An engineered exosome containing an antibody

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an engineered exosome and its applications. This invention is the first to discover that PCDH12 is specifically highly expressed in the trabecular meshwork tissue of glaucoma patients. Based on this target, engineered exosomes loaded with PCDH12 antibodies were successfully developed. These engineered exosomes can achieve non-invasive and precise targeted delivery to the trabecular meshwork tissue. In glaucoma model mouse experiments, they not only significantly reduced intraocular pressure but also effectively protected the optic nerve. The non-invasive and precise delivery strategy proposed in this invention combines potent intraocular pressure reduction with direct neuroprotection, providing a candidate solution with direct clinical translational potential for integrated "pressure reduction-neuroprotection" treatment of glaucoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an engineered exosome and its applications. Background Technology

[0002] Glaucoma is a neurodegenerative disease of the optic nerve, characterized by changes in the optic nerve and visual field damage, caused by various etiologies. According to the World Health Organization, glaucoma is the second leading cause of blindness worldwide. Unlike cataracts and refractive accommodation disorders, glaucoma leads to permanent vision loss and has become the leading cause of irreversible blindness globally. Its core characteristic is the progressive loss of retinal ganglion cells, leading to visual field defects, decreased vision, and ultimately, blindness in its late stages. Primary open-angle glaucoma (POAG) is the most common type of glaucoma, often referred to as the "silent killer," and is extremely dangerous. Research suggests that POAG is caused by abnormalities in the structure and function of the trabecular meshwork (TM), resulting in obstructed aqueous humor outflow and increased intraocular pressure (IOP). The main pathway for aqueous humor outflow is through the TM into Schlemm's canal, where resistance is primarily located within the TM, and the formation of this resistance is closely related to the morphology and function of TM cells (TMCs). Therefore, intervention strategies targeting the regulation of trabecular meshwork function and the improvement of aqueous humor dynamics have become the core direction of glaucoma treatment research.

[0003] Currently, clinical treatment options for glaucoma mainly fall into three categories: drug therapy, laser therapy, and surgical intervention. Drug therapy, due to its non-invasive advantage, remains the preferred treatment for most adult glaucoma patients, and eye drops, as an ideal non-invasive intervention strategy, play a crucial role in clinical application. Commonly used glaucoma medications include prostaglandin analogs and beta-blockers. These drugs primarily lower intraocular pressure by regulating aqueous humor dynamics. For example, prostaglandin analogs mainly increase aqueous humor outflow via the uveal-scleral pathway by activating local FP receptors in the eye; beta-blockers reduce aqueous humor production by inhibiting beta receptors in the ciliary body. However, because the targets of these drugs (such as beta receptors) are also widely distributed throughout the body, they may cause systemic adverse reactions such as cardiovascular or respiratory problems. More importantly, existing drugs are mainly limited to regulating aqueous humor dynamics to lower intraocular pressure and cannot fundamentally improve the dysfunction of trabecular meshwork cells; therefore, their therapeutic effects have significant limitations.

[0004] To overcome the limitations of existing drug therapies, various emerging treatment directions have been explored in related fields. Among them, ROCK inhibitor eye drops target the restoration of physiological aqueous humor outflow, exerting a hypotensive effect by relaxing trabecular meshwork cells, remodeling the extracellular matrix, and enhancing Schlemm's tube permeability. However, clinical studies show that their hypotensive effect is limited and cannot meet the needs of monotherapy, making them more suitable for combination or alternative therapies. Nanoparticle-based sustained-release eye drops, as an innovative direction in drug delivery technology, can effectively improve drug bioavailability and prolong the duration of drug action. However, this system still does not solve the core problem of precise drug targeting of trabecular meshwork cells, thus failing to achieve targeted therapy. Stem cell transplantation and exosome therapy in the field of cell therapy have been research hotspots in recent years, but current administration methods are still limited to injection, failing to achieve drug delivery and therapeutic effects through non-invasive routes, thus restricting their widespread clinical application. Summary of the Invention

[0005] This invention discovered that PCDH12 is specifically expressed in the trabecular meshwork of glaucoma patients. Based on this target, an engineered exosome loaded with a PCDH12 antibody, antiPCDH12-Exo, was developed. This engineered exosome can non-invasively and precisely target the trabecular meshwork, significantly reducing intraocular pressure in glaucoma model mice and effectively protecting the thickness of the retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer (RNFL+GCL+IPL). Based on this, this invention was completed.

