Use of miR-486-3p in preparation of a drug for treating dry age-related macular degeneration

Abstract

The application discloses an application of miR-486-3p in preparation of a medicine for treating dry age-related macular degeneration, and belongs to the technical field of biological medicines. Lcn2 It is proved that mesenchymal stem cell-derived extracellular vesicles (hucMSC-EVs) are naturally rich in miR-486-3p, the miRNA can be directly targeted and combined with the 3'UTR of mRNA, the expression of Lcn2 is inhibited, the downstream ferroptosis pathway is blocked, Gpx4 is up-regulated, the GSH level is restored, and the RPE cell mitochondrial integrity is protected. In a dry AMD mouse model, the miR-486-3p agomir can restore the amplitude of ERG a wave and b wave, and the effect is close to that of complete hucMSC-EVs, thereby providing a highly compliant targeted treatment scheme for dry AMD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of miR-486-3p in the preparation of drugs for treating dry age-related macular degeneration. Background Technology

[0002] Dry age-related macular degeneration (dry AMD) is the leading cause of irreversible blindness in older adults worldwide, affecting approximately 196 million people globally, a number projected to rise to 288 million by 2040. The disease is characterized by progressive degeneration of retinal pigment epithelium (RPE) cells, ultimately leading to photoreceptor death and central vision loss. Currently, there are no effective treatments for dry AMD, especially its late geographic atrophy stage, representing a significant unmet medical need.

[0003] A growing body of research indicates that ferroptosis—an iron-dependent, regulatory form of cell death characterized by lipid peroxidation—is a key driver of renal pelvic floor organ (RPE) cell death in dry AMD. Lipocalin-2 (Lcn2) is a secreted protein closely associated with iron homeostasis and inflammation. Recent studies have found that Lcn2 expression levels are significantly elevated in dry AMD mouse models and human AMD retina, and that it can exacerbate RPE cell damage and death by inhibiting autophagy and activating the inflammasome-ferroptosis pathway.

[0004] Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) possess advantages such as natural drug delivery capabilities, low immunogenicity, and the ability to cross biological barriers, and have shown therapeutic potential in various disease models. In ophthalmology, preliminary studies have shown that MSC-EVs can protect RPE cells from oxidative stress damage by activating Nrf2 signaling. However, the key functional molecules and precise molecular targets of MSC-EVs in inhibiting RPE ferroptosis in dry AMD remain unclear, constituting a core obstacle to the development of MSC-EV-based targeted therapies for dry AMD.

[0005] Therefore, identifying the core effector molecules that inhibit RPE ferroptosis in MSC-EVs and providing targeted therapeutic strategies based on these molecules is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide the application of miR-486-3p in the preparation of drugs for treating dry age-related macular degeneration, in order to solve the problems existing in the prior art. This invention reveals for the first time that MSC-EVs target and inhibit [the disease] by delivering miR-486-3p. Lcn2 The mechanism of gene expression and blocking ferroptosis in retinal pigment epithelial cells provides an effective targeted therapy strategy for dry AMD.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of miR-486-3p or its analogues and agonists in the preparation of medicaments for treating dry age-related macular degeneration.

[0008] Preferably, the nucleotide sequence of miR-486-3p or its analogues comprises a sense strand as shown in SEQ ID NO.3 and an antisense strand as shown in SEQ ID NO.4; the miR-486-3p agonist comprises a nucleotide as shown in SEQ ID NO.7 and an antisense strand as shown in SEQ ID NO.8.

[0009] The present invention also provides the use of extracellular vesicles derived from mesenchymal stem cells in the preparation of medicaments for treating dry age-related macular degeneration, wherein the extracellular vesicles derived from mesenchymal stem cells contain miR-486-3p.

[0010] Preferably, the nucleotide sequence of miR-486-3p comprises the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.4.

[0011] Preferably, the extracellular vesicles derived from mesenchymal stem cells are administered via intravitreal injection or eye drops.

[0012] The present invention also provides a medicament for treating dry age-related macular degeneration, wherein the active ingredient of the medicament comprises any one of the following (a) to (c) and a pharmaceutically acceptable carrier: (a) Extracellular vesicles of mesenchymal stem cells containing miR-486-3p; (b) miR-486-3p; (c) miR-486-3p analogues or miR-486-3p agonists.

