Micelles capable of efficiently delivering recombinant adeno-associated viruses and preparation method of micelles
By using micellar technology containing Plannic and mTOR inhibitors, the gene delivery efficiency of AAV is improved and the production of neutralizing antibodies is reduced, and the problems of poor tissue tropism and difficulty in repeated administration of AAV in clinical applications are solved, achieving more efficient gene therapy effects.
Patent Information
- Application Number
- CN202311578470.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-24
- Publication Date
- 2025-05-27
AI Technical Summary
As a gene therapy vector, AAV faces poor tissue tropism, pre-storage neutralizing antibodies and difficulty in repeated administration in clinical applications, resulting in low transduction efficiency and enhanced immune response.
Using micelles containing Plannic and recombinant adeno-associated virus or Plannic, mTOR inhibitors and recombinant adeno-associated viruses, PF127 or PF127 and mTOR inhibitors were mixed with recombinant AAV by preparation to form micelles to improve the gene delivery capacity of AAV and reduce the production of capsid neutralizing antibodies.
It significantly enhances the infection efficiency of AAV in cells and organisms, improves the muscle site transgene expression by about 10 times, and reduces specific capsid neutralizing antibodies in organisms by 7-10 times, solving the difficulty of repeated administration of AAV and reducing the immunogenicity of AAV.
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Abstract
Description
Technical Field
[0001] The invention relates to a micelle for efficiently delivering recombinant adeno-associated virus and a preparation method thereof. Background Art
[0002] Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA parvovirus. It was first discovered as a byproduct of adenovirus preparation and got its name from it. As a gene therapy delivery vector, AAV has only 145 bp of AAV ITRs in its genome, which are necessary to ensure AAV continuous cell transduction and gene expression. The remaining 96% of the AAV genome can be removed to engineer AAV vectors for gene therapy. It is currently believed that AAV is large enough to accommodate the target gene expression cassette within 4.8 kb. Once it exceeds this, it may lead to a significant decrease in virus yield and transgene expression. At present, 12 natural AAV serotypes and more than 100 serotype mutants have been used in the study of gene delivery vectors. Different AAV serotypes have different tissue tropisms and binding receptors. Currently, several AAV serotypes have been used in clinical trials for patients with various diseases, and many products have been approved for marketing.
[0003] However, as a star vector for gene therapy, AAV also faces many problems in its clinical application. The first is the tissue tropism of AAV capsid. Although people have determined the tissue tropism of some AAV serotypes, there is also the problem that AAV is not efficient in infecting certain tissues. One solution currently adopted is to find mutant capsids. However, the acquisition cost of mutant capsids is high, and mutations may cause other adverse reactions. Secondly, there are pre-existing neutralizing antibodies to AAV in some people, which will affect the effect of AAV in the body. Even if the patient has less pre-existing neutralizing antibodies in the body, after the first administration, the patient will quickly produce a large number of specific neutralizing antibodies against AAV in the body. On the one hand, it makes repeated administration of AAV almost impossible, and on the other hand, the immune response to AAV may produce more adverse reactions. Although the advantage of AAV lies in the stability of long-term expression and low immunogenicity, at present, except for immortalized cells in the body, the lifespan of most cells is between several months and one year. Therefore, except for some indications where AAV-infected cells are immortalized cells, the lifespan of target cells infected by AAV in most other diseases is not long, which are all problems that lead to the inability to repeat administration.
[0004] In order to optimize the clinical application of AAV, in addition to molecular biological modification of the capsid, it is also necessary to solve the problems faced by AAV through formulation. Therefore, it is necessary to design a simple and easy-to-obtain formulation that can improve the transduction efficiency of AAV on the one hand and reduce the production of neutralizing antibodies on the other hand. This can greatly promote the application of AAV as a gene delivery vector in the field of gene therapy from rare diseases to common diseases. Summary of the invention
[0005] The purpose of the present invention is to provide a micelle for efficiently delivering recombinant adeno-associated virus and a preparation method thereof in view of the deficiencies in the prior art. The micelle can improve the gene delivery capacity of the recombinant adeno-associated virus and reduce capsid neutralizing antibodies.
