SiRNA composition for treating Alzheimer's disease

Abstract

The invention belongs to the technical field of biological medicine, and relates to a method for preparing PAPA / siRNA nanoparticles by adopting a delivery carrier PAH-AM-PEG-ApoE (159-167) 2 (PAPA) modified by apolipoprotein E (ApoE) peptide through an electrostatic binding self-assembly method by designing and screening a siRNA sequence aiming at beta-site amyloid precursor protein cutting enzyme 1 (BACE1) and glycogen synthase kinase-3beta (GSK3beta) and adopting a delivery carrier PAH-AM-PEG-ApoE (159-167) 2 (PAPA) modified by ApoE peptide. The siRNA nano delivery system constructed by the invention aims to explore a new way for AD treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to siRNA compositions and their application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD), a neurodegenerative disease with complex etiologies, cannot be effectively treated with single drugs in current clinical practice. Furthermore, these drugs generally have poor targeting and specificity, and pose safety concerns. In recent years, small interfering RNA (siRNA) has gradually become a research hotspot in the field of disease treatment due to its highly efficient gene silencing ability. However, the presence of the blood-brain barrier makes it difficult for siRNA molecules to be delivered to the brain, severely restricting the application and progress of siRNA in the treatment of AD and other central nervous system diseases. To address the problems of single-drug therapy, lack of targeting and specificity, poor stability of siRNA, and the blood-brain barrier hindering drug delivery to the brain, new and effective treatment strategies need to be explored. Summary of the Invention

[0003] Based on this, the present invention uses a novel polymer carrier PAH-AM-PEG4-ApoE(159-167)2(PAPA) to load siRNA in order to construct an siRNA nanodelivery system for the treatment of Alzheimer's disease.

[0004] This invention first designs and screens siRNA sequences targeting β-amyloid precursor protein cleavage enzyme 1 (BACE1) and glycogen synthase kinase-3β (GSK3β). Using a delivery vector PAH-AM-PEG-ApoE(159-167)2 (PAPA) modified with apolipoprotein E (ApoE) peptide, PAPA / siRNA nanoparticles are prepared via electrostatic self-assembly. This invention successfully constructs a novel brain-targeting siRNA delivery system. The prepared nanoparticles exhibit good uniformity, effectively improving the stability of the siRNA.

[0005] In a first aspect, the present invention provides an siRNA composition comprising BACE1-siRNA and GSK3β-siRNA, wherein the nucleic acid sequence of the BACE1 siRNA is shown below:

[0006] The positive-strand nucleic acid sequence is SEQ ID NO:1:5'-CGGACAAGUUCUUCAUCAATT-3'.

[0007] The antisense strand nucleic acid sequence is SEQ ID NO:2:5'-UUGAUGAAGAACUUGUCCGTT-3'; the nucleic acid sequence of the GSK3βsiRNA is shown below:

[0008] The positive strand nucleic acid sequence is SEQ ID NO:3:5'-AGAAAGUUCUACAGGACAATT-3', and the antisense strand nucleic acid sequence is SEQ ID NO:4:5'-UUGUCCUGUAGAACUUUCUTT-3'.

[0009] In some embodiments, the mass ratio of BACE1-siRNA to GSK3β-siRNA is 1:1 to 2, preferably 1:1.

[0010] In some embodiments, the siRNA composition is used to treat Alzheimer's disease.

[0011] In a second aspect, the present invention provides an siRNA nanodelivery system, the nanodelivery system comprising any composition of the first aspect and PAH-AM-PEG4-ApoE(159-167)2.

[0012] In some embodiments, the structure of PAH-AM-PEG4-ApoE(159-167)2 is as follows:

[0013]

[0014] Where x = 15-40, y = 20-30, z = 35-50, l = 10-20, m = 25-35, p = 3-10, x+y+z+l+m+p = 160; the ApoE(159-167)2peptide sequence is SEQ NO:5: LRKLRKRLLLRKLRKRLLC, and the ApoE(159-167)2 is linked to the thiol group on maleamide through the thiol group of cysteine.

