A composition and use thereof for regenerating retinal ganglion cells by utilizing endogenous microglia

Abstract

The application discloses a kind of compositions and applications for regenerating retinal ganglion cells by using endogenous microglia.The present application finds that multiple genes (Brn3b, Sox2, Cbln1 and NP1, referred to as BSCN) can be expressed in microglia to transform into retinal ganglion cells by in vivo microglia fate lineage tracking.The regenerated ganglion cells project axons to the distant brain, rebuild visual neural circuit and restore impaired vision in adult glaucoma mice.In addition, the regenerated ganglion cells can stably survive for up to one year, and the same regeneration strategy is used in old glaucoma mice, which confirms the effectiveness of BSCN reprogramming to regenerate RGCs.Therefore, the present application determines that microglia is a new type of reprogramming seed cell, and finds that four key genes are involved in RGCs regeneration to restore vision, which provides a new therapeutic drug and means for the treatment of glaucoma and related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition and application for regenerating retinal ganglion cells by using endogenous microglia, and belongs to the technical field of biological medicine and retinal nerve cell regeneration. BACKGROUND

[0002] Glaucoma is the most common type of retinal neurodegenerative disease, mainly caused by pathological intraocular pressure rise, and is an irreversible blinding eye disease characterized by retinal ganglion cell apoptosis, optic nerve atrophy and visual loss. Ganglion cells are the only output neurons in the retina, which are difficult to regenerate after injury caused by glaucoma, traumatic optic neuropathy and various genetic, ischemic and neurological diseases, and thus lead to irreversible visual loss or loss. There are many mechanisms for the difficulty in repairing the optic nerve after injury, mainly including: 1) the inhibitory microenvironment of the local optic nerve after injury, the lack of signals stimulating regeneration after injury, and the apoptosis of the ganglion cells themselves after injury, etc. 2) After optic nerve injury, retinal microglia and Muller glia are activated, and activated glial cells secrete various axon growth inhibitors, which promote glial scar formation and thus inhibit the regeneration of retinal ganglion cell axons. 3) The abnormal up-regulation of oxidative stress level of the damaged optic nerve, thereby accelerating the apoptosis of the ganglion cell body.

[0003] Based on the current situation that retinal ganglion cells are difficult to regenerate, the research focus is concentrated on promoting the regeneration of ganglion cells and the repair of optic nerve injury, including drug treatment, cell transplantation and gene therapy. The recent progress in drug treatment mainly promotes the regeneration of ganglion cell axons through appropriate inflammatory stimulation, nerve growth factor protection and activation of target signaling pathways. In recent years, with the continuous development of stem cells and regenerative medicine, cell transplantation has gradually become a new research direction for the treatment of retinal diseases, especially the generation of induced pluripotent stem cells (iPSCs) provides a new way for cell transplantation. Because iPSCs are directly derived from patients, there are no medical ethics and other related problems, so they have advantages in the treatment of retinal diseases. Recent studies have confirmed that iPSCs or embryonic stem cells (ESCs) can differentiate into retinal ganglion cells in vitro, and have certain physiological functions when transplanted into experimental animals. In addition to cell transplantation, gene therapy has made great progress in many fields of regenerative medicine in recent years. Given the unique advantages of retinal vitreous cavity direct injection and immune exemption, gene therapy has also gradually become one of the hotspots in the field of retinal nerve cell regeneration.

[0004] Based on the current research status and a number of research analyses, it is found that the damage repair of the optic nerve mainly includes four steps: inhibiting the apoptosis of the ganglion cells, promoting the axon regeneration of the surviving ganglion cells, the regenerated optic nerve projecting into the brain area responsible for the key nucleus of the visual function, and the repair of the visual function. Although the regeneration strategy of the ganglion cells is well known, it is still challenging to achieve the axon regeneration of the retinal ganglion cell body or promote the transformation of the non-ganglion cell type. The microglia cells, as the glial cell type that can be in vivo proliferation, can regulate the immune response of the retina. Whether the microglia cells have the potential to be transformed into the ganglion cells has not been reported in the relevant literature. Therefore, in-depth research on the strategy of reprogramming the microglia cells to regenerate the retinal ganglion cells is helpful to promote the treatment of glaucoma and optic neuropathy in the future.