[0006] The present invention provides a micelle for efficiently delivering a recombinant adeno-associated virus, characterized in that the micelle contains pluronic and the recombinant adeno-associated virus, or pluronic, an mTOR inhibitor and the recombinant adeno-associated virus.
[0007] The Pluronic described in the present invention includes but is not limited to PF127, preferably PF127.
[0008] The mTOR inhibitors of the present invention include but are not limited to rapamycin, temsirolimus, everolimus, etc., preferably rapamycin (RAPA).
[0009] The present invention provides a micelle for efficiently delivering recombinant adeno-associated virus, wherein the recombinant adeno-associated virus serotype includes all natural and artificially designed adeno-associated virus serotypes, characterized in that the natural adeno-associated virus serotypes include but are not limited to natural AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R and AAVrh10, and the artificially designed adeno-associated virus serotypes include but are not limited to 7m8, preferably AAV6 and AAV8.
[0010] The present invention provides a method for preparing micelles for efficiently delivering recombinant adeno-associated virus, the preparation method comprising the following steps:
[0011] Place PF127 or PF127 and mTOR inhibitor in a round-bottom flask and add appropriate amount of anhydrous ethanol to dissolve;
[0012] The solution obtained in step (1) is placed in a water bath, and ethanol is removed by using a rotary evaporator, and the ethanol is completely evaporated to obtain a film;
[0013] Add an appropriate amount of preheated virus stocking buffer to the film in step (2), and completely hydrate the film under water bath ultrasound to obtain blank micelles;
[0014] The blank micelles in step (3) are vortex-mixed with the recombinant adeno-associated virus and then incubated to obtain the result.
[0015] The virus storage buffer has a formula of: a sterile aqueous solution for injection containing 20mM Tris, 1mM MgCl2, 200mM NaCl, and 0.005% PF68, and the ratio of the recombinant adeno-associated virus, the PF127 and the mTOR inhibitor is in the range of 109vg / ml~1015vg / ml:0.05mM~6mM:0~3mM, preferably 1012vg / ml:0.03mM:0.6mM.
[0016] The present invention provides a method for preparing micelles for efficiently delivering recombinant adeno-associated virus, wherein the water bath temperature in step (2) is preferably 30-50°C, preferably 40°C, and the time for removing ethanol by a rotary evaporator is 10-30 minutes, preferably 20 minutes; the water bath ultrasound conditions in step (3) are 30-50°C, 100w-500w, preferably 40°C, 300w, and the incubation conditions in step (4) are 4-8°C, 1-4h, preferably 4°C, 2h.
[0017] The present invention provides an application of micelles for efficiently delivering recombinant adeno-associated viruses in preparing recombinant adeno-associated virus gene therapy drugs.
[0018] Furthermore, the medicine is a liquid preparation or a solid preparation.
[0019] Furthermore, the administration route of the liquid preparation is one or more of intra-articular injection, subretinal injection, suprachoroidal injection, intramuscular injection, intravenous injection, and transdermal administration. Preferably, the transdermal administration is one or more of subcutaneous injection and microneedle injection.
[0020] Beneficial effects:
[0021] The present invention provides a micelle for efficiently delivering recombinant adeno-associated virus and a preparation method thereof. The preparation method provided by the present invention is simple and fast. The micelle of the present invention has the following advantages: the micelle for efficiently delivering adeno-associated virus can significantly enhance the infection efficiency of AAV in cells and organisms, and the micelle can increase the transgenic expression of muscle parts by about 10 times by intramuscular injection of mice, and can reduce the specific capsid neutralizing antibodies in the organism by 7-10 times, thereby enhancing the effect of single administration of AAV and reducing the amount of AAV used, reducing costs; on the other hand, it is conducive to repeated administration of AAV, thereby better realizing its function as a gene therapy delivery vector; at the same time, it can play the effect of improving the gene delivery ability of recombinant adeno-associated virus and reducing capsid neutralizing antibodies in vivo for a variety of injection methods. The present invention uses micelles for efficiently delivering AAV, so that a low dose of AAV can play an effect equivalent to a high dose of AAV, solving the problem of high cost of AAV, and on the other hand, the micelle reduces the capsid neutralizing antibodies of AAV, solves the problem of repeated administration difficulties caused by capsid neutralizing antibodies, and reduces the adverse reactions caused by AAV immunogenicity. The micelles of this aspect have broad application prospects in the field of gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 .Hydrated particle size of PF127-RAPA-AAV6-GFP.
