CN116688159A - Pharmaceutical composition for treating Alzheimer's disease based on mRNA technology and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of treating Alzheimer's disease, in particular to a pharmaceutical composition and a preparation method for treating Alzheimer's disease based on mRNA technology. The pharmaceutical composition includes lipid nanoparticles and auxiliary materials, and the lipid nanoparticles include a lipid phase and a solution phase. A pharmaceutical composition for treating Alzheimer's disease based on mRNA technology of the present invention, the pharmaceutical composition delivers Olig2-loaded messenger RNA to OPCs, so that OPCs transiently overexpress Olig2, and at the same time, LNPs modified by monoclonal antibody CD140a can effectively Targeted delivery of Olig2 mRNA to target cells CD140a ⁺ OPCs can promote the rapid differentiation and maturation of endogenous OPCs and promote the remyelination of Alzheimer's disease mice. The lipid nanoparticles are more targeted and effective, and can effectively improve the therapeutic effect of Alzheimer's disease.

Description

A pharmaceutical composition for treating Alzheimer's disease based on mRNA technology and its preparation method

technical field

The invention relates to the technical field of treating Alzheimer's disease, in particular to a pharmaceutical composition and a preparation method for treating Alzheimer's disease based on mRNA technology.

Background technique

Myelin sheath is the main component of white matter in the central nervous system and plays an important role in the transmission of nerve signals and communication of information between different regions of the brain. Myelin lesions are the pathological basis of Alzheimer's disease (AD), which can lead to the death of oligodendrocytes in the central nervous system, which in turn causes myelin breakdown.

In the central nervous system of vertebrates, axonal myelination realizes jump-like conduction of nerve impulses and speeds up the conduction speed of nerve impulses. Oligodendrocytes and Schwann cells produce myelin in the central and peripheral nervous systems, respectively. Among them, oligodendrocytes are produced by differentiation of oligodendrocyte precursor cells. Alzheimer's disease causes damage and demyelination of nerve axons, resulting in neurological deficits. Under normal conditions, oligodendrocyte precursor cells stored in the adult brain can be recruited to migrate to the lesion area and differentiate into oligodendrocytes, thereby producing myelin to surround axons, but under pathological conditions, this process is restricted. hinder. Current therapeutic modalities targeting the immune system can reduce immune-mediated damage in demyelinating diseases, but fail to promote remyelination and axonal repair. The endogenous oligodendrocyte progenitor cells (Oligodendrocyte progenitor cells, OPCs) is the main therapeutic target of AD myelin damage. In the early stage, the resident OPCs around the infarcted area in the brain can migrate to the ischemic site, proliferate and differentiate into oligodendrocytes, which partially compensate for the deficiency of myelinating oligodendrocytes. However, the ability of such resident OPCs to differentiate into mature oligodendrocytes and promote myelination is clearly insufficient. To this end, recruiting these resident OPCs and enhancing their differentiation, maturation and myelination capacity is a major therapeutic strategy for the treatment of Alzheimer's disease.

Previous studies have confirmed that the transcription factor Olig2 plays an important role in oligodendrocyte formation and remyelination. In a mouse experiment, activating the expression of Olig2 in PDGFRa+/CD140a-positive OPCs in the brain can induce rapid differentiation and maturation of oligodendrocytes and promote the regeneration of damaged myelin sheaths. Therefore, Olig2 may be an important therapeutic target to promote remyelination in Alzheimer's disease.

In recent years, the highly efficient protein expression mediated by messenger RNA (mRNA) has attracted great attention from academia and industry. Continuous studies have shown that mRNA not only has higher transfection efficiency and higher protein expression levels, but also has greater safety advantages compared with virus-mediated DNA modification. The main component of myelin is lipid, which accounts for about 70% of the dry weight. It can protect axons, realize jumpy conduction of nerve impulses, and accelerate the speed of nerve signal conduction. There are oligodendrocyte precursor cells in the brain of adult individuals, but under the pathological conditions of demyelinating diseases, it is difficult for the oligodendrocyte precursor cells in the brain to differentiate into oligodendrocytes and produce myelin. Therefore, there is a need to provide a pharmaceutical composition for promoting myelin regeneration based on mRNA technology.