[0013] The aforementioned carriers may be selected from at least one of lipid nanoparticles, polymer nanoparticles, extracellular vesicles (e.g., exosomes), adeno-associated virus (AAV) vectors, or cell-penetrating peptides.

[0014] Preferably, the dosage form of the drug includes eye drops or intravitreal injection.

[0015] And / or the nucleotide sequence of miR-486-3p comprises the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.4.

[0016] This invention also provides Lcn2 Its application as a target in screening drugs for the treatment of dry age-related macular degeneration. Lcn2 With the core target as the basis, screening and downregulation were performed. Lcn2 An active substance that expresses and inhibits ferroptosis in retinal pigment epithelial cells, said active substance including miR-486-3p or extracellular vesicles derived from mesenchymal stem cells containing miR-486-3p.

[0017] This invention also provides Lcn2 The application of inhibitors in the preparation of drugs for treating dry age-related macular degeneration. Lcn2 Inhibitors include miR-486-3p or extracellular vesicles derived from mesenchymal stem cells containing miR-486-3p.

[0018] Preferably, the nucleotide sequence of miR-486-3p comprises the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.4.

[0019] The present invention discloses the following technical effects: (1) miR-486-3p was identified and verified for the first time as the core effector molecule in hucMSC-EVs that inhibits the Lcn2 / ferroptosis axis. Through small RNA sequencing, bioinformatics prediction and dual-luciferase reporter gene assay, it was confirmed that miR-486-3p can directly target and bind to the 3'UTR of Lcn2 mRNA, elucidating the mechanism of action of MSC-EVs in treating dry AMD and providing a clear molecular therapeutic target for this disease.

[0020] (2) miR-486-3p mimics or agomir monotherapy can significantly restore visual function in vivo (the amplitude of the a wave of ERG increases by about 6 times and the amplitude of the b wave increases by about 8 times), and the therapeutic effect is close to that of complete MSC-EVs.

[0021] (3) It was first confirmed that hucMSC-EVs could reach the retinal RPE layer within 12 hours of eye drop treatment, and that eye drop administration was as effective as intravitreal injection in retinal thickness recovery, which greatly improved patient compliance.

[0022] (4) By using 3D cell culture technology combined with differential ultracentrifugation and density gradient centrifugation, MSC-EVs with uniform particle size and high purity can be obtained, which is feasible for large-scale production and clinical translation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A) Identification and intraocular distribution of hucMSC-EVs; B) Nanoparticle tracking analysis (NTA) particle size distribution; C) TEM morphology; D) Western blot validation of biomarker proteins; E) Retinal tile and frozen sections at different time points after ophthalmic instillation; E) Distribution at different time points after intravitreal injection. Figure 2 The therapeutic effect of hucMSC-EVs in a NaIO3-induced dry AMD mouse model is shown in Figure A. A schematic diagram of the experimental design is shown in Figure B. Hematoxylin-eosin (HE) stained retinal sections show the retinal structure of different treatment groups. Figures C and D show representative ERG waveforms and quantitative statistics of a-wave and b-wave amplitudes, respectively. Figures E and F show optical coherence tomography (OCT) images and quantitative retinal thickness measurements, respectively. Figures H and G show schematic diagrams of optomotor response (OMR) behavior and statistical data. Figure 3 Targeting exosomal miR-486-3p Lcn2 To enhance the therapeutic effect of dry AMD; A shows the target gene GO analysis of high-abundance miRNAs; B shows the interaction between miR-486-3p and... Lcn2 Schematic diagram of the predicted binding site of 3′UTR; C is the direct binding verified by the dual-luciferase reporter gene experiment; DE are the ERG waveform and amplitude quantification after miR-486-3p agomir treatment, respectively; FG are the OCT image and retinal thickness quantification, respectively; HJ are the TEM image and mitochondrial area quantification, respectively. Figure 4 The molecular and ultrastructural evidence for the inhibition of Lcn2 and ferroptosis by hucMSC-EVs in the retina of dry AMD model mice is presented; A and B represent the detection and quantitative analysis of Lcn2 and Gpx4 protein levels by Western blot, respectively; C represents the total glutathione (GSH) content in the retina; and D and F represent the ferritin light chain (…). Ftl mRNA and Ferritin protein expression levels; GH represents RPE mitochondrial transmission electron microscopy (TEM) images and mitochondrial area quantification, respectively; Figure 5The results show the effects of Lcn2 overexpression on induced retinopathy and the rescue effect of hucMSC-EVs; AB represents Western blot verification (A) and quantification (B) of AAV-mediated Lcn2 overexpression and hucMSC-EVs treatment; CD represents ERG waveforms and quantitative statistics; EF represents OCT images and quantitative retinal thickness; GI represents RPE / retinal TEM images and quantitative mitochondrial area.