[0023] Figure 2 .Fluorescence expression of AAV6-GFP in HEK293T cells at different multiplicity of infection (MOI).
[0024] Figure 3 .Gene expression efficiency of Free-AAV6-GFP, PF127-AAV6-GFP and PF127-RAPA-AAV6-GFP in HEK293T cells.
[0025] Figure 4 .Gene expression efficiency of Free-AAV8-GFP, PF127-AAV8-GFP and PF127-RAPA-AAV8-GFP in ARPE19 cells.
[0026] Figure 5 .Gene expression efficiency of Free-AAV6-mcherry, PF127-AAV6-mcherry and PF68-mcherry in HEK293T cells.
[0027] Figure 6 .In vivo luciferase imaging of mice injected intra-articularly with PF127-RAPA-AAV6-Luc and Free AAV6-Luc at the ankle joint.
[0028] Figure 7 . Quantitative-temporal curve of luciferase expression intensity of PF127-RAPA-AAV6-Luc and Free AAV6-Luc injected into the ankle joint of mice.
[0029] Figure 8 . Specific capsid antibody titers in the serum of mice injected intra-articularly with PF127-RAPA-AAV6-Luc and Free AAV6-Luc.
[0030] Fig. 9 .In vivo luciferase imaging of mice gastrocnemius muscle injected with PF127-RAPA-AAV6-Luc and Free AAV6-Luc.
[0031] Fig.10 . Quantitative-temporal curve of luciferase expression intensity after intramuscular injection of PF127-RAPA-AAV6-Luc and Free AAV6-Luc into the gastrocnemius muscle of mice.
[0032] Fig.11 . Specific capsid antibody titers in the serum of mice injected intramuscularly with PF127-RAPA-AAV6-Luc and Free AAV6-Luc. DETAILED DESCRIPTION
[0033] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these examples are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
[0034] Example 1. Preparation of PF127 micelles
[0035] I. Preparation of PF127-RAPA-AAV, PF127-AAV and PF68-AAV
[0036] 1. Place a certain amount of PF127 and rapamycin in a 50 ml round-bottom flask and dissolve them with an appropriate amount of anhydrous ethanol to obtain a 0.4 mM PF127-0.6 mM rapamycin solution.
[0037] 2. The PF127-rapamycin solution obtained in step 1 was subjected to rotary evaporation for 20 min in a 40° C. water bath to obtain a PF127-rapamycin film.
[0038] 3. Add an appropriate amount of virus storage buffer preheated to 37° C. to the PF127-rapamycin film obtained in step 2, and perform water bath ultrasound (200-300w) at 37° C. for 1 minute to obtain PF127-rapamycin (PF127-RAPA) micelles.
[0039] 4. PF127 micelles were prepared according to the above steps without adding rapamycin.
[0040] 5. Without adding rapamycin, PF68 micelles were prepared using PF68 in the same manner as above.
[0041] 6. Add AAV6-GFP (or AAV8-GFP or AAV6-Luc or AAV6-mcherry) to the PF127-RAPA micelles, PF127 micelles and PF68 micelles obtained in steps 3, 4 and 5, vortex for one minute to mix evenly, incubate at 4°C for 2 hours, and name the final products PF127-RAPA-AAV6-GFP, PF127-AAV6-GFP, PF127-AAV6-mcherry and PF68-AAV6-mcherry (or PF127-RAPA-AAV8-GFP, PF127-AAV8-GFP or PF127-RAPA-AAV6-Luc), where Luc, GFP and mcherry represent recombinant adeno-associated viruses carrying reporter genes Luciferase, GFP and mcherry. The hydrated particle size of PF127-RAPA-AAV6-GFP is as follows: Figure 1 shown.