Contents of the invention

In view of the above technical problems, the present invention provides a pharmaceutical composition and preparation method for treating Alzheimer's disease based on mRNA technology, which can promote the regeneration of myelin sheath and effectively improve the therapeutic effect of Alzheimer's disease.

To achieve the above object, the present invention provides a pharmaceutical composition for treating Alzheimer's disease based on mRNA technology: the pharmaceutical composition includes lipid nanoparticles and excipients, and the lipid nanoparticles include a lipid phase and a solution phase , the lipid phase comprises raw materials in the following molar ratios: cholesterol, 30-45mol; DLink-MC3-DMA, 60-70mol; DMG-PEG2000, 2-4mol; DSPC, 4-6mol; the solution phase consists of the following raw materials Prepare and obtain: loaded Olig2 messenger RNA and citric acid buffer solution, the citric acid buffer solution includes water and ethanol with a volume ratio of 3.0-4.0:1, the pH value of the citric acid buffer solution is 4.0-4.4, the loaded The molar ratio of Olig2 messenger RNA to the DLink-MC3-DMA is 1:10-14.

The present invention also provides a preparation method of the pharmaceutical composition, comprising the following steps:

S1: Prepare the lipid phase: mix and dissolve cholesterol, DLink-MC3-DMA, DMG-PEG2000, and DSPC to obtain the lipid phase;

S2: preparing a solution phase: preparing loaded Olig2 messenger RNA, dissolving the loaded Olig2 messenger RNA in a citrate buffer solution to obtain a solution phase;

S3: preparing lipid nanoparticles: mixing the lipid phase and the solution phase using microfluidic technology to form a nanoparticle solution, performing first dialysis to obtain lipid nanoparticles;

S4: Antibody modification: Carbodiimide salt EDC and CD140a monoclonal antibody were used for the first incubation, and then the CD140a monoclonal antibody was incubated with DSPE-PEG-NH2 for the second time, and the second dialysis was performed to obtain the antibody conjugate. The antibody conjugate and the lipid nanoparticles were incubated for a third time to obtain lipid nanoparticles loaded with Olig2 messenger RNA.

S5: preparing a pharmaceutical composition: mixing lipid nanoparticles loaded with Olig2 messenger RNA and excipients to obtain a pharmaceutical composition.

Using the above preparation method, cationic liposomes and Olig2 mRNA can be passed through a microfluidic device to form lipid nanoparticles loaded with Olig2 messenger RNA. The particle size of the prepared lipid nanoparticles is about 155nm, and the process is stable and controllable. Good performance and reproducibility, easy for industrial production.

In the step of preparing lipid nanoparticles, the first dialysis is performed with a dialysis bag, the molecular weight of the dialysis bag is 2900-3100, and the time of the first dialysis is 25-35 hours.

In the antibody modification step, the concentration of the carbodiimide salt EDC is 0.08-0.20 mg/mL, the pH value is 6-7, and the molar ratio of the DSPE-PEG-NH2 to the CD140a monoclonal antibody is 9-12:1.

Using the above reaction conditions can maximize the activation of the desired monoclonal antibody.

In the antibody modification step, the incubation temperature of the first incubation is 25-30°C, the incubation time of the first incubation is 30-40min; the incubation time of the second incubation is 2-4 hours, and the incubation time of the second incubation is 2-4 hours. The pH value of the second incubation is 7-8, and the second dialysis uses a dialysis membrane with a molecular weight cut-off of 90-130KD.

Using the above reaction conditions, the coupling of monoclonal antibodies can be better achieved.

In the antibody modification step, the incubation temperature of the third incubation is 50-56°C, and the incubation time of the third incubation is 20-30min.

The present invention also provides the application of the pharmaceutical composition in the preparation of medicaments for preventing or treating Alzheimer's disease.