[0006] In a first aspect, the present invention provides an engineered exosome loaded with a PCDH12 antibody, named antiPCDH12-Exo; the method for preparing the engineered exosome is as follows: S1. Select a culture medium containing exosome-free serum to culture mesenchymal stem cells, collect the cell supernatant, and obtain the exosome vector - Exo by ultracentrifugation; S2. The PCDH12 antibody was co-incubated with the DMPE-PEG-MAL conjugate, and the supernatant was discarded after centrifugation to obtain the PCDH12 antibody and DMPE-PEG-MAL conjugate complex. S3. The PCDH12 antibody obtained in S2 and the DMPE-PEG-MAL conjugate complex precipitate were resuspended in the vector exosomes extracted in S1, incubated, and then ultracentrifuged to obtain the engineered exosomes antiPCDH12-Exo.

[0007] Furthermore, in step S3, the mass ratio of the PCDH12 antibody, DMPE-PEG-MAL conjugate, and vector exosomes is 1:1:1.

[0008] In a second aspect, the present invention provides the application of the engineered exosomes described in the second aspect in the preparation of drugs targeting trabecular meshwork tissue; wherein the engineered exosomes are antiPCDH12-Exo.

[0009] Furthermore, the drug can be used alone or in combination with other drugs.

[0010] Furthermore, the drug dosage form includes eye drops or injections.

[0011] Furthermore, the administration methods of the injection include intravitreal injection, subretinal injection, peribulbar injection, retrobulbar injection, or Tenon capsule injection.

[0012] Thirdly, the present invention provides the use of the engineered exosomes described in the second aspect in the preparation of a medicament for treating glaucoma, wherein the engineered exosomes are antiPCDH12-Exo; and the medicament has at least one of the following functions: (1) Targeted trabecular meshwork organization; (2) Reduce intraocular pressure in glaucoma animals; (3) Improves the thickness of the retinal nerve fiber layer, ganglion cell layer and inner plexiform layer (RNFL+GCL+IPL).

[0013] Furthermore, the glaucoma includes primary glaucoma, secondary glaucoma, congenital glaucoma, and developmental glaucoma.

[0014] Furthermore, the primary glaucoma includes primary open-angle glaucoma and primary angle-closure glaucoma.

[0015] Furthermore, the drug can be used alone or in combination with other drugs.

[0016] Furthermore, the drug dosage form includes eye drops or injections.

[0017] Furthermore, the administration methods of the injection include intravitreal injection, subretinal injection, peribulbar injection, retrobulbar injection, or Tenon capsule injection.

[0018] Fourthly, this invention provides the application of PCDH12 as a target in the preparation of drugs targeting trabecular meshwork.

[0019] Furthermore, the drug includes a PCDH12 inhibitor or a PCDH12 antibody.

[0020] Preferably, the drug is a PCDH12 antibody.

[0021] Beneficial effects The engineered exosome antiPCDH12-Exo constructed in this invention exhibited significant therapeutic advantages in a dexamethasone-induced glaucoma model. Administered non-invasively via eye drops, this exosome precisely targets the trabecular meshwork region, demonstrating superior intraocular pressure reduction and optic nerve protection compared to the control group.

[0022] Exosome tracing results showed that, after daily eye drop administration, samples taken at different time points revealed that unmodified exosomes (Exo group) were widely distributed throughout the entire corneal layer, while the antiPCDH12-Exo group was more concentrated in the trabecular meshwork region to exert its effect. This result confirms that the engineering strategy employed in this invention can achieve non-invasive and precise drug delivery.

[0023] Regarding therapeutic effects, although both the Exo group and the antiPCDH12-Exo group showed some ability to lower intraocular pressure, the antiPCDH12-Exo group exhibited a more significant effect. Simultaneously, retinal thickness measurements indicated that antiPCDH12-Exo was more effective in protecting the optic nerve function of glaucoma model mice and slowing its thinning trend.