[0005] In addition to mature nerve cells such as photoreceptor cells and ganglion cells in the mammalian retina, there are non-neuronal cell types such as Muller glial cells and microglia cells. At present, the pluripotency of Muller glial cells has been well known (it can be transformed into photoreceptor cells and ganglion cells through different reprogramming strategies), but the reprogramming strategy will reduce the number of endogenous Muller glial cells to a certain extent, which will affect the function of the retina to different degrees. Our previous research has confirmed that the reprogramming of amacrine cells can achieve the regeneration of ganglion cells. However, in this process, we also found that the number of endogenous amacrine cells, as intermediate neurons connecting bipolar cells and ganglion cells, significantly decreased after reprogramming, which may induce new retinal dysfunction in the future. Therefore, finding an endogenous proliferative cell type in the retina for reprogramming to regenerate ganglion cells is a more ideal cell reprogramming strategy.

[0006] Microglia cells are derived from the yolk sac during the embryonic period, and they play different roles in various retinal diseases as immune cells. Current research mainly focuses on the role of microglia cells in regulating the immune response of the retina, but the pluripotency of microglia cells has not been reported in the relevant literature. If the microglia cells are targeted for reprogramming, they can be transformed into stable and surviving retinal ganglion cells in vivo and reconstruct the visual neural circuit from the eye to the brain, which will help to promote the clinical treatment of glaucoma in the future. SUMMARY

[0007] The purpose of the present application: in view of the deficiencies of the existing retinal nerve cell regeneration technology, the present application provides a composition and application for regenerating retinal ganglion cells by using endogenous microglia cells.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a composition comprising an agent that promotes expression or biological activity of Brn3b, Sox2, Cbln1 and NP1 genes in microglia cells, for use in any of the following:

[0010] a) for the manufacture of a product for retinal ganglion cell regeneration;

[0011] b) for the manufacture of a product for inducing microglia reprogramming into ganglion cells;

[0012] b) for the manufacture of a medicament for treating retinal ganglion cell apoptosis-related diseases;

[0013] c) for the manufacture of a medicament for treating retinal neurodegenerative diseases.

[0014] Preferably, the agent is a nucleotide or nucleic acid construct that targets or increases the biological activity of Brn3b, Sox2, Cbln1 and NP1 genes expressed by microglia cells.

[0015] Preferably, the nucleic acid construct comprises the coding sequences of Brn3b, Sox2, Cbln1 and NP1, and a promoter associated with each coding sequence for driving efficient and stable expression of exogenous genes in mammalian cells.

[0016] Preferably, the nucleic acid construct comprises a DIO-AAV expression plasmid of Brn3b, Sox2, Cbln1 and NP1 genes or an AAV viral vector thereof.

[0017] Preferably, the DIO-AAV expression plasmid sequences of Brn3b, Sox2, Cbln1 and NP1 genes are shown in SEQ ID NO: 1-4, respectively.

[0018] Preferably, the retinal ganglion cell apoptosis-related diseases include glaucoma and traumatic optic neuropathy.

[0019] Preferably, the retinal neurodegenerative diseases include glaucoma.

[0020] Preferably, the product is a kit, an injection or a medicament.

[0021] The present application also provides a method for inducing microglia reprogramming into ganglion cells, comprising at least: transfecting a nucleotide or nucleic acid construct encoding Brn3b, Sox2, Cbln1 and NP1 genes into microglia cells to transform them into retinal ganglion cells.