[0042] Example 2. Experiment on improving the expression efficiency of recombinant adeno-associated virus gene at the cellular level using PF127 micelles
[0043] 1. Gene expression efficiency of PF127-RAPA-AAV6-GFP in HEK293T cells
[0044] The PF127-RAPA-AAV6-GFP and PF127-RAPA-AAV8-GFP prepared in Example 1 were subjected to the following experiments respectively:
[0045] 1. Determine the optimal multiplicity of infection (MOI)
[0046] HEK293T cells were infected with free AAV6-GFP at a multiplicity of infection (MOI) of 0, 700, 1400, 3500, 10500, and 35000. After 24 h of infection, laser confocal microscopy was performed to determine the GFP expression intensity. The results are shown in Figure 2The results showed that obvious GFP expression could be observed at MOI35000. At the same time, the fluorescence intensity decreased significantly with the decrease of MOI, indicating that MOI35000 did not reach the saturation point. Therefore, if the micelles have an effect on improving the efficiency of AAV gene expression, the fluorescence intensity should be enhanced. Selecting an appropriate amount of virus will help observe the results.
[0047] 2. Gene expression efficiency of PF127-RAPA-AAV6-GFP / PF127-AAV6-GFP in HEK293T cells
[0048] HEK293T cells were seeded in a 12-well plate at 1E+5 cells / well, and PF127-RAPA-AAV6-GFP was obtained according to the method in Example 1 at an MOI of 35000 (i.e., the amount of virus used was 3.5E+9 vg / well). HEK293T cells were infected with free AAV6-GFP, FP127-AAV6-GFP, and PF127-RAPA-AAV6-GFP, respectively, and 3 wells were operated in parallel. After 3 days of infection, the cells were observed under a laser confocal microscope, and the positive cell rate was quantified by flow cytometry. The results are shown in Figure 3 The results showed that the gene expression efficiency of PF127-AAV6-GFP and PF127-RAPA-AAV6-GFP was significantly higher than that of free AAV6-GFP, proving that PF127-RAPA and PF127 micelles can significantly enhance the gene expression efficiency of AAV6.
[0049] II. Gene expression efficiency of PF127-RAPA-AAV8-GFP / PF127-AAV8-GFP in ARPE19 cells
[0050] ARPE19 cells were seeded in a 12-well plate at 1E+5 cells / well, and PF127-RAPA-AAV8-GFP was obtained according to the method in Example 1 at an MOI of 75000 (i.e., a virus dosage of 7.5E+9 vg / well). ARPE19 cells were infected with free AAV8-GFP, PF127-AAV8-GFP, and PF127-RAPA-AAV8-GFP, respectively, and 3 wells were operated in parallel. After 3 days of infection, the cells were observed under a laser confocal microscope, and then the cells were collected and counted by flow cytometry to quantify the positive cell rate. The results are shown in Figure 4 The results showed that the gene expression efficiency of PF127-AAV8-GFP and PF127-RAPA-AAV8-GFP was significantly higher than that of free AAV8-GFP, proving that PF127-RAPA micelles and PF127 micelles can significantly enhance the gene expression efficiency of AAV8.
[0051] III. Gene expression efficiency of PF127-AAV6-mcherry and PF68-AAV6-mcherry in HEK293T cells
[0052] HEK293T cells were seeded in a 12-well plate at 1E+5 cells / well. PF127-AAV6-mcherry and PF68-AAV6-mcherry were obtained according to the method in Example 1 at an MOI of 35000 (i.e., the amount of virus used was 3.5E+9 vg / well). Free AAV6-mcherry, FP127-AAV6-mcherry and PF68-AAV6-mcherry were used to infect HEK293T cells, respectively, and three wells were operated in parallel. After 3 days of infection, the cells were observed under a laser confocal microscope, and the positive cell rate was quantified by flow cytometry. The results are shown in Table 1. Figure 5 The results showed that the expression ability of PF68 micelles loaded with adeno-associated virus was lower than that of free adeno-associated virus, and the expression ability of PF127 micelles loaded with adeno-associated virus was significantly stronger than that of free adeno-associated virus and PF68 micelles.