[0024] In summary, the non-invasive and precise delivery strategy proposed in this study has both a powerful effect in lowering intraocular pressure and direct neuroprotection, providing a candidate solution with direct clinical translational potential for integrated "pressure-lowering and neuroprotection" treatment of glaucoma. Attached Figure Description

[0025] Figure 1 A technical solution for the preparation of engineered exosomes antiPCDH12-Exo.

[0026] Figure 2 To screen for genes specifically expressed in the trabecular meshwork in the anterior segment (a) and PCDH12, whose expression was significantly increased after POAG disease (b).

[0027] Figure 3 Electron micrographs of exosomes and engineered exosomes (top), nanoparticle tracking analysis (middle), and zeta potential (bottom).

[0028] Figure 4 Staining of sections after administration of PBS, fluorescently labeled exosomes, and engineered exosomes to eye drops.

[0029] Figure 5 The intraocular pressure-lowering effects of PBS, exosomes, and engineered exosomes.

[0030] Figure 6 Comparison of retinal RNFL+GCL+IPL thickness in mice treated with PBS, exosomes, and engineered exosomes. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0033] Example 1: Transcriptomic sequencing of trabecular meshwork tissues from POAG patients and normal individuals GSE138125 and GSE27276 from the GEO database were selected as transcriptomic data comparing POAG patients and healthy individuals. The files were read in using R 4.5.1, and probe IDs were mapped to EntrezGene identifiers according to the latest platform annotations provided by GEO. If multiple probes corresponded to the same gene, the probe with the largest mean absolute deviation (MAD) among all samples was retained.

[0034] To remove technical differences between platforms and preserve biological differences within each study, the two sets of data were independently standardized at the gene level using Z-scores: Z = (Xg - μg) / σg (g = 1…G genes); Where Xg is the log2 expression vector of gene g, and μg and σg are the mean and standard deviation of the gene in all samples of the same GEO series.

[0035] The mRNA expression microarray datasets GSE27276 contain 19 POAGs and 17 control samples, while GSE138125 contains 4 POAGs and 4 control samples. Normalization between arrays was performed using the R "limma" package to eliminate batch-to-batch effects. DEGs in the GSE27276 and GSE138125 datasets were analyzed using R v3.64.3 with the "limma" package. The cutoff criteria were adjusted p-value (adj.p.val) < 0.05 and |logFC| > 0.5. The two expression matrices were merged along the gene (row) direction without further cross-dataset scaling; only the relative expression levels within each dataset were compared.

[0036] Merging the two datasets revealed 30 upregulated genes. To better target specific genes, 13 more genes expressing surface proteins were selected from these 30.

[0037] In normal individuals, the expression of 13 genes expressing surface proteins was detected in the cornea, iris, trabecular meshwork (TM), and lens structures. This allowed for the screening of genes specifically expressed in the trabecular meshwork, resulting in the selection of three genes (PCDH12, MXRA8, and LTC4S) (see...). Figure 2 a).

[0038] PCR validation was performed on the trabecular meshwork of normal individuals and patients with porcine angina pectoris (POAG). Genes showing significantly elevated levels in the trabecular meshwork of POAG patients were screened, and PCDH12 was ultimately identified. Therefore, PCDH12 antibody was chosen as a targeted therapy tool (see [link to relevant documentation]). Figure 2 b).

[0039] Example 2: Preparation of engineered exosomes 1. Preparation of exosomes: When mesenchymal stem cells (MSCs) have grown and proliferated to about 80%, they are cultured in a medium containing exosome-free serum for 48 hours. The cell supernatant is then collected and exosomes (Exo) are extracted by ultracentrifugation. 2. Preparation of PCDH12 antibody and DMPE-PEG-MAL conjugate complex: 1 μg / μL of PCDH12 antibody (bs-11109R; Beijing Bio-Science Biotechnology Co., Ltd.) and 1 μg / μL of conjugate DMPE-PEG-MAL (R-DM-0125; Xi'an Ruixi Biotechnology Co., Ltd.) were co-incubated at 4℃ for 12 h-18 h. After centrifugation, the supernatant was discarded to obtain the PCDH12 antibody and DMPE-PEG-MAL conjugate complex complex. 3. Preparation of engineered exosomes: The precipitate was resuspended in 1 μg / μL of MSC-derived exosomes and incubated at room temperature for 1 hour. The mixture was then ultracentrifuged again to collect the engineered exosomes antiPCDH12-Exo.