[0022] The application also provides a recombinant cell, which is obtained after introducing nucleotides or nucleic acid constructs capable of expressing Brn3b, Sox2, Cbln1 and NP1 genes into microglial cells.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] (1) The application first confirms that microglial cells can be transformed into ganglion cells, and finds four key genes Brn3b, Sox2, Cbln1 and NP1 that play an effect, thereby providing a reliable theoretical basis for the regeneration research of retinal ganglion cells;

[0025] (2) Cell gene reprogramming can promote the regeneration of other types of cells, but it is not clear whether the number of seed cells will be sharply reduced to induce new retinal dysfunction; the application explicitly states a new gene therapy strategy for BSCN reprogramming microglial cells to regenerate retinal ganglion cells, and further explains the maintenance of the innate immune homeostasis of microglial cells after transformation, thereby providing a safety guarantee for the new strategy of ganglion cell regeneration;

[0026] (3) The stability maintenance of the visual neural circuit plays an important role in promoting the vision repair process of glaucoma animals, but it is still not clear whether the regenerated ganglion cells of the transformed microglial cells can survive for a long time and reconstruct the visual circuit; the application fully confirms the feasibility of the stability maintenance of retinal ganglion cell regeneration and visual circuit reconstruction by lineage tracing technology, and the application fully confirms the new gene therapy strategy for reprogramming microglial cells to regenerate retinal ganglion cells by in vivo experiments, which is helpful to provide a new method and theoretical basis for the treatment of clinical glaucoma and optic neuropathy in the future, and has important clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : BSCN reprogramming microglial cells to regenerate retinal ganglion cells; wherein, a. Construction strategy of AAV plasmid expression vector: Brn3b, Sox2, Cbln1 and NP1 are subcloned into DIO vectors respectively; b. Detailed experimental strategy for reprogramming microglial cells to regenerate retinal ganglion cells in vivo; c-d. Representative images of RBPMS co-localization with EGFP and tdTomato in the retinas of the control group (c) and the BSCN treatment group (d); e-f. Representative images of Brn3a co-localization with EGFP and tdTomato in the retinas of the control group (e) and the BSCN treatment group (f); g-h. Representative images of tdTomato + or tdTomato + RBPMS +Quantitative analysis of cell number; i-j. tdTomato + or tdTomato + Brn3a + Quantitative analysis of cell number; arrowheads indicate regenerated retinal ganglion cells in GCL; (c-f) scale bar = 50 μm.

[0028] Figure 2 Transformed retinal ganglion cells make synaptic contacts with downstream brain areas through axons; where a-b. tdTomato distribution in microglia in the optic nerve of Vehicle group; c-d. tdTomato regenerated projections in the optic nerve of AAV-treated group; e. tdTomato regenerated projections in the areas of OPC, OPT, dLGN and SC; (a, c) scale bar = 500 μm, (b, d, e) scale bar = 50 μm.

[0029] Figure 3 BSCN reprograms microglia to regenerate retinal ganglion cells in glaucomatous mouse retina; where a. Experimental procedure of retinal ganglion cell (RGC) regeneration in glaucomatous mouse model; b-c. Representative images of co-localization of RBPMS with EGFP and tdTomato in I / R retina of control (b) and BSCN-treated (c) groups; d-e. Representative images of co-localization of Brn3a with EGFP and tdTomato in I / R retina of control (d) and BSCN-treated (e) groups; f-g. tdTomato + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell number; arrows indicate regenerated ganglion cells in GCL; h-i. tdTomato + (h) or tdTomato + Brn3a + (i) Quantitative analysis of cell number; j-l. Representative PERG waveforms in response to reversing contrast checkerboard stimuli in normal control (j), I / R control (k) and BSCN-treated I / R mouse retina (l); (b-e) scale bar = 50 μm.