[0015] In some embodiments, the mass ratio of the PAH-AM-PEG4-ApoE(159-167)2 and siRNA composition is 1:1 to 5:1; preferably 2:1.

[0016] In some embodiments, the nanodelivery system is prepared as follows: A tube containing 1 OD of siRNA powder is placed in a centrifuge tube and centrifuged at 3000 rpm for 3 min to allow the powder to settle to the bottom of the tube. Then, 125 μL of DEPC water is added to the centrifuge tube and thoroughly mixed by vortexing to ensure complete dissolution, thereby preparing a 20 μM siRNA stock solution for later use. 1 mg of PAH-AM-PEG4-ApoE (PAPA) polymer is weighed and dissolved in 1 mL of DEPC water, ensuring complete dissolution by vortexing. The resulting solution is filtered through a 0.22 μm polyethersulfone filter. Subsequently, the PAPA polymer solution is mixed with the prepared siRNA solution at a specific mass ratio and incubated at room temperature for 30 min.

[0017] In some embodiments, the molecular weight of the PEG fragment is 400–500 Da.

[0018] In some implementations, the nanodelivery system is used to treat Alzheimer's disease.

[0019] Thirdly, the present invention provides a pharmaceutical composition comprising any composition of the first aspect or any nanodelivery system of the second aspect and a pharmaceutically acceptable carrier.

[0020] Fourthly, the present invention provides the use of any composition of the first aspect or any nanodelivery system of the second aspect in the preparation of a medicament for treating Alzheimer's disease.

[0021] Beneficial effects

[0022] This study first designed and screened siRNA sequences targeting β-amyloid precursor protein cleavage enzyme 1 (BACE1) and glycogen synthase kinase-3β (GSK3β). PAPA / siRNA nanoparticles were prepared using a delivery vector PAH-AM-PEG-ApoE(159-167)2 (PAPA) modified with apolipoprotein E (ApoE) peptide, with a carrier-to-siRNA mass ratio of 2:1 and a BACE1-siRNA to GSK3β-siRNA ratio of 0.5:0.5. Gel electrophoresis experiments showed that the siRNA could be completely bound when the delivery vector-to-siRNA mass ratio was 2:1. Overall, under the same total siRNA dose (100 nM), compared with the single siRNA group (100 nM), the combined application of two siRNA sequences (total siRNA dose of 100 nM, namely 50 nM siBACE1 and 50 nM siGSK3β) can synergistically reduce the expression levels of two target genes and proteins, and its knockdown effect is better than that of the single group, while not having an adverse effect on the myelin regeneration process. Attached Figure Description

[0023] Figure 1 PAPA NMR Spectroscopy

[0024] Figure 2 Characterization of particle size, polydispersity index, and potential of nanoparticles of the present invention with different mass ratios.

[0025] Figure 3 In vitro release results of naked siRNA and the nanoparticles of this invention

[0026] Figure 4 Anti-enzymatic degradation effect of naked siRNA and nanoparticles of the present invention

[0027] Figure 5 Serum stability of the nanoparticles of this invention

[0028] Figure 6 Gene level results of Neuro-2a cells treated with nanoparticles of this invention (n=3)

[0029] Figure 7 Results of target protein and internal control protein bands in Neuro-2a cells treated with nanoparticles of this invention (n=3) Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of this invention clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Unless otherwise specified, the reagents or instruments used are all commercially available conventional products.

[0031] I. Example 1: Preparation and Characterization of Nanoparticles

[0032] 1.1 Preparation of Nanoparticles: The siRNA sequence in this study is as follows (the supplier of the siRNA sequence is General Biotechnology (Anhui) Co., Ltd.):

[0033] BACE1 siRNA:

[0034] The positive strand nucleic acid sequence is SEQ ID NO:1:5'-CGGACAAGUUCUUCAUCAATT-3', and the antisense strand nucleic acid sequence is SEQ ID NO:2:5'-UUGAUGAAGAACUUGUCCGTT-3';

[0035] GSK3βsiRNA:

[0036] The positive strand nucleic acid sequence is SEQ ID NO:3:5'-AGAAAGUUCUACAGGACAATT-3', and the antisense strand nucleic acid sequence is SEQ ID NO:4:5'-UUGUCCUGUAGAACUUUCUTT-3'.

