver, spleen, lung, and kidney were removed for Western Blot and immunofluorescence staining analysis, and blood was collected from the orbits to prepare serum for ELISA testing. The results showed that the brain-targeted lipid nanoparticle-loaded siRNA drug of the present invention can significantly inhibit the expression of the circHECTD1 gene in the mouse brain, alleviate brain damage after stroke in mice, and reduce brain neuroinflammatory response, and it is statistically significant. The experimental results show that siRNA targeting the expression of the circHECTD1 target gene is expected to be a drug for the treatment of stroke.
[0022] 3. In the mouse ischemic stroke model, intranasal administration of Cy5 si-circHECTD1@LNP and Cy5After si-circHECTD1@MLNP, brain tissue was taken at a series of time points for fluorescence imaging. The results showed that the circHECTD1@MLNP with brain targeting function of the present invention can significantly improve the distribution of siRNA drugs in the mouse brain, indicating that mannose-modified LNP has significant blood-brain barrier penetration function. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0024] Figure 1 Physicochemical characterization of lipid nanoparticles (particle size, potential, coverage)
[0025] Figure 2 To verify the delivery efficiency and cytotoxicity of lipid nanoparticles-encapsulated si-circHECTD1 to mouse cells in vitro, as well as the silencing effect on circ HECTD1;
[0026] Figure 3 To verify the in vivo delivery efficiency of lipid nanoparticle-encapsulated si-circHECTD1 to the mouse brain;
[0027] Figure 4 To verify the effect of lipid nanoparticle-encapsulated si-circHECTD1 on the sensorimotor function of mice after stroke at the in vivo level;
[0028] Figure 5 To detect the regulatory effect of lipid nanoparticle-encapsulated si-circHECTD1 on proinflammatory factors, microglia, astrocytes and neurons in the mouse brain at the in vivo level. DETAILED DESCRIPTION
[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Example
[0031] 1. CircHECTD1 sequence and siRNA design
[0032] circ HECTD1 was sequenced and analyzed to obtain the circ HECTD1 sequence fragment, and the DICER substrate sequence was designed and chemically modified based on the fragment to synthesize the siRNA structure. The specific information is shown below.
[0033] The sense strand of the circHECTD1 target has a nucleotide sequence of 5'-GAGCUCCACAAUGGUCAACTT-3' (SEQ ID NO. 3 in the sequence listing) or a homologous sequence thereof, and the antisense strand has a nucleotide sequence of 5'-GUUGACCAUUGUGGAGCUCTT-3' (SEQ ID NO. 4 in the sequence listing) or a homologous sequence thereof.
[0034] 1. Verify the safety and lysosomal release performance of si-circHECTD1@MLNP at the cellular level.
[0035] 2.1 Preparation process of mLNP-coated si-circHECTD1
[0036] (1) Take si-circHECTD1 and use sodium citrate buffer (pH = 5.0) to prepare a si-circHECTD1 sodium citrate solution with a pH of 5.0, a si-circHECTD1 concentration of 0.2 mg / mL, and a sodium citrate concentration of 25 mM;
[0037] (2) Ionizable lipids, DSPC, cholesterol, and PEG lipids were prepared into an oil phase lipid mixture with a total lipid concentration of 8-10 mg / mL using anhydrous ethanol; 0.1 mL of the oil phase lipid mixture was injected into 0.3 mL of sodium citrate buffer using a 1 mL BD Shurui insulin syringe at a volume ratio of 1:3 to prepare empty lipid nanoparticles (LNP vehicle);
[0038] (3) 0.1 mL of the si-circHECTD1 sodium citrate solution prepared in step (1) was injected into 0.3 mL of the oil phase lipid mixture prepared in step (2) using a 1 mL BD Shurui insulin syringe, and incubated under vortex stirring for 1 minute to obtain the initial solution of lipid nanoparticles loaded with si-circHECTD1 (si-circHECTD1@MLNP). The above steps (1) to (3) can be repeated to obtain more si-circHECTD1@MLNP;
[0039] If anhydrous ethanol is contained in the preparation process, the ethanol in the MLNP is removed by ultrafiltration at a centrifugal force of 13000g using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and the si-circHECTD1 concentration is adjusted to 0.1 mg / mL with denuclease water, and the pH is adjusted to 7.0 with 0.1 M sodium hydroxide or hydrochloric acid solution to obtain a si-circHECTD1@MLNP suspension.
[0040] 2.2. Investigation of biocompatibility, cellular entry and lysosomal release properties of si-circHECTD1@MLNPs at the cellular level
[0041] The MLNP-encapsulated si-circHECTD1 suspension prepared above was taken and the cell viability was detected by CCK8 assay.
[0042] Take the MLNP encapsulated Cy5 si-circHECTD1 suspension, and confocal microscopy was used to observe the cellular entry and lysosomal escape of nucleic acid drugs.
[0043] The results are as follows Figure 2 As shown in the figure, after MLNP is taken up by cells, it can achieve efficient lysosomal escape.