[0030] Figure 4: Regenerated retinal ganglion cells establish synaptic connections with inferior brain regions in glaucomatous mice; a-b. Distribution of tdTomato-labeled endogenous microglia and regenerated ganglion cell axons in the optic nerve of control glaucomatous mice (a) or BSCN-treated glaucomatous mice (b); left, whole optic nerve images; right, magnified images of the rectangular area; scale bars = 400 pm (left), 100 pm (right); c. Distribution of tdTomato-labeled microglia in the OPT, dLGN, APN and SC in EGFP-treated I / R control mice brains; d. Regenerated ganglion cell axons project through the OPC to the OPT, dLGN, APN and SC in BSCN-treated glaucomatous mice brains; (c-d) scale bars = 100 pm.

[0031] Figure 5 : Regeneration of retinal ganglion cells presents time dependence; where a. Detailed experimental strategy for regeneration of retinal ganglion cells in vivo; b. Representative images of RBPMS immunostaining in the retina of normal control group; c. Representative images of RBPMS immunostaining in the retina after 2 weeks of BSCN reprogramming; d. Representative images of whole-mount RBPMS immunostaining in the retina of control group; e. Representative images of whole-mount RBPMS immunostaining in the retina after 2 weeks of BSCN reprogramming; f-g. Quantitative analysis of cell number in the GCL (ganglion cell layer) of control group retina; h-i. Quantitative analysis of cell number in the GCL of retina after 2 weeks of BSCN reprogramming; (a-d) scale bars = 50 pm. + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell number; h-i. tdTomato + (h) or tdTomato + RBPMS + (i) Quantitative analysis of cell number; (a-d) scale bars = 50 pm.

[0032] Figure 6 : Regenerated ganglion cell axon projections present time dependence; where a. tdTomato-labeled regenerated optic nerves at 2 weeks of microglia BSCN reprogramming; b. EGFP-labeled regenerated optic nerves at 2 weeks of microglia BSCN reprogramming; c. Regenerated optic nerves have not established synaptic connections with the OPC, OPT and LGN and SC. + - tdTomato +

[0033] Figure 7 ​​: Regenerated retinal ganglion cells and axonal projections exhibit time dependence; wherein, a. Detailed experimental strategy for in vivo regeneration of retinal ganglion cells; b. Representative images of RBPMS immunostaining in the retinas of control group; c. Representative images of RBPMS immunostaining in the retinas of BSCN reprogrammed for 2 weeks; d. Representative images of RBPMS whole-mount immunostaining in the retinas of control group; e. Representative images of RBPMS whole-mount immunostaining in the retinas of BSCN reprogrammed for 2 weeks; f-g. tdTomato + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell number; h-i. tdTomato + (h) or tdTomato + RBPMS + (i) Quantitative analysis of cell number; (a-d) scale bar = 50 pm.

[0034] Figure 8 : Regeneration of retinal ganglion cells in aged glaucomatous mice; a-b. Immunostaining images of RBPMS in the retinas of EGFP-treated aged glaucomatous mice; c-d. Representative immunostaining images of RBPMS in the retinas of BSCN-treated aged mice; e-f. tdTomato + (e) or tdTomato + RBPMS + (f) Quantitative analysis of cell number; arrows indicate regenerated RGC-like cells in the GCL, and arrowheads indicate retinal ganglion cells migrating from the INL (inner nuclear layer) to the GCL; (a-d) scale bar = 50 pm.

[0035] Figure 9 : Regenerated ganglion cells in aged glaucomatous mice establish visual circuits with subcortical brain regions; wherein, a-b. Retinas of EGFP-treated aged glaucomatous mice, tdTomato-labeled microglia cells distributed in the ZI (zone of unknown identity), dLGN and SC; c-d. In the brains of BSCN-treated mice, tdTomato-labeled ganglion cell axonal projections to OPC, ZI, dLGN and SC; (a, c) scale bar = 50 pm; (b, d) scale bar = 50 pm.