Compared with prior art, the beneficial effect of the present invention is:

A pharmaceutical composition for treating Alzheimer's disease based on mRNA technology of the present invention, the pharmaceutical composition delivers Olig2-loaded messenger RNA to OPCs, so that OPCs transiently overexpress Olig2, and at the same time, LNPs modified by monoclonal antibody CD140a can effectively Targeted delivery of Olig2 mRNA to target cells CD140a ⁺ OPCs can promote the rapid differentiation and maturation of endogenous OPCs and promote the remyelination of Alzheimer's disease mice. The lipid nanoparticles are more targeted and effective, and can effectively improve the therapeutic effect of Alzheimer's disease.

Description of drawings

Fig. 1 is the schematic diagram of the Olig2 lipid nanoparticle of synthetic targeting antibody modification in embodiment 1;

Fig. 2 is the result figure (scale bar 100nm) of the Olig2 lipid nanoparticle of antibody modification in embodiment 1;

Fig. 3 is the I-Olig2 and C-Olig2 agarose gel electrophoresis figure of embodiment 1;

Fig. 4 is the I-Olig2 of embodiment 1 and C-Olig2 encapsulation rate result figure;

Fig. 5 is the result graph of the in vitro transfection efficiency of C-Olig2 of different concentrations detected in embodiment 2 in OPCs;

6 is a schematic diagram of the cell strategy for flow cytometry sorting CD140a ⁺ and CD140a ⁺ OPCs in the mouse brain in Example 3;

7 is a graph showing the results of Western blot analysis of CD140a ⁺ and CD140a ⁺ OPCs in the mouse brain after 24 hours of intracranial injection of C-Olig2 in the expression of Olig2 in Example 3;

Fig. 8 is the neurological deficit scoring result figure in embodiment 4;

Fig. 9 is a result diagram of the effect of a single injection of C-Olig2 on the number of MBP-positive oligodendrocytes in the hippocampus of mice detected by tissue immunofluorescence in Example 4; wherein, Fig. 9A is the expression of MBP in the hippocampus of mice detected by immunofluorescence The result map (scale bar 100 μm), Fig. 9B is the quantitative statistical result map of MBP expression (*p<0.05, ##p<0.01);

Figure 10 is the result of a single C-Olig2 intracranial injection in Example 4 on the formation of myelin in the mouse hippocampus, Figure 10A is a transmission electron microscope detection of myelinated axons, and Figure 10B is the quantification of myelinated axons stat(*p<0.05, ###p<0.001);

Fig. 11 is the result figure of the effect of a single intracranial injection of C-Olig2 in Example 4 on improving the learning and memory function of MCAO mice, wherein, Fig. 11A is a schematic diagram of the water maze experiment; Fig. 11B is after removing the platform, the mouse Find the latency period of the platform area for the first time (*p<0.05, ##p<0.01); Figure 11C is the percentage of the time that the mouse is in the quadrant of the platform within 60s (*p<0.05, ## #p<0 .01); the upper row of Fig. 11D: representative trajectory diagrams of each group of mice during training, lower row: representative trajectory diagrams of each group of mice during testing;

Among them, I-Olig2 is the modified control antibody IgG Olig2 mRNA/LNPs, and C-Olig2 is the modified targeting antibody CD140a Olig2 mRNA/LNPs.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the associated drawings. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

definition:

DLink-MC3-DMA: refers to a new type of cationic lipid, the chemical name is 4-(N, N-dimethylamino) butyric acid (6Z, 9Z, 28Z, 31Z)-heptanthyroxycarbon-6,9, 28,31-Tetraene-19-yl fat, molecular formula C43H79NO2, is a colorless to pale yellow oily liquid.

DSPC: refers to 1,2-distearoyl-sn-glycero-3-phosphocholine.

DMG-PEG2000: refers to 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.

Carbodiimide salt EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

DSPE-PEG-NH2: refers to distearoylphosphatidylethanolamine PEG amine.

source:

Anti-CD140a mouse monoclonal antibody (135902, BioLegend Company), DLink-MC3-DMA (Shanghai Shunna Biotechnology Co., Ltd., purity 98%), DSPC, cholesterol, DSPE-PEG2000, carbodiimide salt EDC, DSPE- PEG-NH2 (both purchased from Germany Lipoid company).