[0037] This invention uses ApoE peptide-modified maleimide polyethylene glycol to replace polyallylamine hydrochloride PAH-AM-PEG4-ApoE(159-167)2, i.e., PAPA, as a carrier to co-load two siRNA drugs.

[0038] 1.1.1 Preparation of PAH-AM-PEG4

[0039] The structural formula of PAH-AM is shown below:

[0040]

[0041] The structural formula of PAH-AM-PEG4 is shown below:

[0042]

[0043] The molecular weight of PEG is 345.35.

[0044] The peptide sequence of ApoE(159-167)2 is shown in SEQ ID NO:5:

[0045] LRKLRKRLLLRKLRKRLLC.

[0046] The synthesis method of PAH-AM-PEG, and the specific reaction steps are as follows:

[0047] (1) Weigh 5g of PAH-AM into a 500mL glass beaker, add 60mL of MeOH to dissolve it, and then transfer the solution to a 250mL four-necked jacketed bottle. Rinse the beaker with 15mL of MeOH and add it to the jacketed bottle. Add 1.405g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride (DMTMM, CAS:3945-69-5) to the jacketed bottle and stir to dissolve. Weigh 1.538 g of Mal-PEG4-COOH (molecular weight: 400.47, of which PEG molecular weight is 345.35) into a 10 mL centrifuge tube, add 6 mL of MeOH to dissolve, and add the Mal-PEG4-COOH solution dropwise to the PAH-AM solution at a rate of not less than 6 mL / min under normal stirring conditions. Rinse with 9 mL of MeOH and add the solution. Add 250 μL of 6N HCl and stir until homogeneous. Keep the jacketed bottle connected to a low-temperature circulating bath (set to 3-5℃, circulating medium is ethanol) until the temperature reaches 3-5℃, then add 152 mg of triethylamine. Stir the reaction at 3-5℃ for 22 h, then stop the reaction.

[0048] (2) After the reaction stops, add 3 mL of 6N HCl and stir until homogeneous. Transfer the reaction system to a 250 mL round-bottom flask, remove the solvent by rotary evaporation and dry under vacuum. Crush the dried sample into a fine powder, add 100 mL of acetonitrile and stir for 10 min. Transfer to a suitable centrifuge tube and centrifuge for 3 min (10000 r / min). Discard the supernatant. Wash the precipitate with acetonitrile 3-8 times. Dry the precipitate under vacuum overnight to obtain a yellow solid, which is the crude product of maleimide polyethylene glycol substituted polyallylamine hydrochloride (PAH-AM-PEG).

[0049] (3) Dissolve the crude product in 50 mL MeOH. Dialyze the solution in dilute hydrochloric acid aqueous solution (3 mL 6N HCl, 4.5 L H2O) for 48 h (with 6 water changes) using a dialysis bag with a molecular weight cutoff of 8000 Da. Then, filter the liquid in the dialysis bag through a 0.45 μm PES syringe filter (PES, 0.45 μm, 25 mm) into a 1 L single-necked flask. Rotate and freeze the flask in a dry ice ethanol bath until no liquid flow occurs. Then freeze-dry the flask until no ice is present to obtain a light yellow, fluffy, purified PAH-AM-PEG4 product.