[0025] Figure 6 Transcriptome analysis identified Lcn2 as a key therapeutic target; A is a volcano plot showing differentially expressed genes between the intravitreal EV injection group and the saline control group; B is a Venn plot showing genes downregulated by both administration routes; C is a heatmap of genes downregulated by both routes; DE shows RNA-seq data. Lcn2 , Gfap FPKM values ​​of isogens; F represents RT-qPCR validation results; GI represents the effects of different doses of hucMSC-EVs on ERG and Lcn2 The effect of mRNA. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] The sequences involved in the following embodiments are shown in Table 1: Table 1 Sequence Information In the table above, (mA), (mC), (mG), (mU) and The numbers following the numbers indicate the number of bases corresponding to the modification; the modification methods involved in some sequences are listed in Table 2.

[0032] Table 2 List of Modified Codes Example 1: Isolation, preparation and identification of hucMSC-EVs The hucMSC-EVs were purchased from HUAMEI BIOTECH, which produced them using 3D cell culture technology and purified them using differential ultracentrifugation combined with density gradient centrifugation.

[0033] Particle size and concentration were determined using a nanoparticle tracking analyzer (NTA). The results showed that the EVs exhibited a uniform population distribution with an average particle size of 152.4 nm, a polydispersity index (PDI) of 0.18 ± 0.03, and a concentration of 7.8 × 10⁻⁶. 11 particles / mL (see particles / mL) Figure 1 (A)

[0034] Transmission electron microscopy (TEM) confirmed that EVs exhibited a typical cup-shaped morphology, with a diameter ranging from 80 to 180 nm, and that the lipid bilayer membrane structure remained intact (see [link]). Figure 1 (B)

[0035] Western blot analysis showed that EVs were significantly enriched in transmembrane biomarkers CD9 and CD63 and intraluminal biomarker TSG101, while the endoplasmic reticulum biomarker Calnexin was undetectable in the EV components (see [link to data]). Figure 1 (C)

[0036] The protein concentration was determined to be 1.5 μg / μL using the BCA method, and the particle-to-protein ratio was calculated to be 5.2 × 10⁻⁶. 8 The protein content is measured in particles / μg, meeting the standards for high-quality exosomes.

[0037] The above orthogonal identification data collectively confirm that high-purity hucMSC-EVs, which can be used for subsequent functional studies, were successfully isolated.

[0038] Example 2: The therapeutic effect of hucMSC-EVs in a NaIO3-induced dry AMD mouse model 1. Experimental Methods A dry AMD model was established using C57BL / 6 mice via intraperitoneal injection of 15 mg / kg sodium iodate (NaIO3). Experimental design is described below. Figure 2 In the middle group (A), hucMSC-EVs were administered via two routes: intravitreal injection (on days 0 and 3, twice a week) and ophthalmic drops (twice daily). The NaIO3 model group served as a control and received an equal volume of physiological saline.

[0039] Intravitreal injection: Mice were anesthetized with a mixture of 1 mL ketamine, 500 μL xylazine and 8.5 mL NaCl (dose of 10 mg / kg). Mydriasis was achieved with compound tropicamide eye drops. A 30 G needle was used to make a hole at the equatorial edge of the mouse eyeball, and exosomes (2 μL) were injected using a microsyringe. After the injection, the needle was left in the eye for 10 seconds before being removed.

