Application of rAAV-mediated PEDF gene in treatment of uveitis

Abstract

The invention relates to an application of an rAAV-mediated PEDF gene in treatment of uveitis, specifically, a recombinant adeno-associated virus is used as a gene delivery carrier, the PEDF gene is efficiently introduced into a vitreous cavity, and continuous and stable expression of PEDF in eyes is realized, so that the PEDF is used for treating autoimmune uveitis, and a new thought is provided for development and treatment of new clinical drugs for uveitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of rAAV-mediated PEDF gene in treating uveitis. Background Art

[0002] Autoimmune uveitis is a common blinding eye disease with a complex pathogenesis, frequent recurrences, and difficult-to-control inflammation, which can lead to serious complications and visual impairment. Uveitis affecting the posterior segment, in particular, requires systemic treatment with glucocorticoids, immunotherapies, and biologics, which can cause systemic toxic side effects such as gastrointestinal reactions and liver and kidney damage. Therefore, the search for safe and effective therapeutic agents remains a key research priority in the treatment of uveitis.

[0003] Pigment epithelium-derived factor (PEDF) is a 50kDa secretory glycoprotein that belongs to the serine protease inhibitor gene superfamily. PEDF is involved in the occurrence and development of various eye diseases, exerting anti-inflammatory, anti-oxidative stress, anti-angiogenesis and neuroprotective effects. It has important potential value in the treatment of diseases such as dry eye, corneal damage, age-related macular degeneration (ARMD), and diabetic retinopathy (DR).

[0004] Adeno-associated virus (AAV) is the simplest, non-enveloped, single-stranded DNA virus discovered to date. It consists of a protein capsid and a 4.7kb single-stranded DNA genome, with the capsid measuring 20 to 25 nanometers in length. It belongs to the parvovirus family. The AAV genome is terminated by inverted terminal repeats (ITRs), which serve as the starting point for viral DNA replication and the signal that triggers viral packaging. AAV cannot replicate on its own and must rely on other viruses, such as adenoviruses, herpes viruses, and baculoviruses, for replication. Over 80% of the human population carries AAV, and AAV has not been associated with any disease.

[0005] Recombinant adeno-associated virus (rAAV) is an engineered AAV vector that has had all of the Rep or Cap gene sequences removed from the AAV genome, leaving only the trans-acting DNA sequences (i.e., ITRs) at either end that serve as packaging signals. The protein capsid carried by rAAV used as a gene therapy vector is nearly identical to that of wild-type AAV, but the portion of the genome that encodes viral proteins has been completely replaced by the therapeutic transgene.

[0006] Given this utility, there is a need to develop new agents and methods for treating autoimmune uveitis. Summary of the Invention

[0007] To overcome the above-mentioned drawbacks, the present invention provides an application of rAAV-mediated PEDF in the preparation of a drug for treating uveitis. rAAV-PEDF is administered at a certain concentration via intravitreal injection to treat uveitis, providing a new approach for the development of new clinical drugs for the treatment of uveitis. The details are as follows:

[0008] In a first aspect, the present invention provides a use of a fusion construct in preparing a drug for treating uveitis, wherein the fusion construct comprises rAAV and PEDF.

[0009] Preferably, the adeno-associated virus capsid in the rAAV includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 capsids or their variants.

[0010] In one embodiment, the adeno-associated virus capsid is AAV2.

[0011] Preferably, the uveitis includes autoimmune uveitis, and further preferably, the uveitis is experimental autoimmune uveitis.

[0012] Preferably, the drug is a topically administered drug, more preferably, the topically administered drug is an ocular drug, and more preferably, the topically administered drug includes intravitreal injection.

[0013] Preferably, the concentration of the fusion construct is no higher than 10 9 vg / μl, further preferably, the concentration of the fusion construct includes 10 8 -10 9 vg / μl.

[0014] Preferably, the rAAV in the fusion construct promotes the expression of PEDF.

[0015] Preferably, the fusion construct modulates the expression of helper T cells.

[0016] Preferably, the helper T cells include Treg cells, Th1 cells or Th17 cells.

[0017] Preferably, the fusion construct promotes the expression of Treg cells and inhibits the expression of Th1 cells and Th17 cells.