[0050] The synthesis reaction equation is as follows:

[0051]

[0052] 1.1.2 Preparation of PAH-AM-PEG-ApoE (PAPA)

[0053] The cationic polymer-targeted nanomaterial peptide-grafted polyallylamine hydrochloride peptide derivative PAH-AM-PEG-ApoE is obtained by reacting the maleimide group on PAH-AM-PEG with the thiol group on the peptide protein ApoE, thus grafting ApoE onto PAH-AM-PEG. The specific reaction steps for the synthesis of PAH-AM-PEG-ApoE are as follows:

[0054] Weigh 3.0 g of PAH-AM-PEG4 into a 100 mL round-bottom flask and dissolve it in 20 mL of MeOH. Weigh 3.342 g of ApoE into a 100 mL beaker, dissolve it in 12.5 mL of PBS by stirring, and then add 12.5 mL of MeOH and mix well. While stirring, add the ApoE solution to the PAH-AM-PEG4 solution at a rate of 12 mL / min. Rinse the beaker with 5 mL of MeOH and add it to the solution. Stir the reaction at room temperature for 43 h, and then stop the reaction.

[0055] After the reaction stopped, the reaction system was transferred to an 8000 Da dialysis bag and dialyzed in pure water for 48 h (with 6 water changes). The liquid in the dialysis bag was then filtered through a 0.45 μm needle filter (PES, 0.45 μm, 25 mm) into a 1 L single-necked flask. The flask was then frozen in a dry ice ethanol bath until no liquid flow occurred. Finally, it was freeze-dried in a freeze dryer until no ice was present, yielding a pale yellow, fluffy, purified polypeptide grafted with polyallylamine hydrochloride derivative (PAH-AM-PEG-ApoE(159-167)2), which is PAPA.

[0056] The synthesis reaction equation is as follows:

[0057]

[0058] Nuclear magnetic resonance characterization such as Figure 1 As shown.

[0059] 1.1.3 Preparation of Nanoparticles

[0060] A tube containing 1 OD of siRNA powder was placed in a centrifuge tube and centrifuged at 3000 rpm for 3 min to allow the powder to settle to the bottom of the tube. Then, 125 μL of DEPC water was added to the centrifuge tube and vortexed to thoroughly dissolve the powder, thus preparing a 20 μM siRNA stock solution for later use. 1 mg of PAH-AM-PEG4-ApoE (PAPA) polymer was weighed and dissolved in 1 mL of DEPC water, ensuring complete dissolution by vortexing. The resulting solution was filtered through a 0.22 μm polyethersulfone filter. Subsequently, the PAPA polymer solution was mixed with the prepared siRNA solution at a specific mass ratio and incubated at room temperature for 30 min.

[0061] 1.2 Characterization of nanoparticles

[0062] Nanoparticles were precisely prepared according to a polymer-siRNA mass ratio of 2:1 (all nanoparticles used in subsequent experiments were based on this mass ratio). Key physical parameters of the nanoparticles were characterized using a nanoparticle size potentiometry analyzer. Figure 2 As shown, when the mass ratio reaches 2, the average particle size of the nanoparticles is approximately 135 nm, the Zeta potential is approximately +23 mV, and the PDI is at its lowest level. This indicates that the nanoparticles possess excellent dispersibility and uniformity at this point, effectively suppressing particle aggregation and thus maintaining good physical stability in the solution system.

[0063] II. Example 2: In vitro dialysis experiment and stability evaluation experiment of nanoparticles

[0064] 2.1 In vitro release characteristics of siRNA in nanoparticles of the present invention