The reagents, materials, and equipment used in this example are all commercially available sources unless otherwise specified; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

Example 1

Preparation and characterization of Olig2 mRNA-targeting antibody-loaded cationic lipid nanoparticles.

1. Preparation of LNPs modified by Olig2mRNA targeting antibody.

The preparation process is shown in Figure 1.

(1) Construct a plasmid expressing mouseOlig2, and synthesize Olig2 mRNA in vitro.

(2) lipid phase DLink-MC3-DMA: DSPC: cholesterol: DMG-PEG2000 (molar ratio is 55:5:38.5:1.5) dissolved in 90% ethanol; Volume ratio = 3:1; citric acid to adjust pH = 4), wherein the molar ratio of loaded Olig2 messenger RNA and DLink-MC3-DMA is 1:10, injected and mixed with NanoAssemblrBenchtop microfluidic device to form a nanoparticle solution of about 100nm ; Then use a 3000 molecular weight dialysis bag to dialyze with DEPC water (pH7.4) for 24 hours to prepare LNPs loaded with Olig2 messenger RNA.

(3) Use carbodiimide salt EDC and CD140a monoclonal antibody at a concentration of 0.1 mg/mL, incubate at 28°C for 30 minutes, pH=7, activate the carboxyl group of CD140a monoclonal antibody, and then react with 10 of CD140a monoclonal antibody The double molar amount of DSPE-PEG-NH2 was co-incubated for 4 hours (PH=7.5), and the unlinked DSPE-PEG-NH2 was removed through a 100kd dialysis membrane to obtain a DSPE-PEG-antibody conjugate. After incubating the conjugate with the LNPs loaded with Olig2 messenger RNA at 50°C for 20 minutes, the antibody-modified mRNA carrier (modified with green fluorescence) formed was stored at 4°C for future use.

2. Detect the particle size and Zeta potential of mRNA/LNPs.

At room temperature, dissolve and dilute the mRNA/LNPs solution and the blank control with 50 times distilled water, add them to a quartz dish, and measure the particle size distribution and Zeta potential of the samples through a Malvern laser particle size analyzer. The results are shown in the table below.

Table 1 Nano-characterization of lipid nanoparticles

LNPS Average particle size (nm) Potential (mV) PDI I-Olig2 107.4 34.8 0.275 C-Olig2 157.5 18.2 0.21

The results showed that the average particle size of the modified control antibody IgG Olig2 mRNA/LNPs (I-Olig2) was 107.4nm, and the Zeta potential was 34.8mV; the average particle size of the modified targeting antibody CD140a Olig2 mRNA/LNPs (C-Olig2) was 157.5nm, Zeta potential at 18.2mV.

3. The ultrafine morphology of mRNA/LNPs was observed by transmission electron microscope (TEM).

After diluting the two mRNA/LNPs in an appropriate amount, 10 μL of the liquid was dropped on the copper grid of the carbon film, dried and fully infiltrated with 2% phosphotungstic acid for negative staining, and the morphology of the nanoparticles was observed under TEM.

The results are shown in Figure 2. The TEM images of the two kinds of mRNA/LNPs were all circular, with a particle size of about 100 nm.

4. Determination of the encapsulation efficiency of mRNA/LNPs.

Add mRNA lipid nanoparticles containing 1 μg (mRNA quality) and corresponding mRNA to 4% (w/v) agarose gel sample wells, electrophoresis at 55V for 60 minutes, EB color development, and bioelectrophoresis images The analysis system observes the plasmid bands and takes pictures. As shown in Figure 3, it was found that Olig2mRNA ran out of the corresponding molecular weight band, while the mRNA in the nanocarrier stayed in the sample hole (the molecular weight was too large to move), indicating that the mRNA was successfully and stably loaded in the lipid nanocarrier without additional mRNA Stripes are produced, and the encapsulation efficiency is above 95%, as shown in Figure 4.