[0018] Preferably, the drug may further contain other vision-improving ingredients. Further preferably, the other vision-improving ingredients include anthocyanidins, lutein, docosahexaenoic acid, astaxanthin, lycopene, taurine, panthenol, potassium aspartate, chondroitin sulfate, zinc, calcium or magnesium, etc.

[0019] In a second aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises rAAV, PEDF and pharmaceutically acceptable excipients.

[0020] Preferably, the adeno-associated virus capsid in the rAAV includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 capsids or their variants.

[0021] In one embodiment, the adeno-associated virus capsid is AAV2.

[0022] Preferably, the pharmaceutically acceptable excipients include but are not limited to diluents, adhesives, lubricants, wetting agents, etc.

[0023] Preferably, the drug may further contain other vision-improving ingredients. Further preferably, the other vision-improving ingredients include anthocyanidins, lutein, docosahexaenoic acid, astaxanthin, lycopene, taurine, panthenol, potassium aspartate, chondroitin sulfate, zinc, calcium or magnesium, etc.

[0024] In a third aspect, the present invention provides a method for treating uveitis, wherein the method comprises administering a fusion construct or the above-mentioned pharmaceutical composition to a subject, wherein the fusion construct comprises rAAV and PEDF.

[0025] Preferably, the adeno-associated virus capsid in rAAV includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 capsids or variants thereof.

[0026] In one embodiment, the adeno-associated virus capsid is AAV2.

[0027] Preferably, the uveitis includes autoimmune uveitis, and further preferably, the uveitis is experimental autoimmune uveitis.

[0028] Preferably, the administration method is topical administration, more preferably, the topical administration is ocular administration, and more preferably, the topical administration includes intravitreal injection.

[0029] Preferably, the administration concentration is not higher than 10 9 vg / μl, further preferably, the administration concentration includes 10 8 -10 9 vg / μl.

[0030] The subject in the present invention can be a mammal, such as a human, monkey, dog, rabbit, mouse, rat, etc.

[0031] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0032] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0033] Beneficial effects of the present invention:

[0034] This study used intravitreal injection of recombinant adeno-associated virus (rAAV)-mediated pigment epithelium-derived factor (PEDF) gene to treat uveitis, which has the following advantages:

[0035] 1. Local effect: Intravitreal injection is a local drug delivery method that can deliver therapeutic genes directly to ocular tissues and reduce systemic side effects.

[0036] 2. Efficient expression: Introducing the PEDF gene into the eye through the rAAV vector can achieve continuous and efficient expression of PEDF in the eye, thus meeting treatment needs.

[0037] 3. High safety: rAAV vectors have low immunogenicity and good biosafety, and can reduce immune responses and cytotoxicity.

[0038] 4. Easy to operate: Intravitreal injection is a relatively simple and safe surgical method that is easy for patients to accept.

[0039] 5. Potential multiple therapeutic effects: PEDF not only has anti-inflammatory effects, but also anti-angiogenesis and neurotrophic effects. It can treat multiple pathological processes of uveitis, thereby improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : The expression of PEDF protein was detected by Western blot, with GPDH as the internal control.

[0041] Figure 2 : A. Representative fundus images of mice in different concentration groups 7, 14, and 21 days after intravitreal injection of rAAV-PEDF. B. Representative OCT images of mice in each group.

[0042] Figure 3 : A. Representative fundus images of mice in different concentration groups 7, 14, and 21 days after intravitreal injection of rAAV-GFP. B. Representative OCT images of mice in each group. White arrows represent inflammatory cells in the vitreous cavity.

[0043] Figure 4 : Representative HE staining images of mice in different concentration groups 21 days after intravitreal injection. The black arrows represent inflammatory cells in the vitreous cavity.

[0044] Figure 5 : The expression of GFP green fluorescence in the retina of mice was detected under a fluorescence microscope. No fluorescence expression was observed in the retinal sections of the mice in the PBS group; while in the retinal sections of the mice in the rAAV-GFP group, EGFP green fluorescence was observed in the retina, mainly distributed in the inner and outer nuclear layers (ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer).