[0040] Hematoxylin-eosin (HE) staining: Hematoxylin stains the cell nucleus blue-purple, while eosin stains the cytoplasm and extracellular matrix pink. After dewaxing and hydration, eyeball sections are first stained with hematoxylin, then differentiated and blued with hydrochloric acid alcohol, counterstained with eosin, and finally dehydrated, cleared, and mounted to provide a clear color contrast for microscopic observation.

[0041] Electroretinography (ERG) under scorching conditions: After mice have undergone sufficient dark adaptation, the electrophysiological responses of the retina to a series of low- or high-intensity flashes of light are recorded using corneal contact lens electrodes to assess the function of the retinal system. The a-wave, representing photoreceptor activity, and the b-wave, reflecting the activity of bipolar cells and Müller cells, accurately reflect the state of individual retinal cells.

[0042] OCT (Optical Coherence Tomography): Under isoflurane anesthesia, retinal images were acquired using Heidelberg Spectralis HRA-OCT to measure the thickness of the RGC layer.

[0043] OMR (Optic-Motion Response) behavioral assessment: Mice are placed on a platform surrounded by multiple screens, and their head movement trajectories are recorded in real time by an overhead camera. Software algorithms analyze the relationship between tracking performance and visual stimulus parameters to calculate quantitative indicators such as visual acuity and contrast sensitivity.

[0044] 2. Experimental Results HE staining results showed that the outer nuclear layer (ONL) of the retina in the saline-treated model group mice was severely thinned and structurally disordered. In contrast, the hucMSC-EVs-treated groups (regardless of whether administered via intravitreal injection or eye drops) significantly preserved retinal structure and ONL thickness, reduced RPE vacuolation, and orderly cellular layers (see...). Figure 2 (B)

[0045] Dark-vision ERG analysis revealed that NaIO3 treatment resulted in a significant reduction in the amplitudes of both the a-wave (reflecting photoreceptor function) and the b-wave (reflecting inner retinal function). Following intravitreal injection of hucMSC-EVs, the a-wave amplitude recovered from -14.14 ± 4.622 μV to -30.28 ± 5.77 μV, and the b-wave amplitude recovered from -3.521 ± 1.439 μV to 64.92 ± 8.728 μV. Similar significant functional recovery was achieved via ophthalmic administration (see [link to article]). Figure 2 (CD), and ERG recovery showed a dose-dependent trend, with consistency across multiple flash intensities.

[0046] OCT examination showed that the total retinal thickness in the model group was 141.9 ± 28.1 μm, with disordered retinal structures. The retinal thickness recovered to 198.9 ± 10.1 μm in the intravitreal injection EVs group and to 205.3 ± 16.4 μm in the eye drop group. P <0.001 vs model group, see Figure 2 In the EF layer, both the ONL and the kernel layer (INL) are well preserved.

[0047] OMR behavioral assessment showed a trend toward improvement in the EVs treatment group, but the difference did not reach statistical significance (see [link to OMR study]). Figure 2 (Zhong GH).

[0048] The above results indicate that hucMSC-EVs can effectively alleviate retinal degeneration and improve visual function through both administration routes, with intravitreal injection showing slightly better results, possibly due to direct delivery to the posterior segment of the eye and higher bioavailability.

[0049] Example 3: Transcriptome analysis identifies Lcn2 as a key therapeutic target 1. Transcriptome analysis Mice in both the control and exosome-treated groups were euthanized by cervical dislocation, and retinal tissue was rapidly dissected to extract total RNA. RNA sequencing was performed using the Illumina NovaSeq 6000 platform. Raw sequencing data were processed using the BMKCloud online analysis platform (www.biocloud.net). First, the raw data (raw reads) underwent quality control, removing reads containing adapter sequences, large amounts of unknown bases (poly-N), and low-quality sequences to obtain high-quality data (clean reads). Subsequent bioinformatics analyses were performed based on clean reads. The clean reads were aligned to the mouse reference genome downloaded from the UCSC database using HISAT2 software (v2.0.4). Differential expression analysis was performed using DESeq2 software (v1.30.1), with an adjusted P-value <0.01 and a fold change ≥2 as the screening criteria for differentially expressed genes (DEGs). Functional annotation and enrichment analysis were performed on differentially expressed genes using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