[0036] Figure 10: Regenerated retinal ganglion cells can survive long-term in the retina; wherein, a-b. Staining images of RBPMS in the retina of glaucomatous mice after 12 months of control (a) and BSCN treatment (b); c-d. Staining images of Brn3a in the retina of glaucomatous mice after 12 months of control (c) and BSCN treatment (d); e-f. tdTomato + (e) or tdTomato + RBPMS + (f) Quantitative analysis of cell number; g-h. tdTomato + (g) or tdTomato + Brn3a + (h) Quantitative analysis of cell number; (a-d) Scale bar = 50 pm.

[0037] Figure 11 : Regenerated retinal ganglion cell axons can maintain visual circuit integrity long-term; wherein, a. tdTomato labeled regenerated axons in the optic nerve of mice after 12 months of BSCN treatment; b. tdTomato labeled endogenous microglia in the retina of glaucomatous mice; c. Representative images of regenerated ganglion cell axons projecting through OPC nerves to target brain regions (OPT, ZI and dLGN); d. Magnified images of OPC, OPT, ZI and dLGN regions in the brain of BSCN treated glaucomatous mice; e. Magnified images of OPC, OPT, ZI and dLGN regions in the brain of control glaucomatous mice; (a-b) Scale bar = 400 pm, (c) Scale bar = 400 pm, (d-e) Scale bar = 100 pm.

[0038] Figure 12 : BSCN reprogramming strategy does not reduce endogenous microglia number; wherein, a. In normal retina, EGFP + tdTomato + labeled microglia; b. EGFP + tdTomato + labeled microglia at 1 m of microglia BSCN reprogramming; c. Statistical analysis of EGFP + tdTomato + microglia number; (a-b) Scale bar = 50 pm.

[0039] Figure 13 : CX3CR1 CreERT2-2A-EGFP Knock In mouse construction strategy. DETAILED DESCRIPTION

[0040] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail below with the accompanying drawings.

[0041] In the embodiments of the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the embodiments of the present application, the technical means used are conventional means well known to those skilled in the art, unless otherwise specified.

[0042] Example 1

[0043] Sox2, Oct4, Klf4 and c-Myc are four Yamanaka factors which can effectively induce fibroblasts to reverse into dedifferentiated pluripotent stem cells (iPSCs), thus having strong transformation prospects in the field of regenerative medicine. Lu et al. found that in a mouse optic nerve injury model, OSK (OCT4; Sox2; Klf4) combined therapy can restore aged ganglion cells to a young DNA methylation pattern and promote axon regeneration of ganglion cell bodies. Thomas et al. found that specific initiation of OSKM (OCT4; Sox2; Klf4; c-Myc) gene expression can promote myocardial cell regeneration in a mouse model of myocardial infarction. In order to explore the potential role of Yamanaka factors in promoting microglia to transform into retinal ganglion cells, we used the Cre-dependent DIO (Direction inverted open reading frame) system (which can achieve specific expression of target genes in microglia) to construct four expression plasmids of the above-mentioned four genes (pAAV-Cre, pAAV-Sox2, pAAV-Brn3b, pAAV-Klf4) and packaged them into AAV viruses respectively, and injected them into the vitreous cavity of CX3CR1-tdTomato mice to analyze whether microglia can be transformed into ganglion cells. The results showed that two months after AAV injection, no tdTomato positive regenerated ganglion cells were detected in the retinal ganglion cell layer, suggesting that Yamanaka factors cannot effectively promote microglia to transform into retinal ganglion cells, and therefore it is necessary to redesign the reprogramming strategy of microglia. Figure 1 a) and packaged into AAV viruses respectively, and injected into the vitreous cavity of CX3CR1-tdTomato mice to analyze whether microglia can be transformed into ganglion cells. The results showed that two months after AAV injection, no tdTomato positive regenerated ganglion cells were detected in the retinal ganglion cell layer, suggesting that Yamanaka factors cannot effectively promote microglia to transform into retinal ganglion cells, and therefore it is necessary to redesign the reprogramming strategy of microglia.