[0065] This study used phosphate buffer (pH 7.4) as a simulated body fluid medium to conduct in vitro dialysis experiments, aiming to simulate the release behavior of nanoparticles in vivo. The experimental groups were Free FAM-siRNA (FAM-labeled free siRNA) and PAPA / FAM-siRNA NPs (FAM-labeled siRNA nanoparticles). FAM is a green fluorescent dye specifically for siRNA (FAM is attached to the 5' end of the siRNA using a phosphate ester bond to form 5'-FAM-labeled siRNA, which is then purified by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to remove unreacted raw materials). During the experiment, 2 mL of each sample containing 200 nM siRNA was placed in a dialysis bag with a molecular weight cutoff of 15000 Da at room temperature. The dialysis bag was then placed in a container containing 20 mL of pH 7.4 PBS buffer for dialysis. During dialysis, 100 μL samples were collected from the external medium of the dialysis bag at different time points (0h, 1h, 2h, 4h, 8h, 12h, 24h). The fluorescence intensity of FAM-siRNA and PAPA / FAM-siRNA in the samples was measured using an ELISA reader to detect the release of siRNA from the nanoparticles, and an in vitro release curve of the nanoparticles was plotted. The cumulative release rate of siRNA at each sampling point was calculated using the following formula: Cumulative release rate = (A1-A0) / (A2-A0)×100%. Where A0 represents the fluorescence intensity of the dialysate at the start of dialysis, A1 represents the fluorescence intensity of the dialysate at the sampling time point, and A2 represents the total fluorescence intensity after complete release of FAM-siRNA or PAPA / FAM-siRNA from the nanocomposite. The experimental results are shown in the cumulative release results, such as... Figure 3 At 4 hours, the free siRNA was almost completely released, and its release amount was about 1.5 times that of the nanoparticles. The release rate of siRNA in the siRNA nanoparticle group was significantly slower than that in the free siRNA group, and it took until 12 hours to reach nearly 100% cumulative release, which was about 3 times longer than that in the free siRNA group.

[0066] 2.2 Stability assessment of nanoparticles under enzyme conditions

[0067] Because siRNA is readily degraded and loses its activity by abundant nucleases in vivo, this study focused on investigating the protective efficacy of PAPA against siRNA under enzymatic conditions. The experimental groups consisted of a Free siRNA group and a PAPA / siRNA NPs group, with siRNA concentrations of 0.25 μg / μl in both groups. The experimental groups were co-incubated with 0.5 mg / ml ribonuclease A (RNase A), with the Free siRNA group incubated for 0 and 15 min, and the PAPA / siRNA NPs group incubated for 1 h, 2 h, 4 h, and 8 h, respectively. After the corresponding incubation times, the samples were co-incubated with 12.5 IU / μL heparin sodium for 10 min. Subsequently, 1.5% agarose gels were prepared, and the samples were thoroughly mixed with 6× DNA loading buffer and accurately pipetted into the gel wells (10 μl per well). The electrophoresis parameters were set to 100V and 25min for detection. After electrophoresis, the gel bands were observed using a blue light gel cutter, and gel imaging was performed using a molecular imaging system. The experimental results are as follows: Figure 4 As shown, free siRNA was completely degraded in just 15 minutes after contact with RNase A enzyme; siRNA nanoparticles still had siRNA fluorescent bands after co-incubation for up to 8 hours, which fully proves that the siRNA was not completely degraded at this time.

[0068] 2.3 Stability assessment of nanoparticles in serum environment

[0069] This study focused on investigating the protective efficacy of PAPA against siRNA in 100% fetal bovine serum (FBS). The experimental groups consisted of a free siRNA group and a PAPA / siRNA NPs group, with siRNA concentrations of 0.16 μg / μl in both groups. The free siRNA group served as a baseline reference, while the PAPA / siRNA NPs group was used to investigate the stability changes of siRNA nanoparticles after co-incubation with 100% FBS at different time points. After the corresponding incubation time, the samples in the experimental groups were co-incubated with 12.5 IU / μL heparin sodium for 10 min to simulate possible in vivo interactions. Subsequently, 1.5% agarose gels were prepared, and the samples were thoroughly mixed with 6× DNA loading buffer and accurately loaded into the gel wells using a pipette (10 μl per well). Electrophoresis parameters were set to 100 V for 25 min, and electrophoresis results were obtained. After electrophoresis, the gel bands were observed using a blue light gel cutter, and gel imaging was performed using a molecular imaging system. The experimental results are as follows: Figure 5As shown: siRNA fluorescent bands were present at each of the above time points, and the nanoparticles continuously released siRNA within 72 hours without complete degradation. This indicates that the prepared nanoparticles have good stability in 100% fetal bovine serum and can enhance the stability of siRNA in body fluids. III. Example 3: Verification of the gene and protein silencing effect of nanoparticles.