[0050] Considering that the microRNAs carried by exosomes mainly exert their biological functions by inhibiting the expression of target gene mRNAs, differentially expressed genes that were significantly downregulated in the intravitreal injection exosome group and the ophthalmic administration exosome group were screened separately, and intersection analysis was performed to obtain a total of 87 candidate target genes that were downregulated by both administration routes. Figure 6 (AB). GeneOntology (GO) functional enrichment analysis showed that these co-downregulated genes were mainly enriched in biological processes such as inflammatory response, immune system process, and response to oxidative stress, suggesting that MSC-derived exosomes may exert neuroprotective effects by regulating inflammation and oxidative stress-related pathways. Further differential expression level ranking analysis of the co-downregulated genes was performed (AB). Figure 6 The results showed that Lipocalin-2 (Lcn2) was among the top 10 genes with the most significant downregulation. Combined with existing research indicating that Lcn2 is involved in neuroinflammation, immune regulation, and oxidative stress damage, it has been identified as an important effector molecule for exosomes to exert therapeutic effects and will be a key target for future targeted regulation research.

[0051] 2. qPCR detection 2.1 RNA extraction using the Trizol method (1) Tissue samples: The isolated mouse retinal tissue was placed in an enzyme-free centrifuge tube, 1 mL of Trizol lysis buffer was added, and the tissue was thoroughly ground using a tissue homogenizer to ensure complete lysis.

[0052] (2) Cell samples: Discard the old culture medium in the culture dish and wash the cells three times with PBS buffer to remove residual serum. Then add 1 mL of Trizol to each well for pipetting and lysis, and transfer the lysate to a 1.5 mL enzyme-free centrifuge tube.

[0053] (3) RNA isolation and precipitation: Add 200 μL of chloroform to the above lysis buffer, vortex vigorously to mix, and let stand at room temperature for 15 min to allow for sufficient reaction. Then place in a 4 ℃ low-temperature centrifuge and centrifuge at 12000 rpm for 15 min. After centrifugation, the mixture will show a three-layer separation (colorless aqueous phase on top, white protein layer in the middle, and red organic phase on the bottom). Carefully aspirate the upper aqueous phase and transfer it to a new enzyme-free EP tube, taking care to avoid aspirating the middle layer. Add an equal volume of isopropanol, invert to mix, and incubate at room temperature for 20 min to precipitate RNA.

[0054] (4) Washing and reconstitution: Centrifuge again at 4 ℃ and 12000 rpm for 15 min, and discard the supernatant. Wash the precipitate twice with 75% ethanol (after adding ethanol, gently tap the tube wall and centrifuge), and then let it air dry at room temperature until the precipitate becomes transparent. Finally, add 100 μL of DEPC-treated water to dissolve the RNA precipitate and store it in a -80 ℃ refrigerator for long-term storage.

[0055] (5) Quality control: Strictly maintain low-temperature operation on ice throughout the entire process. Take a small amount of the thawed RNA sample and use a Nano-Drop spectrophotometer to determine its concentration and purity, ensuring that A... 260 / A 280 The ratio is between 1.8 and 2.0, which meets the requirements for subsequent experiments.

[0056] 2.2 Reverse Transcription PCR Reverse transcription PCR was performed using the HiScript III cDNA synthesis kit. The reaction mixture was prepared according to Table 3 and incubated at 42 °C for 2 min.

[0057] Table 3 Genomic DNA Removal Reaction System Take clean, enzyme-free centrifuge tubes and strictly follow the proportions in Table 4 to add the components to construct the reaction system for reverse transcription. The specific conditions are set as follows: react at 50 ℃ for 15 min, followed by treatment at 85 ℃ for 5 s. After the program completes, cDNA synthesis is complete, and the sample can be used for subsequent experiments.

[0058] Table 4 cDNA synthesis reaction system 2.3 Real-time quantitative PCR (1) Based on the system composition in Table 5, prepare the reaction solution using the PerfectStart® Green qPCR SuperMix (+Dye II) kit. Add the mixed solution to the wells of the qPCR plate.