Example 2

The in vitro uptake effect of LNPs modified by Olig2 mRNA targeting antibody on OPCs derived from mouse neural stem cells was verified.

In order to explore the transfection effect of different concentrations of Olig2mRNA/LNPs, OPCs were seeded in 12-well plates at a density of 5x10 ⁵ per well. The next day, 5-carboxyfluorescein FAM-labeled C-Olig2 was used to incubate with OPCs for 6 hours at final concentrations of 1, 2, 4, 6 and 8 μg mL ^-1 . The uptake efficiency was detected by flow cytometry. The results are shown in AB of FIG. 5 . It can be seen that 6 μg mL ^-1 of C-Olig2 can achieve a transfection effect of more than 92%.

Example 3

The uptake effect of LNPs loaded with Olig2mRNA targeting antibody modified by OPCs in mice was verified.

In order to investigate the target expression level of Olig2mRNA/LNPs in the CD140a ⁺ OPCs cells after intracranial injection into the mouse brain, the mice received intracranial injection of C-Olig2 (0.05mg/kg). After 24 hours, the mice were sacrificed, and CD140a ⁺ , CD140a ^- OPCs were isolated from brain tissue ( FIG. 6 ). The expression of Olig2 protein was detected by Western Blot. As shown in Figure 7, C-Olig2 successfully targeted CD140a ⁺ OPC in the mouse brain and overexpressed Olig2 protein.

The specific operation is as follows:

1. Middle cerebral artery embolism (middle cerebral artery occlusion, MCAO) model mice received (0.05mgkg ^-1 ) C-Olig2 intracranial injection, 24 hours later, the mouse brain was removed. Using Adult Brain Dissociation Reagent, after dissociation, red blood cells and cell debris are removed to make a single cell suspension. The anti-CD140a sorting antibody was added to the single cell suspension, ^and the CD140a ^- and CD140a ⁺ cells in the mouse glial cells were sorted by flow cytometry. CD140a ⁺ .

2. The sorted CD140a ⁺ , CD140a ^- OPCs were lysed and sorted with 100uL RIPA, the total protein of cells was extracted, and the protein concentration was determined by BCA method.

3. Add 5x loading buffer, denature the protein at 100°C for 5 minutes, and separate the samples by electrophoresis on 10% polyacrylamide gel (SDS-PAGE). Add 6uL of protein maker, CD140a ⁺ , CD140a ^- OPCs protein to the sample well in sequence. Electrophoresis was performed at a constant voltage of 200V for 1 hour.

4. After electrophoresis, methanol activates the PVDF membrane, takes out the gel after electrophoresis, cuts it into a suitable size, and puts it in the transfer buffer to balance. Prepare the transfer membrane cartridge, in order from negative electrode to positive electrode, sponge, filter paper, gel, PVDF membrane, filter paper and sponge. Put the installed transfer cartridge into the tank, add the transfer buffer, and put it in the ice box at the same time. The membrane was transferred at a constant current of 300mA for 2 hours at 4°C.

5. After the membrane transfer, block the PVDF membrane in blocking solution (TBS containing 5% skimmed milk, 0.05% tween-20) at room temperature for 1 hour, and then put it into the diluted primary antibody, at 4°C Incubate overnight. The next day, wash 3 times with TBST, 5 minutes each time, then add horseradish peroxidase-labeled secondary antibody and incubate at room temperature for 1 hour. Wash 3 times with TBST, 5 minutes each time, and finally develop with a chemiluminescent instrument. The results are shown in Figure 7. After 24 hours of intracranial injection of C-Olig2, Olig2 was successfully expressed in CD140a ⁺ OPCs, thus confirming the targeted expression of C-Olig2 in CD140a ⁺ OPCs in the mouse brain.

Example 4

The therapeutic effect of intracranial injection of C-Olig2 on MCAO mice.