[0040] 4. Identification of exosomes: 4.1 Transmission electron microscopy identification: (1) Adsorption: Take the prepared exosome suspension onto the Parafilm sealing film, place the copper mesh of the carrier film face down, and let it naturally adsorb the suspension droplets. Then use filter paper strips to remove excess droplets and let it dry slightly. (2) Staining: Take 2% phosphotungstic acid solution onto the sealing film, place the copper mesh with the front side facing the staining solution, and invert it to stand; (3) Drying: Use filter paper strips to absorb excess liquid droplets and air dry; (4) Observation and photography: Observe and photograph under a transmission electron microscope.

[0041] 4.2 Nanoparticle Tracking Analysis (NTA) (Instrument: ZetaView_Particle Metrix): (1) Clean the sample pool; (2) After cleaning, use standard products (100nm PS beads, polystyrene microspheres) to calibrate the instrument. After calibration, perform subsequent testing. (3) Clean the sample pool with PBS; (4) After diluting the sample with PBS (see Dilution Factor in the PDF report for the dilution factor), add it to the sample pool and observe the real-time dynamic image of exosome particles on the computer screen. (5) Collect information and issue corresponding test reports; (6) After the test is completed, use PBS to clean the sample injection cell and then test the next sample.

[0042] 4.3 Zeta potential (instrument: Malvern laser particle size analyzer): (1) Measure the Zeta potential of the blank buffer as the background value (it should be close to 0 mV). (2) Dilute the exosome solution with deionized water to a suitable concentration, and add the diluted exosome solution to a dedicated Zeta potential cuvette; (3) Set the detection parameters: 25°C, solvent: pure water, refractive index (~1.39) and absorbance (0), select “ZetaPotential” mode, equilibration time: 1 minute; (4) Start the measurement. The instrument analyzes the electrophoretic mobility of particles by electrophoretic light scattering (ELS) and calculates the Zeta potential. (5) Check whether the phase diagram is stable and whether the decay curve conforms to a single exponential fit; (6) Record the average value and dispersion (PDI) of the Zeta potential, and save the original data and phase diagram for subsequent analysis.

[0043] Depend on Figure 3 The results showed that the engineered treatment did not alter the stability of the exosomes. Electron microscopy images showed that the morphology of the engineered exosomes remained largely unchanged, retaining the saucer-like vesicles. NTA analysis revealed that antiPCDH12-Exo particles remained within the normal particle size range (30–200 nm). The engineered treatment also did not significantly alter the zeta potential of the exosomes, which remained within the normal range of -10 to -30 mV. All of these findings indicate that the engineered exosomes remained stable.

[0044] Example 3: Validation of the targeting effect of engineered exosomes After dexamethasone (DEX) modeling, C57BL / 6 mice were randomly divided into PBS, Exo, and antiPCDH12-Exo groups. The PBS group served as the solvent control group and was not fluorescently labeled. The Exo group served as the negative control group and was labeled with PKH26 red fluorescence at a concentration of 0.5 μg / μL. The antiPCDH12-Exo group served as the experimental group; PCDH12 antibody was labeled with FITC green fluorescence, and Exo was labeled with PKH26 red fluorescence. AntiPCDH12-Exo was prepared according to the protocol in Example 2 at a concentration of 0.5 μg / μL.

[0045] All experimental groups were treated with eye drops, 2 μL each time, twice a day, morning and evening. Treatment lasted for 14 consecutive days, and the targeting of the trabecular meshwork tissue was observed on days 1, 7, and 14.