[0059] Table 5 qPCR reaction system The primers used are shown in Table 6. Table 6 Primers (2) To ensure the reaction system is homogeneous and free of bubbles, the reaction plate was briefly centrifuged (1000 rpm, 2 min). Then it was immediately placed in the instrument and fluorescence signal was collected according to the temperature cycling program set in Table 7.

[0060] Table 7 qPCR reaction procedure (3) Experimental data were normalized using β-actin as an internal reference, and analyzed by 2... -ΔΔCt Using relative quantitative formulas, the differences in transcriptional levels of each gene were calculated and analyzed.

[0061] 2.4 Results and Analysis RNA-seq was performed on the retinas of dry AMD mice treated with exosomes via intravitreal injection and ocular drops, respectively. Differentially expressed genes were analyzed. Following intravitreal injection of exosomes, a total of 819 upregulated genes and 1,336 downregulated genes were identified. Figure 6 (A) After ophthalmic administration, 284 and 306 genes were upregulated and downregulated, respectively. A total of 87 genes were downregulated by both administration methods. Figure 6 (China B), among which Lcn2 ranks highly ( Figure 6 (C). Both RNA-seq and RT-qPCR results confirmed that exosome treatment significantly downregulated Lcn2 expression (C). Figure 6 (Middle DF). Compared with the saline control group, MSC-EVs treatment caused retinal effusion. Lcn2mRNA (reduced by 1.5 ± 0.1 times) Gfap (Reduced by 2.3 ± 0.3 times) Steap4 (reduced by 2.15 ± 1.9 times) and Serpina3n (Reduced by 1.3 ± 0.2 times) Significantly downregulated ( Figure 6 To investigate the dose-dependent effects of hucMSC-EVs, NaIO3-induced AMD-like mice were treated with intravitreal injections of different concentrations of hucMSC-EVs. RT-qPCR analysis showed that, compared with the NaIO3 group, retinal Lcn2 expression was significantly reduced in all EVs treatment groups (F). Figure 6 (I). However, ERG analysis showed that improvements in a-wave and b-wave amplitudes were observed only in mice receiving undiluted hucMSC-EVs, while diluted EV formulations had limited effects on retinal function recovery. Figure 6 (Medium GI). These results indicate that undiluted EVs exhibit the greatest therapeutic effect in restoring retinal function.

[0062] Example 4: Identification of miR-486-3p and verification of its function in inhibiting ferroptosis by targeting Lcn2. Small RNA sequencing was performed on purified hucMSC-EVs, identifying 1157 known miRNAs with a total of 3.1 × 10⁻⁶ reads. 7 Among them, miR-486-3p had the highest abundance.

[0063] GO analysis of the target genes of the most abundant miRNAs showed that their target genes were enriched in multiple biological processes such as transcriptional regulation, apoptosis, and iron homeostasis (see [link to study]). Figure 3 (A), revealing its broad regulatory potential.

[0064] Bioinformatics prediction using mirDB revealed a conserved binding site for miR-486-3p in the 3′ UTR of Lcn2 mRNA (see [link to mirDB bioinformatics prediction]). Figure 3 (B), with a binding free energy of -18.7 kcal / mol.

[0065] The assay was validated using a dual-luciferase reporter gene assay. Wild-type (WT) Lcn2 3′UTR fragments and mutant (MUT) Lcn2 3′UTR fragments were cloned into the pmirGLO vector, respectively. HEK-293T cells were co-transfected with 0.2 μg of the reporter plasmid and miR-486-3p mimics or negative control (NC) mimics at a final concentration of 50 nM. The results showed that miR-486-3p mimics significantly inhibited the luciferase activity of the WT Lcn2 3′UTR reporter plasmid. P<0.05), but had no inhibitory effect on the MUT reporter plasmid (see Figure 3 (C). The experimental results confirm that miR-486-3p directly targets and binds to Lcn2 3′UTR.