[0070] 3.1 Verification of gene silencing effect at the nanoparticle level

[0071] Neuro-2a cells were seeded in 12-well plates (5 × 10⁶ cells per well). 5 Add 1 mL of culture medium to each well and incubate overnight. Neuro-2a cells were treated according to the following experimental groups: PBS blank control group (Group A), PAPA / siScr NPs negative control group (Group B), PAPA / siBACE1 NPs group (Group C), PAPA / siGSK3β NPs (Group D), PAPA / (siBACE1+siGSK3β) NPs (Group E, siBACE1+siGSK3β are encapsulated together in PAPA vector), and a mixture of PAPA / siBACE1 NPs and PAPA / siGSK3β NPs (Group F). The total siRNA content in all groups was 100 nM, with the siRNA ratio in groups E and F being siBACE1:siGSK3β = 50 nM:50 nM. Cells were incubated in fresh culture medium containing the above samples and cultured and treated according to the manufacturer's instructions. Total RNA was extracted using a total RNA extraction kit. Reverse transcription was performed using PrimeScript. TM cDNA was synthesized using the RT reagent kit with gDNA Eraser, and qPCR was performed. Premix Ex Taq gene expression detection protocol. Melting curve analysis confirms the specificity of PCR products, identifying the presence of impurities and primer dimers. The Ct values ​​of each sample are used to calculate the relative mRNA expression level of each sample using formula 2-ΔΔCt.

[0072] mRNA silencing results are as follows Figure 6As shown, gene-level analysis revealed that compared to group A, the expression level of BACE1 mRNA in group C cells was significantly reduced by approximately 50%, and the expression level of GSK3β mRNA in group D cells was significantly reduced by approximately 64%. In group E cells, the expression levels of BACE1 mRNA and GSK3β mRNA were significantly reduced by approximately 60% and 65%, respectively. In group F cells, the expression levels of BACE1 mRNA and GSK3β mRNA were significantly reduced by approximately 55% and 62%, respectively. However, the expression levels of the target genes in group B showed no significant change. Therefore, under the same total siRNA dose (100 nM), compared to the single siRNA group (100 nM), the combined application of two siRNA sequences (total siRNA dose of 100 nM, consisting of 50 nM siBACE1 and 50 nM siGSK3β) synergistically reduced the expression levels of both target genes, and its knockdown effect was superior to the single group. Thus, the combination of siBACE1 and siGSK3β can produce a synergistic effect.

[0073] 3.2 Verification of the silencing effect of nanoparticles at the protein level

[0074] Neuro-2a cells were seeded in 12-well plates (5 × 10⁶ cells per well). 5 Cells were cultured overnight in 1 mL medium per well. Neuro-2a cells were treated according to the following experimental groups: PBS blank control group (Group A), PAPA / siScr NPs negative control group (Group B), PAPA / siBACE1 NPs group (Group C), PAPA / siGSK3β NPs (Group D), PAPA / (siBACE1+siGSK3β) NPs (Group E), and a mixture of PAPA / siBACE1 NPs and PAPA / siGSK3β NPs (Group F). The siRNA content in all groups was 100 nM, with the siRNA ratio in groups E and F being siBACE1:siGSK3β = 50 nM:50 nM. Cells were incubated in fresh medium containing the above samples and cultured and treated according to the manufacturer's instructions. The total protein concentration in the supernatant was determined using a BCA protein assay kit. SDS-PAGE electrophoresis was then performed, with the stacking gel at 80V for 30 min and the separating gel at 120V for 90 min. After electrophoresis, the proteins were transferred to a PVDF membrane (0.22 μm), blocked with 5% skim milk powder, and incubated with primary and secondary antibodies. Then, chemiluminescence imaging was performed using Tanon ECL to compare the expression levels of BACE1, GSK3β, P-tau, and MBP proteins among the groups. The samples were standardized using β-actin protein.

[0075] Protein inhibition such as Figure 7At the protein level, using β-actin as an internal control, compared with group A, the BACE1 protein levels in groups C, E, and F were significantly reduced; the GSK3β protein levels in groups D, E, and F were significantly reduced. In group B, the expression levels of BACE1, GSK3β, P-tau, and MBP proteins were not significantly reduced, while the P-tau protein levels in groups C, D, E, and F were significantly reduced, with the knockdown rates in groups E and F being higher than those in groups C and D. Meanwhile, the MBP protein levels in groups C, D, E, and F were unaffected and similar to the blank control PBS group.