[0053] Example 3. Experiment on improving the gene expression efficiency of recombinant adeno-associated virus in mice after intra-articular injection of PF127 micelles
[0054] PF127-RAPA-AAV6-Luc was obtained according to the method in Example 1 and the following experiment was performed:
[0055] Female 6-week-old Balb / c mice weighing about 20 g (purchased from Jiangsu Jicui Pharmaceutical) were selected and injected with free AAV6-Luc or PF127-RAPA-AAV6-Luc (virus amount was 1E+9vg / ankle) through the bilateral ankle joints using a Hamilton syringe. The mice were housed in separate cages to ensure normal food and water intake. The mice were observed by in vivo imaging 4, 7, and 14 days after injection. At each time point, each mouse was intraperitoneally injected with 200ul D-luciferin potassium salt substrate.
[0056] The luciferase expression intensity was measured by small animal in vivo imaging system ( Lumina LT Series III, PerkinElmer, Germany) analysis, the results are shown in Figure 6 At the same time, the curve of fluorescence intensity changing with time is Figure 7 The results showed that PF127-RAPA micelles can still quickly enhance the efficiency of AAV gene expression in vivo, with strong expression on the 7th day and high expression efficiency for a long time, while the expression of free adenovirus was very low in the early stage due to less uptake, and as time went on, a small amount of free virus also had a certain expression.
[0057] Example 4. Effect of PF127 micelles injected into ankle joint cavity on neutralizing antibodies against AAV capsid in mice
[0058] PF127-RAPA-AAV6-Luc was obtained according to the method in Example 1 and the following experiment was performed:
[0059] Female 6-week-old Balb / c mice weighing about 20 g (purchased from Jiangsu Jicui Pharmaceutical) were selected and injected with free AAV6-Luc or PF127-RAPA-AAV6-Luc (virus amount of 1E+9 vg / ankle) through the bilateral ankle joints using a Hamilton syringe. The mice were housed in separate cages to ensure normal food and water intake. Blood was collected from the mouse orbits on the 28th and 54th days after injection, and serum was collected for capsid-specific antibody detection by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 8 The results showed that PF127-RAPA micelles significantly reduced AAV capsid-specific antibodies in mice, facilitating repeated administration of AAV.
[0060] Example 5. Experiment on improving the gene expression efficiency of recombinant adeno-associated virus in mice after intramuscular injection of PF127 micelles
[0061] PF127-RAPA-AAV6-Luc was obtained according to the method in Example 1 and the following experiment was performed:
[0062] Female 6-week-old Balb / c mice weighing about 20 g (purchased from Jiangsu Jicui Pharmaceutical) were selected, and free AAV6-Luc or PF127-RAPA-AAV6-Luc (virus amount of 2E+9vg / leg) was injected into the bilateral gastrocnemius muscles. The mice were housed in separate cages to ensure normal food and water intake. The mice were observed by live imaging 4, 7, 14, 21, and 30 days after injection. At each time point, each mouse was intraperitoneally injected with 200ul D-luciferin potassium salt substrate.
[0063] The luciferase expression intensity was measured by small animal in vivo imaging system ( Lumina LT Series III, PerkinElmer, Germany) analysis, the results are shown in Fig. 9 The fluorescence intensity versus time curve is Fig.10Indicated. The results show that PF127-RAPA micelles still have the effect of enhancing the efficiency of AAV gene expression through intramuscular injection. The results of Examples 3 and 5 show that the micelles can effectively improve the efficiency of AAV actively infecting cells in the body in vivo, while the current methods mainly limit the distribution of AAV in the body through gel preparations, so that more AAV stays in place, thereby enhancing the local infection efficiency of AAV. Due to its physical properties, the micelles do not limit the distribution of AAV in the body, so they are suitable for more scenarios and can better promote AAV infection of parts that are difficult to reach by conventional injections.