[0045] Figure 6 : Clinical score curves of EAU mice after intravitreal injection of PBS, rAAV-GFP, and rAAV-PEDF, **P<0.001, **P<0.01, *P<0.5, Kruskal-Wallis test.

[0046] Figure 7 : Clinical evaluation of the therapeutic effect, A. Representative fundus images of EAU mice in each group 18 days after immunization, B. Representative OCT images of EAU mice in each group 18 days after immunization, the white arrows represent inflammatory cells in the vitreous cavity.

[0047] Figure 8: Histopathological evaluation. A. Representative HE-stained images of EAU mice in each group 21 days after immunization. Black arrows indicate retinal wrinkles and detachment around the optic disc. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; Choroid, choroid. B. Retinal histopathological scores of EAU mice in each group. **P<0.01, *P<0.5, Kruskal-Wallis test.

[0048] Figure 9 : Flow cytometry was used to detect the proportions of CD4+ T cells in the cervical draining lymph nodes and spleen of EAU mice in each group. A. Representative images of the proportions of CD4+Foxp3+, CD4+IFN-γ+, and CD4+IL-17A+ cells in the cervical draining lymph nodes and spleen of mice in each group. B. The proportions of CD4+Foxp3+ cells in the cervical draining lymph nodes of mice in each group. C. The proportions of CD4+Foxp3+ cells in the spleen of mice in each group. D. The proportions of CD4+IFN-γ+ and CD4+IL-17A+ cells in the cervical draining lymph nodes of mice in each group. E. The proportions of CD4+IFN-γ+ and CD4+IL-17A+ cells in the spleen of mice in each group. **P<0.01, *P<0.5, one-way analysis of variance. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0051] Example 1 Expression of rAAV vectors in the retina after intravitreal injection and evaluation of safe dose

[0052] Experimental methods:

[0053] 1. Expression of AAV vectors in the retina and evaluation of safe doses after intravitreal injection

[0054] 1.1 Intravitreal injection

[0055] Mice were anesthetized with tropicamide compound eye drops (0.2 ml / 10 g) injected intraperitoneally to dilate the pupils. After anesthesia, the mice were placed on a microscope stage, the eyeballs exposed, and one drop of proparacaine hydrochloride eye drops was instilled. The microscope focus knob was adjusted to ensure clear visualization of the fundus, with the optic disc centered in the field of view. A sterile 34-gauge needle was used to create an incision 1 mm below the limbus. A Hamilton 34-gauge microinjection needle was inserted along the incision, avoiding the lens, and advanced to the anterior aspect of the optic disc. An assistant then slowly advanced the needle to inject 1 μl each of rAAV-GFP and rAAV-PEDF into each eye. After injection, the eyes were covered with tobramycin ointment to prevent infection and dryness, and the mice were placed on a heating blanket until fully awake. Fundus photography and optical coherence tomography (OCT) examinations were performed 1, 2, and 3 weeks after injection.

[0056] 1.2 Western blotting to detect PEDF protein expression in the retina

[0057] Three weeks after intravitreal injection, some mice in each group were killed, their eyes were removed, and the retinas were dissected and placed in 1.5 ml EP tubes. A mixture of RIPA and PMSF (100:1) was added to lyse the retinas and extract the proteins. The protein was quantified by the BCA method, and an equal amount of protein was loaded for Western blot detection of PEDF expression in the retina.

[0058] 1.3 Fundus imaging and OCT testing

[0059] Fundus examinations were performed using a Phoenix Micron IV fundus module and a Heidelberg Spectralis OCT at 7, 14, and 21 days after intravitreal injection. A +25D lens was added to the standard OCT lens to capture images and scans centered on the optic disc and lesion area. The aim was to assess whether injection of different concentrations of rAAV-GFP and rAAV-PEDF induced intraocular inflammation.

[0060] 1.4 Histopathological evaluation

[0061] Twenty-one days after intravitreal injection, mice were sacrificed and their eyeballs removed. The removed eyeballs were fixed in fixative and then embedded in paraffin. The tissue blocks were sectioned at 4 μm thickness and stained with hematoxylin and eosin. The mouse retinal structure was observed and photographed under a light microscope. The injection of different concentrations of rAAV-GFP and rAAV-PEDF was evaluated to determine whether they induced structural changes in the retina. A safe dose for subsequent experiments was determined based on fundus imaging, optical coherence tomography (OCT), and histopathological evaluation. This safe dose was then considered the appropriate injection dose for subsequent experiments.