[0066] To verify in vivo whether miR-486-3p alone could reproduce the therapeutic effect of intact hucMSC-EVs, miR-486-3p agomir (2 μg / eye) was injected intravitreally in a NaIO3 model. Results showed that agomir treatment significantly restored ERG waveform and amplitude, from -26.15 ± 11.57 μV to -43.21 ± 20.13 μV, and b-wave amplitude from 39.20 ± 41.48 μV to 106.2 ± 48.29 μV (see [link to study]). Figure 3 (Delta retinal thickness). OCT showed a significant increase in retinal thickness (see [link to OCT]). Figure 3 (FG). TEM observation showed that agomir effectively rescued NaIO3-induced RPE / retinal ultrastructural damage and abnormal mitochondrial morphology (see [reference]). Figure 3 (HJ).

[0067] Western blot results showed that Lcn2 protein expression was significantly downregulated (reduced by 1.7 ± 0.1-fold) in the retina of a NaIO3-induced dry AMD mouse model treated with hucMSC-EVs. P< 0.01), Gpx4 protein expression was upregulated (increased by 3.6 ± 0.1 times). P <0.01) (see Figure 4 (AB). GSH levels were significantly elevated (see...) Figure 4 (C) Ftl mRNA decreased by 2.7 ± 0.5 times ( P < Ferritin protein decreased by 1.7 ± 0.0 times (0.05). P< 0.01) (see) Figure 4 (Dermatologic Defibrillation). TEM observation of RPE mitochondria showed that the model group exhibited characteristic changes of ferroptosis, such as cristae condensation, membrane rupture, and vacuolation, while the mitochondrial morphology of the MSC-EVs-treated group was basically normal (see [reference needed]). Figure 4 (Zhong GH).

[0068] The above results collectively demonstrate that hucMSC-EVs target and inhibit [the virus] by delivering miR-486-3p. Lcn2 It expresses and thus blocks the downstream ferroptosis pathway, playing a protective role for the retina.

[0069] Example 5: Lcn2 Construction of overexpression model and verification of the rescue effect of hucMSC-EVs For direct verification Lcn2 Whether they are sufficient to drive dry AMD-like pathological changes, and whether EVs can counteract Lcn2-mediated damage. Mice were directly synthesized by Virgin Laboratories. Lcn2 The full-length cDNA sequence was used to initiate gene expression using the CMV promoter. Lcn2 The Kozak sequence GCCACC was added before the ATG, and a Flag tag was added to the C-terminus of the target gene. The target gene and GFP were expressed via a P2A non-fusion pathway. The sequences were then ligated to construct the pAAV-CMV-Lcn2-P2A-GFP recombinant plasmid, which was co-transfected with the packaging plasmid into 293T cells. The packaged virus was collected after 48 h. AAV2 adeno-associated virus expressing only GFP served as a control. The final viral concentration was 1 × 10⁻⁶. 13 The viral genome was stored in balanced salt solution (BSS) containing 0.014% Tween-20 (Alcon, Forth Worth, Texas). Lcn2 was then successfully overexpressed in the retina of C57BL / 6 mice via intravitreal injection.

[0070] Fourteen days after injection, Western blot confirmed significant overexpression of retinal Lcn2 protein (see...). Figure 5 (AB). ERG detection showed a significant decrease in the amplitude of both a-wave and b-wave (see...). Figure 5 (Medium CD). OCT showed retinal thinning and impaired latticeal integrity, with total thickness decreasing from 216.8 ± 18.7 μm in the control group to 189.8 ± 27.2 μm (see...). Figure 5 (EF). The above data confirms that simply Lcn2 Overexpression can induce retinal degeneration and visual function impairment, and a study has been established. Lcn2 Causal relationship with AMD-like pathology.

[0071] AAV-Lcn2-OE mice were then intravitreally injected with hucMSC-EVs (2 μL / eye). TEM observation showed that EVs treatment effectively restored the function of the endothelial cells. Lcn2 Overexpression disrupts RPE and retinal structural integrity. Lcn2 The abnormal morphology of mitochondrial cristae condensation, swelling, and membrane rupture observed in the overexpression group was significantly improved (see [link to article]). Figure 5 Medium GI).

[0072] The above results provide strong evidence for both sides, confirming... Lcn2 It is a pathogenic factor of retinal degeneration, and hucMSC-EVs can effectively combat Lcn2-mediated pathological damage.