[0044] 3. Detection of brain delivery efficiency of si-circHECTD1@MLNP
[0045] C57BL / 6 mice were divided into 2 groups: Cy5 si-circHECTD1@LNP group and Cy5 In the si-circHECTD1@MLNP group, there were 5 mice in each group, and the C57BL / 6 mice were nasally administered with a dose of 0.06 mg / kg. 4, 12, and 24 hours after injection, the mice were anesthetized with isoflurane and the fluorescence intensity after MLNP and LNP delivery was observed with an IVIS Spectrum imaging instrument. After imaging, the mice were killed and the organs (brain, heart, liver, spleen, lung, and kidney) were taken for ex vivo fluorescence imaging again.
[0046] Depend on Figure 3 It can be seen that compared with LNP, MLNP has a stronger ability to penetrate the blood-brain barrier and can make more Cy5 si-circHECTD1 was delivered to the brain.
[0047] 4. In vivo verification of the therapeutic effect of si-circHECTD1@MLNP on ischemic stroke
[0048] First, the mice included in the behavioral experiment were trained, and then the mice were modeled with ischemic stroke tMCAO and randomly divided into groups. The C57BL / 6 stroke model mice were divided into 4 groups (12 mice in each group), namely sham group, tMCAO+empty MLNP group, and tMCAO+si-circHECTD1@MLNP group. The frequency of administration was once a day for a total of three times. After the treatment, behavioral tests were performed to verify the therapeutic effect of si-circHECTD1@MLNP on ischemic stroke. The specific behavioral test items include: cylinder test, grid-walking test, and adhesive removal test.
[0049] The results are as follows Figure 4-5 shown.
[0050] As can be seen from the figure, compared with the mice in the group that were not given si-circHECTD1@MLNP after modeling, si-circHECTD1@MLNP significantly improved the sensory and motor function of mice after stroke in the cylinder, stepping in the air and adhesion removal experiments, and had a significant regulatory effect on the pro-inflammatory factors and microglial phenotype in the mouse brain, and was statistically significant.
[0051] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0052] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. A siRNA for inhibiting circHECTD1 gene expression, characterized in that: The siRNA comprises a sense chain and an antisense chain, wherein the nucleotide sequence of the sense chain includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:1; the nucleotide sequence of SEQ ID NO:1 is: 5'-GAGCUCCACAAUGGUCAACTT-3'; the nucleotide sequence of the antisense chain includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:2, and the nucleotide sequence of SEQ ID NO:2 is: 5'-GUUGACCAUUGUGGAGCUCTT-3'.
2. The siRNA for inhibiting circHECTD1 gene expression according to claim 1, characterized in that: The modification includes substitution of deoxyribonucleotides at the 3'-end overhang of the sense strand and / or the antisense strand; or addition of deoxyribonucleic acid dTdT or addition of ribonucleotide UU at the 3'-end overhang of the sense strand and / or the antisense strand.
3. The siRNA for inhibiting circHECTD1 gene expression according to claim 1, characterized in that: The modification also includes fluorine substitution modification, methoxy modification or thio backbone modification of the nucleotide.
4. A brain-targeted delivery system based on lipid nanoparticles loaded with siRNA, characterized in that: The delivery system comprises the siRNA described in any one of claims 1 to 3 and a lipid component, wherein the nitrogen-phosphorus ratio of the lipid component to the siRNA is (3 to 10):1; the composition of the lipid component comprises DOTAP, charged lipids, DSPC, cholesterol, PEG lipids and mannose-modified PEG lipids, and the molar ratio of the ionizable lipids, DSPC, cholesterol, PEG lipids and mannose-modified PEG lipids is (40 to 65):(5 to 15):(30 to 50):(1 to 5):(0.5 to 1).
5. A lipid nanoparticle-based siRNA delivery system according to claim 4, characterized in that: The ionizable lipid is at least one of DLin-MC3-DMA, NT1-O14B, and SM-102.
6. A method for constructing a lipid nanoparticle-based siRNA delivery system as claimed in claim 4 or 5, characterized in that: The construction method comprises the following steps: S1. Prepare siRNA sodium citrate solution using sodium citrate buffer; S2, taking ionizable lipids, DSPC, cholesterol, PEG lipids and mannose-modified PEG lipids, and preparing a lipid mixed solution; S3, injecting the lipid mixed solution into the siRNA sodium citrate buffer to obtain an initial LNP solution loaded with siRNA; S4. Ultrafiltration is performed to remove ethanol in the LNP initial solution to obtain the brain-targeted delivery system based on lipid nanoparticles carrying siRNA.
7. The method for constructing a delivery system based on lipid nanoparticles carrying siRNA according to claim 6, characterized in that: The buffer in step S1 is a sodium citrate buffer, the concentration of the sodium citrate is 25 mM, and the pH of the configured siRNA buffer is 5.
0.
8. The method for constructing a lipid nanoparticle-based siRNA delivery system according to claim 6, characterized in that: The total lipid concentration in the lipid mixture prepared in step S2 is 8-10 mg / mL.
9. According to the method for constructing a delivery system based on lipid nanoparticles for siRNA delivery in claim 6, the buffer in step S3 is sodium citrate buffer, the volume ratio of the lipid mixture to the buffer is 1:3, and the adjusted pH value in step S4 is about 7.2 to 7.
5.
10. A brain-targeted delivery system based on lipid nanoparticles loaded with siRNA as claimed in claim 4 or 5, used for preparing a pharmaceutical composition for down-regulating circular RNA gene expression.