[0064] Example 6. Effect of PF127 micelles on neutralizing antibodies against AAV capsid in mice after intramuscular injection
[0065] PF127-RAPA-AAV6-Luc was obtained according to the method in Example 1 and the following experiment was performed:
[0066] Female 6-week-old Balb / c mice weighing about 20 g (purchased from Jiangsu Jicui Pharmaceutical) were selected and injected with free AAV6-Luc or PF127-RAPA-AAV6-Luc (virus amount of 2E+9 vg / leg) into the bilateral gastrocnemius muscles. The mice were housed in separate cages to ensure normal food and water intake. On the 33rd day after injection, blood was collected from the mouse orbits, and serum was collected for capsid-specific antibody detection by enzyme-linked immunosorbent assay (ELISA). The results are shown in Fig.11 The results showed that PF127-RAPA micelles could also significantly reduce AAV capsid-specific antibodies by intramuscular injection, which is helpful for repeated administration of AAV.
[0067] Summarize
[0068] In summary, the results of the present invention suggest that PF127 micelles can effectively improve the infection efficiency of AAV in vivo and in vitro and reduce its specific capsid antibodies. On the one hand, it can reduce the dosage of AAV and thus reduce costs, making it possible for expensive drugs such as AAV to be available to a wider range of patients. At the same time, both intra-articular injection and intramuscular injection can improve the infection ability of AAV, indicating that the micelles are suitable for a variety of administration methods and benefit more patients. On the other hand, PF127 micelles can reduce AAV capsid neutralizing antibodies, indicating that it is conducive to AAV immune escape and reduce its immunogenicity, thereby helping to solve the problem of difficulty in secondary administration of AAV required for some common diseases, and also avoiding adverse reactions caused by AAV immunogenicity. It can be seen that the micelles of the present invention have the potential to optimize recombinant adeno-associated viruses as gene therapy drugs.
[0069] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. A micelle for efficient delivery of recombinant adeno-associated virus, It is characterized in that The micelles contain pluronic and recombinant adeno-associated virus, or pluronic, an mTOR inhibitor and a recombinant adeno-associated virus.
2. The micelle according to claim 1, Features The Pluronic includes but is not limited to PF127, preferably PF127.
3. The micelle according to claim 1, Features The mTOR inhibitor includes but is not limited to one or more of rapamycin, temsirolimus, and everolimus, preferably rapamycin. RAPA).
4. The micelle according to claim 1, It is characterized in that The recombinant adeno-associated virus serotypes include all natural and artificially designed adeno-associated virus serotypes, characterized in that the natural adeno-associated virus serotypes include but are not limited to natural AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R and AAVrh10, and the artificially designed adeno-associated virus serotypes include but are not limited to 7m8.
5. A method for efficiently delivering micelles of recombinant adeno-associated virus according to claim 1, Features The following steps are involved: (1) Place pluronic or pluronic and mTOR inhibitor in a round-bottom flask and add appropriate amount of anhydrous ethanol to dissolve; (2) removing ethanol from the solution obtained in step (1) using a rotary evaporator under water bath conditions, and completely evaporating the ethanol to obtain a film; (3) adding an appropriate amount of preheated virus storage buffer to the film of step (2), and using water bath ultrasound to completely hydrate the film to obtain blank micelles; (4) Take the blank micelles of step (3) and vortex mix with the recombinant adeno-associated virus to obtain.
6. The preparation method according to claim 5, It is characterized in that The ratio of the recombinant adeno-associated virus, the pluronic and the mTOR inhibitor is 10 9 vg / ml~10 15 vg / ml:0.05mM~6mM:0~3mM.
7. Use of the micelle for efficiently delivering recombinant adeno-associated virus as claimed in any one of claims 1 to 4 in the preparation of recombinant adeno-associated virus gene therapy drugs.
8. The use according to claim 7, It is characterized in that The medicine is a liquid preparation or a solid preparation.
9. The use according to claim 7, It is characterized in that The administration route of the liquid preparation is one or more of intra-articular injection, subretinal injection, suprachoroidal injection, intramuscular injection, intravenous injection, and transdermal administration. Preferably, the transdermal administration is one or more of subcutaneous injection and microneedle injection.