[0062] 1.5 Eyeball frozen section and staining

[0063] The rAAV-GFP dose selected for subsequent experiments was injected into the vitreous cavity of mice. Three weeks later, the mice were sacrificed and the eyeballs were removed. The eyeballs were embedded in OCT embedding medium and placed in liquid nitrogen for quick freezing. The slices were set to 8 μm thick in a freezing microtome and evenly sliced along the cornea-optic nerve axis. 4% paraformaldehyde was added to the cut eyeball slices for 30 minutes, and the slides were washed with PBS three times for 5 minutes each. DAPI staining solution was then added and stained at room temperature for 10 minutes. The slides were washed with PBS three times for 5 minutes each and then mounted with anti-fluorescence quencher. The expression of DAPI blue-stained cell nuclei and GFP green fluorescence in the interretinal layers was observed under a confocal microscope.

[0064] Experimental results:

[0065] 1. Expression of rAAV vectors in the retina and evaluation of safe doses after intravitreal injection

[0066] 1.1 Western blotting to detect PEDF protein expression in the retina

[0067] Mouse retina western blot analysis results are as follows Figure 1 As shown in the figure, three weeks after injection, the total PEDF protein expression in the rAAV-PEDF group was significantly higher than that in the rAAV-GFP group, and with the increase of rAAV-PEDF injection dose, the expression of PEDF protein in the retina also increased accordingly.

[0068] 1.2 Fundus imaging and OCT testing

[0069] Fundus and OCT images were taken 7, 14, and 21 days after intravitreal injection. Figure 2 、 3 Slight inflammation was observed in the fundus of mice in the rAAV-GFP and rAAV-PEDF groups with a titer of 1E9 vg / μl on day 21 after injection.

[0070] 1.3 Histopathological evaluation

[0071] Pathological tissue sections were taken 21 days after different concentrations of rAAV-PEDF and rAAV-GFP were injected into the vitreous cavity of each group of mice. The results showed that there was almost no difference in the retinal structure among the three dose groups of rAAV-PEDF and rAAV-GFP. However, obvious inflammatory cells were observed in the vitreous cavity of the high-dose group ( Figure 4 ).

[0072] 2.5 Eyeball frozen section and staining

[0073] 1 μl rAAV-GFP (titer: 5E8 vg / μl) was injected into the vitreous cavity of mice. The mice were killed 3 weeks after injection, and the eyeballs were removed, fixed and embedded in OCT, and frozen sections were made. The GFP green fluorescence expression in the sections was observed under a confocal microscope. It was mainly concentrated in the outer nuclear layer and the inner nuclear layer of the retina ( Figure 5 ).

[0074] Example 2: Use of rAAV-PEDF in the treatment of experimental autoimmune uveitis

[0075] Experimental methods:

[0076] 1. Study on the treatment of experimental autoimmune uveitis by intravitreal injection of rAAV-PEDF

[0077] 1.1 Research subjects and groups

[0078] 4-5 week old C57BL6J mice were randomly divided into 3 groups:

[0079] (1) PBS group: 1 μl of PBS was injected into the vitreous cavity of both eyes;

[0080] (2) rAAV-GFP group: 1 μl of rAAV-GFP was injected into the vitreous cavity of both eyes;

[0081] (3) rAAV-PEDF group: 1 μl of rAAV-PEDF was injected into the vitreous cavity of both eyes.

[0082] 1.2 Induction of EAU model in mice

[0083] Three weeks after intravitreal injection, mice were injected subcutaneously with IRBP 651-670 Induce EAU model. 651-670 The peptide (300 μg / mouse) was mixed with equal volumes of complete Freund's adjuvant containing Mycobacterium tuberculosis (3.5 mg / ml) in two 5 ml syringes connected by a three-way valve. The mixture was thoroughly mixed and emulsified on ice until it became white and milky and dissolved slowly when dropped into water. After general anesthesia, mice were injected subcutaneously with 200 μl of the emulsion bilaterally in the back and groin. 0.5 μg of pertussis toxin was injected intraperitoneally 30 minutes before and 24 hours after model establishment.