[0076] Besides target gene proteins, MBP refers to myelin basic protein, whose main function is to maintain the stability of myelin structure and function. When myelin is damaged, MBP can be released into cerebrospinal fluid or blood, and its concentration changes can serve as a biomarker for assessing the degree of damage to the central nervous system. The lack of BACE1 impairs the myelin regeneration process, thereby affecting cognitive function. Therefore, the above results fully demonstrate that the siBACE1 and siGSK3β sequences used in this invention possess specific gene silencing activity. Overall, under the same total siRNA dose (100 nM), compared with the single siRNA group (100 nM), the combined application of the two siRNA sequences (total siRNA dose of 100 nM, namely 50 nM siBACE1 and 50 nM siGSK3β) synergistically reduces the expression levels of the two target genes and proteins, and its knockdown effect is better than that of the single group, without adversely affecting the myelin regeneration process.

[0077] Regarding sequences: If there is a conflict between the sequence list and the specific sequence recorded in the instruction manual, the sequence actually recorded in the instruction manual shall prevail.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A siRNA composition, wherein the siRNA comprises BACE1-siRNA and GSK3β-siRNA, and the nucleic acid sequence of the BACE1 siRNA is shown below: The positive-strand nucleic acid sequence is SEQ ID NO:1:5'-CGGACAAGUUCUUCAUCAATT-3'. The antisense strand nucleic acid sequence is SEQ ID NO:2:5'-UUGAUGAAGAACUUGUCCGTT-3'; the nucleic acid sequence of the GSK3βsiRNA is shown below: The positive-strand nucleic acid sequence is SEQ ID NO:3:5'-AGAAAGUUCUACAGGACAATT-3'. The antisense strand nucleic acid sequence is SEQ ID NO:4:5'-UUGUCCUGUAGAACUUUCUTT-3'.

2. The siRNA composition according to claim 1, wherein the mass ratio of BACE1-siRNA to GSK3β-siRNA is 1:1 to 2, preferably 1:

1.

3. The siRNA composition of claim 1, for the treatment of Alzheimer's disease.

4. An siRNA nanodelivery system, wherein the nanodelivery system comprises any one of the compositions of claims 1-3 and PAH-AM-PEG4-ApoE(159-167)2.

5. The nanodelivery system according to claim 4, wherein the mass ratio of the PAH-AM-PEG4-ApoE(159-167)2 and siRNA composition is 1:1 to 5:1; preferably 2:

1.

6. The nanodelivery system according to any one of claims 4-5, wherein the preparation method of the nanodelivery system is as follows: a tube containing 1D of siRNA powder is placed in a centrifuge tube and centrifuged at 3000 rpm for 3 min to allow the powder to precipitate to the bottom of the tube; then, 125 μL of DEPC water is added to the centrifuge tube and thoroughly mixed by vortexing to completely dissolve the powder, thereby preparing a 20 μM siRNA storage solution for later use; 1 mg of PAH-AM-PEG4-ApoE (PAPA) polymer is weighed and dissolved in 1 mL of DEPC water, and the polymer is completely dissolved by vortexing; the resulting solution is filtered through a 0.22 μm polyethersulfone filter membrane; then, the PAPA polymer solution is mixed with the siRNA solution prepared above in a specific mass ratio and incubated at room temperature for 30 min to obtain the final product.

7. The nanodelivery system according to any one of claims 4-6, wherein the nanodelivery system is used to treat Alzheimer's disease.

8. A pharmaceutical composition comprising any one of the compositions of claims 1-3 or any one of claims 4-7 and a pharmaceutically acceptable carrier.

9. Use of any composition of claims 1-3 or the nanodelivery system of any one of claims 4-7 in the preparation of a medicament for treating Alzheimer's disease.

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