[0046] Depend on Figure 4 The results showed that the red fluorescently traced exosomes in the Exo group were distributed throughout the entire corneal layer, except for the trabecular meshwork, and could not precisely target the trabecular meshwork. In contrast, in the antiPCDH12-Exo group, the green-labeled PCDH12 antibody and red-labeled exosomes aggregated near the trabecular meshwork. These results indicate that, when administered via eye drops to glaucoma mice, antiPCDH12-Exo can precisely target the trabecular meshwork tissue.

[0047] Example 4: Verification of the effects of engineered exosomes After dexamethasone (DEX) modeling, C57BL / 6 mice were randomly divided into three groups: PBS group, Exo group, and antiPCDH12-Exo group. The PBS group served as the solvent control group; the Exo group served as the negative control group with a concentration of 0.5 μg / μL; and the antiPCDH12-Exo group served as the experimental group with a concentration of 0.5 μg / μL.

[0048] All experimental groups were treated with eye drops, 2 μL each time, twice a day, morning and evening. Treatment lasted for 14 consecutive days, during which intraocular pressure was measured. On day 14, the thickness of the retinal RNFL+GCL+IPL was measured to observe the protective effect in each group.

[0049] Depend on Figure 5 As shown in Table 1, both Exo and antiPCDH12-Exo can effectively reduce intraocular pressure, with antiPCDH12-Exo showing better efficacy.

[0050] Table 1. Intraocular pressure-lowering effects of PBS, exosomes, and engineered exosomes. Depend on Figure 6As shown in Table 2, the thickness of mice after DEX modeling was significantly reduced compared to normal mice. After 14 days of treatment with PBS, Exo, and antiPCDH12-Exo, the thickness of the mice in the antiPCDH12-Exo group was significantly improved, while the change in the Exo group was not statistically different.

[0051] Table 2 Comparison of RNFL+GCL+IPL thickness in PBS, exosomes, and engineered exosomes

Claims

1. An engineered exosome loaded with PCDH12 antibody, characterized in that, The engineered exosomes were named antiPCDH12-Exo; the preparation method of the engineered exosomes is as follows: S1. Select a culture medium containing exosome-free serum to culture mesenchymal stem cells, collect the cell supernatant, and obtain the exosome vector - Exo by ultracentrifugation; S2. The PCDH12 antibody was co-incubated with the DMPE-PEG-MAL conjugate, and the supernatant was discarded after centrifugation to obtain the PCDH12 antibody and DMPE-PEG-MAL conjugate complex. S3. The PCDH12 antibody obtained in S2 and the DMPE-PEG-MAL conjugate complex precipitate were resuspended in the vector exosomes extracted in S1, incubated, and then ultracentrifuged to obtain the engineered exosomes antiPCDH12-Exo.

2. The engineered exosomes as described in claim 1, characterized in that, In step S3, the mass ratio of the PCDH12 antibody, DMPE-PEG-MAL conjugate, and vector exosomes is 1:1:

1.

3. The application of engineered exosomes as described in claim 1 in the preparation of drugs targeting trabecular meshwork tissue, characterized in that, The engineered exosome is antiPCDH12-Exo.

4. The application as described in claim 3, characterized in that, The drug can be used alone or in combination with other drugs.

5. The application as described in claim 3, characterized in that, The drug dosage forms include eye drops or injections.

6. The use of the engineered exosomes as described in claim 1 in the preparation of a medicament for treating glaucoma, characterized in that, The engineered exosome is antiPCDH12-Exo; the drug has at least one of the following functions: (1) Targeted trabecular meshwork organization; (2) Reduce intraocular pressure in glaucoma animals; (3) Improves the thickness of the retinal nerve fiber layer, ganglion cell layer and inner plexiform layer.

7. The application as described in claim 6, characterized in that, Glaucoma includes primary glaucoma, secondary glaucoma, congenital glaucoma, and developmental glaucoma.

8. The application as described in claim 6, characterized in that, The drug can be used alone or in combination with other drugs.

9. The application as described in claim 6, characterized in that, The drug dosage forms include eye drops or injections.

10. The application of PCDH12 as a target in the preparation of drugs targeting trabecular meshwork, characterized in that, The drug includes PCDH12 inhibitors or PCDH12 antibodies.

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