[0084] 1.3 Evaluation of therapeutic effect: clinical scoring

[0085] Mice were observed every other day starting on day 9 after immunization. Tropicamide compound eye drops were first used to dilate the pupils. After pupil dilation, binocular indirect ophthalmoscopes were used to observe the fundus of the mice for pathological changes. Clinical scores were recorded according to the Caspi criteria. The Caspi scoring criteria are shown in Table 1.

[0086] Table 1. Clinical scoring criteria for Caspi EAU mice

[0087]

[0088] 1.4 Assessment of therapeutic effect: optical coherence tomography

[0089] On the 18th day after immunization, the mice were anesthetized, their pupils were dilated, artificial tears were instilled, levofloxacin gel was applied to the eye, and contact lenses were worn. The eyes were photographed using an optical coherence tomography scanner, with the mouse optic disc as the center of the scan.

[0090] 1.5 Evaluation of therapeutic effect: histopathological scoring

[0091] On the 21st day after the model was established, the mice were killed and their eyeballs were removed. The removed eyeballs were fixed in a fixative and then embedded in paraffin tissue. The tissue wax blocks were sliced to a thickness of 4 μm and then stained with HE.

[0092] The mouse retinal structure was observed and photographed under an optical microscope. Histopathological scoring was performed according to the CASPI criteria. The scoring criteria are shown in Table 2.

[0093] Table 2. Histopathological scores of Caspi EAU mice

[0094]

[0095] 1.6 Evaluation of therapeutic effects: Flow cytometry analysis of CD4+ T cell subsets in the lymph nodes and spleen of EAU mice

[0096] Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17A+) cells are the main pathogenic T cells in autoimmune diseases, while Treg cells (CD4+CD25+Foxp3+) play an important role in reducing autoimmune reactions. To further verify the therapeutic effect of intravitreal rAAV-PEDF on EAU mice, we used flow cytometry to detect the effects on CD4+ T cell subsets in the cervical draining lymph nodes and spleen of EAU mice at the peak of disease.

[0097] Preparation of cervical draining lymph node and spleen cell suspensions: On day 18 after immunization, mice were killed by cervical dislocation, the skin was cut open along the midline of the abdomen, and the cervical draining lymph nodes and spleen of the mice were removed and immersed in sorting buffer (buffer configuration: 50 ml PBS + 0.05 g BSA) and ground with a grinding rod. Both tissue suspensions were filtered through a 200-mesh filter and centrifuged at 1800 rpm / min for 5 min. The supernatant was discarded and 1 ml of 1640 complete culture medium was added to completely suspend the cells for flow cytometric analysis.

[0098] Treg cell detection: Add the above cells into the flow cytometry tube and prepare Brilliant Viloet TM 711-anti-CD4 and FITC-anti-CD25 antibody dilutions were incubated at 4°C in the dark for 30 minutes, 1 ml of PBS was added, the cells were centrifuged at 1800 r for 5 minutes, the supernatant was discarded, cell fixative was added for 60 minutes, the cells were centrifuged at 1800 r for 5 minutes, the supernatant was discarded, and then permeabilization solution was added, the cells were centrifuged at 1800 r for 5 minutes, the supernatant was discarded, and finally PE-anti-Foxp3 antibody dilutions were added and incubated at 4°C in the dark for 30 minutes, and then centrifuged. An appropriate amount of PBS was taken to resuspend the stained cells and the proportion of CD4+CD25+Foxp3+ cells was detected on a flow cytometer.

[0099] Th1 and Th17 cell assay: Prepare 1640 full culture stimulation solution containing 50ng / ml PMA, 1μg / ml ionomycin and 1μg / ml BFA. Use this stimulation solution to stimulate the above cells in a 96-well plate at 37℃ for 5 hours, then collect the cells and add Brilliant Viloet TM 711-anti-CD4 antibody dilution solution was incubated at 4°C in the dark for 30 minutes, 1 ml of PBS was added, the cells were centrifuged at 1800r for 5 minutes, the supernatant was discarded, cell fixative was added for 60 minutes, the cells were centrifuged at 1800r for 5 minutes, the supernatant was discarded, and then permeabilization solution was added, the cells were centrifuged at 1800r for 5 minutes, the supernatant was discarded, and finally FITC-anti-IFN-γ and PE-anti-IL-17 antibody dilution solution was added and incubated at 4°C in the dark for 30 minutes, and then centrifuged. An appropriate amount of PBS was taken to resuspend the stained cells and the proportion of CD4+IFN-γ+ and CD4+IL-17+ cells was detected on a flow cytometer.