1. Construction of MCAO model mice.

The mice were fasted for 24 hours before the operation, and the mice were anesthetized with isoflurane inhalation using an anesthesia machine (3-4% induction anesthesia, 2-2.5% maintenance anesthesia, isoflurane flow rate 0.5-0.7L/min), The anesthetized mouse was fixed supine on the stage of a stereomicroscope, the neck was cut midline, the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA) were separated, and the important branches of the pterygopalatine artery were ligated. , ligate the CCA, ligate and free a section of the ECA trunk, clamp the base of the ICA with a non-invasive arterial clip, make a slipknot at the bifurcation of the CCA near the ICA, cut a "V"-shaped incision, and thread the thread along the CCA Insert the "V"-shaped incision, loosen the arterial clamp on the ICA, and pass the thread plug through the ICA until the middle cerebral artery (MCA). Stop when resistance is encountered, and pass the tip of the thread through the origin of the MCA to the thinner anterior cerebral artery. At this point, the middle cerebral artery occlusion (MCAO) on one side was completed, the suture was fixed, and the incision was sutured layer by layer. In the sham operation control group, no suture was inserted. After 1 hour of cerebral ischemia in mice, the thread plug was withdrawn to the external carotid artery stump to form reperfusion injury. After the operation, an appropriate amount of penicillin sodium aqueous solution for injection was applied locally, put into a cage and raised at 37°C, and 200,000 U/mouse of penicillin sodium was injected intraperitoneally every day for 3 consecutive days (in case of infection in mice, can be extended to 1 week). The experimental animals were divided into 4 groups, 15 in each group, the above 4 groups were: sham operation (sham) group, model group (MCAO), injection of C-Olig2, injection of I-Olig2. The injection dose of lipid nanoparticles is 0.05mg .kg ^-1 .

2. Changes of neurological function (Menzies) score of MCAO mice after a single intracranial injection of C-Olig2.

On day 21 after a single injection of Olig2 mRNA/LNP, neurological deficits were scored in MCAO mice. The scoring standard is Menzies: normal without injury, both limbs are symmetrically stretched to the opposite side (0); the contralateral forelimb continues to adduct (1 point); the contralateral forelimb grip strength decreases (2 points); the mouse turns in circles after being stimulated ( 3 points); the mice turned in circles autonomously (4 points). The results are shown in Figure 8. Compared with the MCAO model mice, the mice treated with lipid nanoparticles can significantly reduce the neurological deficit score, suggesting the improvement of neurological function. It is worth noting that the targeted modified C-Olig2 Show better neurological function improvement effect.

3. The effect of a single intracranial injection of C-Olig2 on the differentiation, maturation and myelination of hippocampal OPCs in mice.

MCAO mice received intracranial injection of C-Olig2, and after 4 weeks, the brain was perfused with paraformaldehyde and PBS, and the brain was fixed for embedding. The expression of MBP in the hippocampus was detected by tissue immunofluorescence and the ultrastructure of myelin was detected by transmission electron microscopy. The results of tissue immunofluorescence detection of the effect of a single injection of C-Olig2 on the number of MBP-positive oligodendrocytes in the mouse hippocampus are shown in Figure 9A-B. Compared with MCAO model mice, the treatment of lipid nanoparticles can Thereby promoting the differentiation and formation of MBP-positive oligodendrocytes in the brain; and C-Olig2, which has a targeting effect, shows a more obvious effect of promoting the formation of oligodendrocytes. In addition, transmission electron microscopy further showed that a single injection of C-Olig2, the increased oligodendrocytes may further cause the regeneration of myelin in the mouse hippocampus, as shown by a significant increase in myelinated axons (Figure 10A-B ). Based on the above results, a single intracranial injection of C-Olig2 can significantly promote the differentiation and maturation of hippocampal OPCs in MCAO mice, and eventually lead to the regeneration of myelin sheath.