[0073] Example 6: Preliminary preparation and intraocular tracing of eye drop formulation hucMSC-EVs were labeled with PKH26 fluorescent dye: 10 μg of EVs were mixed with 100 μL of PKH26 labeling solution and incubated at room temperature in the dark for 5 minutes. The reaction was then terminated by adding an equal volume of stop solution.

[0074] The labeled EVs were resuspended in PBS (pH 7.4) to a final concentration of approximately 10 μg EVs / μL, and 0.01% benzalkonium chloride was added as a preservative. The EVs were then dispensed into sterile eye drop bottles and stored at 4°C in the dark for later use.

[0075] Two μL of the above-mentioned eye drops were instilled into each eye of normal C57BL / 6 mice. The eyeballs were then removed at 0, 3, 6, 12, and 24 hours to prepare retinal slides and frozen sections.

[0076] Confocal microscopy revealed no fluorescence signal on the retina at 0 hours after eye drops; punctate red signals appeared at 3 hours; and a large amount of red fluorescence aggregation was visible on a retinal retinal surfacing film at 12 hours (see...). Figure 1 (Middle D, top image). Frozen sections confirmed that the signal penetrated to the ganglion cell layer (GCL) and the inner nuclear layer (INL), and a positive signal in the RPE / choroid region could be observed under high magnification (see...). Figure 1 (See figure below). This non-invasive administration route greatly improves patient compliance, especially suitable for the treatment of chronic diseases requiring long-term medication. Furthermore, the intravitreal injection of exosomes in this invention also detected PKH26 positive signals on the retina on days 1, 3, and 6 after exosome injection; frozen section and retinal smear results showed that the signal peaked on day 3, and the signal could reach the RPE / choroid region (see figure below). Figure 1 The result (E) indicates that intravitreal injection is more effective than eye drops.

[0077] The above data demonstrate that the non-invasive eye drop administration route can effectively deliver hucMSC-EVs to the posterior segment of the retina, providing a feasible basis for the development of eye drop formulations for dry AMD.

[0078] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of miR-486-3p or its analogues and agonists in the preparation of drugs for treating dry age-related macular degeneration.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of miR-486-3p or its analogues includes the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.4; the miR-486-3p agonist includes the nucleotides as shown in SEQ ID NO.7 and the antisense strand as shown in SEQ ID NO.

8.

3. The application of extracellular vesicles derived from mesenchymal stem cells in the preparation of drugs for treating dry age-related macular degeneration, characterized in that... The extracellular vesicles derived from mesenchymal stem cells contain miR-486-3p.

4. The application as described in claim 3, characterized in that, The nucleotide sequence of miR-486-3p includes the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.

4.

5. The application as described in claim 3, characterized in that, The mesenchymal stem cell-derived extracellular vesicles are administered via intravitreal injection or eye drops.

6. A drug for treating dry age-related macular degeneration, characterized in that, The active ingredient of the drug comprises any one of (a) to (c) below and a pharmaceutically acceptable carrier: (a) Extracellular vesicles of mesenchymal stem cells containing miR-486-3p; (b) miR-486-3p; (c) miR-486-3p analogues or miR-486-3p agonists.

7. The drug as described in claim 6, characterized in that, The dosage form of the drug includes eye drops or intravitreal injection; And / or the nucleotide sequence of miR-486-3p includes the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.

4.

8. Lcn2 Its application as a target in screening drugs for the treatment of dry age-related macular degeneration is characterized by, by Lcn2 With the core target as the basis, screening and downregulation were performed. Lcn2 An active substance that expresses and inhibits ferroptosis in retinal pigment epithelial cells, said active substance including miR-486-3p or extracellular vesicles derived from mesenchymal stem cells containing miR-486-3p.

9. Lcn2 The application of inhibitors in the preparation of drugs for treating dry age-related macular degeneration is characterized by, Lcn2 Inhibitors include extracellular vesicles derived from mesenchymal stem cells that contain miR-486-3p or miR-486-3p.

10. The application as described in claim 8 or 9, characterized in that, The nucleotide sequence of miR-486-3p includes the sense strand as shown in SEQ ID NO.3 and the antisense strand as shown in SEQ ID NO.4.

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