[0100] Experimental results:

[0101] 1. Local therapeutic effect of intravitreal injection of rAAV-PEDF on EAU in mice

[0102] Clinical scoring results showed that ( Figure 6 ), on days 15-21 after immunization, the clinical scores of the rAAV-PEDF group were lower than those of the PBS group and the rAAV-GFP control group, and the differences were statistically significant; when photographing the fundus of mice at the peak of EAU onset, a large number of linear fusion lesions were observed in the fundus of mice in the PBS group and the rAAV-GFP group, while the lesions in the rAAV-PEDF treatment group were relatively limited, with only a few small punctate lesions around the optic disc ( Figure 7 A). OCT images taken at the same time point showed that there were fewer inflammatory cells in the vitreous of mice treated with rAAV-PEDF, and there was almost no damage to the retinal structure ( Figure 7 B). 21 days after immunization, histopathological evaluation was performed. The results showed that rAAV-PEDF could effectively protect the retinal structure, with no obvious retinal wrinkles. The score of the rAAV-PEDF treatment group was significantly lower than that of the PBS group and the rAAV-GFP control group, and the difference was statistically significant (P < 0.05). ( Figure 8 ).

[0103] Effects of intravitreal injection of rAAV-PEDF on the immune response of EAU mice

[0104] The results of flow cytometry analysis showed that ( Figure 9 ), compared with the PBS group and rAAV-GFP control group, the proportion of Treg (CD4+Foxp3+CD25+) cells in the cervical draining lymph nodes and spleen of the mice in the rAAV-PEDF group was increased and higher than that in the control group, and the difference was statistically significant (P>0.05); the proportions of Th1 (CD4+IFN-γ) and Th17 (CD4+IL-17A+) cells also showed a downward trend (P>0.05).

[0105] Experimental conclusion:

[0106] 1. rAAV-PEDF can be stably expressed in vivo, and the expression level is positively correlated with the amount of injected virus.

[0107] 2. Excessive doses of rAAV can cause inflammation in the eye, so an appropriate dose of rAAV should be selected for intraocular injection.

[0108] 3. rAAV-PEDF showed good therapeutic effects in the EAU model. Compared with the PBS and rAAV-GFP groups, it can significantly reduce EAU in mice. The use of rAAV-PEDF for local precision treatment can reduce the number and frequency of injections in patients with autoimmune uveitis, and also reduce the side effects of long-term systemic application. This patent provides new ideas for the development of new clinical drugs for the treatment of uveitis.

[0109] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. Use of a fusion construct in the preparation of a drug for treating uveitis, characterized in that: The fusion construct includes rAAV and PEDF.

2. The use according to claim 1, characterized in that The adeno-associated virus capsid in the rAAV includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 capsids or their variants.

3. The use according to claim 1 or 2, characterized in that The uveitis includes autoimmune uveitis.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is a topical drug, and preferably, the topical drug is ocular drug.

5. The application according to claim 4, characterized in that: The local administration includes intravitreal injection.

6. The application according to any one of claims 1 to 5, characterized in that: The concentration of the fusion construct is no more than 10 9 vg / μl.

7. The use according to any one of claims 1 to 6, characterized in that: The rAAV in the fusion construct promotes the expression of PEDF.

8. The use according to any one of claims 1 to 7, characterized in that: The drug further comprises other vision-improving ingredients. Preferably, the other vision-improving ingredients include anthocyanidins, lutein, docosahexaenoic acid, astaxanthin, lycopene, taurine, panthenol, potassium aspartate, chondroitin sulfate, zinc, calcium or magnesium.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises rAAV, PEDF and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The adeno-associated virus capsid in the rAAV includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 capsids or their variants.

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