4. The effect of a single intracranial injection of C-Olig2 on improving the learning and memory function of MCAO mice.

Many previous studies have shown that myelination in the hippocampus plays an important role in learning and learning. It has been observed that a single intracranial injection of C-Olig2 induces the differentiation, maturation and remyelination of hippocampal OPCs in mice. Next, it will be further studied whether a single injection of C-Olig2 can improve the long-term learning and memory deficits of MCAO mice. On day 21 of lipid nanoparticle treatment, the Morris water maze was used to detect the learning and memory abilities of MCAO mice ( FIG. 11A ). The detection indicators adopted are the time spent by MCAO mice entering the original platform area for the first time after removing the platform, and the percentage of the time spent in the quadrant of the target platform when looking for the platform to the test time of 60s. As shown in Figure 11B, compared with MCAO model mice and mice treated with no targeted I-Olig2, mice treated with targeted C-Olig2 had less first latency in finding the platform , indicating that the area where the original platform is located can be memorized and recognized more quickly. Furthermore, mice treated with targeted C-Olig2 spent a greater proportion of time in the platform quadrant (Fig. 11C). Representative training and testing path diagrams are shown in Figure 11D. Based on the above results, it was shown that a single intracranial injection of C-Olig2 could significantly improve the learning and memory deficits of MCAO mice.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (7)

1. A pharmaceutical composition for treating Alzheimer's disease based on mRNA technology, characterized in that, the pharmaceutical composition comprises lipid nanoparticles and adjuvants, and said lipid nanoparticles comprise a lipid phase and a solution phase, said The lipid phase includes raw materials in the following molar ratios: cholesterol, 30-45mol; DLink-MC3-DMA, 60-70mol; DMG-PEG2000, 2-4mol; DSPC, 4-6mol; the solution phase is prepared from the following raw materials: Load Olig2 messenger RNA and citric acid buffer solution, the citric acid buffer solution includes water and ethanol with a volume ratio of 3.0-4.0:1, the pH value of the citric acid buffer solution is 4.0-4.4, and the load Olig2 messenger RNA The molar ratio with the DLink-MC3-DMA is 1:10-14. 2. the preparation method of pharmaceutical composition according to claim 1, is characterized in that, comprises the following steps: S1: Prepare the lipid phase: mix and dissolve cholesterol, DLink-MC3-DMA, DMG-PEG2000, and DSPC to obtain the lipid phase; S2: preparing a solution phase: preparing loaded Olig2 messenger RNA, dissolving the loaded Olig2 messenger RNA in a citrate buffer solution to obtain a solution phase; S3: preparing lipid nanoparticles: mixing the lipid phase and the solution phase using microfluidic technology to form a nanoparticle solution, performing first dialysis to obtain lipid nanoparticles; S4: Antibody modification: Carbodiimide salt EDC and CD140a monoclonal antibody were used for the first incubation, and then the CD140a monoclonal antibody was incubated with DSPE-PEG-NH2 for the second time, and the second dialysis was performed to obtain the antibody conjugate. Carrying out the third incubation of the antibody conjugate and the lipid nanoparticles to obtain lipid nanoparticles loaded with Olig2 messenger RNA; S5: preparing a pharmaceutical composition: mixing lipid nanoparticles loaded with Olig2 messenger RNA and excipients to obtain a pharmaceutical composition. 3. The preparation method according to claim 2, characterized in that, in the step of preparing lipid nanoparticles, the first dialysis is carried out using a dialysis bag, the molecular weight of the dialysis bag is 2900-3100, and the second dialysis One dialysis time is 25-35h. 4. The preparation method according to claim 2, characterized in that, in the antibody modification step, the concentration of the carbodiimide salt EDC is 0.08-0.20 mg/mL, the pH value is 6-7, and the The molar ratio of DSPE-PEG-NH2 to the CD140a monoclonal antibody is 9-12:1. 5. The preparation method according to claim 2, characterized in that, in the antibody modification step, the incubation temperature of the first incubation is 25-30°C, and the incubation time of the first incubation is 30-40min; The incubation time of the second incubation is 2-4 hours, the pH value of the second incubation is 7-8, and the second dialysis adopts a dialysis membrane with a molecular weight cut-off of 90-130KD. 6. The preparation method according to claim 2, characterized in that, in the antibody modification step, the incubation temperature of the third incubation is 50-56°C, and the incubation time of the third incubation is 20-30min. 7. The application of the pharmaceutical composition according to claim 1 in the preparation of a medicament for preventing or treating Alzheimer's disease.