[0044] Sox2 is a member of the SoxB1-HMG box transcription factor family, and is one of the core transcription factors for forming induced pluripotent stem cells (iPSCs). At the same time, it plays a key role in regulating retinal ganglion cell development, neural progenitor cell maintenance and cell fate determination together with Brn3b. Therefore, we explored the possibility of reprogramming microglia into retinal ganglion cells by combining Sox2 and Brn3b. The results showed that after combined Sox2 and Brn3b gene therapy, tdTomato + RBPMS +regenerating retinal ganglion cells, however, when we analyzed the visual neural circuit, we found that the microglia-converted ganglion cells could not transmit signals through the optic chiasm to the distal brain to establish synaptic connections via axons, which suggested that the method of retinal ganglion cell regeneration still needs to be optimized.

[0045] Cerebellin-1 (Cbln1) and Neuronal Pentraxin-1 (NP1) bind to the presynaptic membrane and postsynaptic membrane in excitatory synapses, respectively, to stabilize the conduction of nerve impulses. Kunimichi et al. found that injecting Cbln1 and NP1 into the hippocampus of Alzheimer's mice can induce the generation of a large number of excitatory synapses and significantly improve the spatial memory ability of mice. In order to achieve the reprogramming of microglia to regenerate retinal ganglion cells and reconstruct the synaptic connections between the retina and the brain area, we constructed Brn3b, Sox2, Cbln1 and NP1 into DIO plasmid expression vectors (a), respectively, and packaged them into AAV viruses. The specific experimental design, experimental methods and results are as follows: Figure 1

[0046] (I) Experimental design, grouping and detection index

[0047] Randomly selected healthy 8-week-old adult CX3CR1-tdTomato mice (half male and half female) were uniformly raised in the Experimental Animal Center of Shanghai Medical College. All animal experiments were conducted in accordance with the relevant regulations of the American Society for Vision and Ophthalmology (ARVO) and were approved by the Experimental Animal Ethics Committee.

[0048] The specific design is as follows: After sufficient anesthesia and mydriasis, the retinal ischemia-reperfusion surgery was performed on the CX3CR1-tdTomato mice, and the left eye was used as a blank control group and the right eye as a model group. Gene therapy was performed one week after glaucoma surgery, and AAV-BSCN (Brn3b+Sox2+Cbln1+NP1) was injected into the vitreous cavity. The regeneration phenotype of ganglion cells, reconstruction of visual neural circuit and physiological function of regenerated ganglion cells were analyzed at different time points after treatment (see Experimental Methods for details of the construction of the retinal ischemia-reperfusion model), and the experimental process is shown in Figure 1 b.

[0049] Experimental grouping:

[0050] 1) Normal control group

[0051] 2) Glaucoma model group

[0052] 3) Glaucoma model + AAV-EGFP negative control group

[0053] 4) Glaucoma model + AAV-BSCN (Brn3b+Sox2+Cbln1+NP1) treatment group​

[0054] Detection index:

[0055] 1) Assess the damage of ischemia-reperfusion to retinal ganglion cells and optic nerve of CX3CR1-tdTomato mice using lineage tracing, immunofluorescence (RBPMS, Brn3a).

[0056] 2) Analyze the distribution and localization of regenerated ganglion cells in the glaucoma model of CX3CR1-tdTomato mice after gene therapy using immunofluorescence and lineage tracing.

[0057] 3) Analyze the visual nerve circuit reconstruction in the glaucoma model of CX3CR1-tdTomato mice after gene therapy using immunofluorescence and lineage tracing techniques.

[0058] 4) Evaluate the physiological function of regenerated retinal ganglion cells in the glaucoma model of CX3CR1-tdTomato mice using Pattern ERG.

[0059] 5) Analyze whether the regeneration of retinal ganglion cells and the reconstruction of visual circuit show time dependence at different time points (2 weeks, 3 weeks, 1 month, and 2 months) after microglia reprogramming using immunofluorescence and lineage tracing techniques.

[0060] 6) Evaluate whether the regenerated ganglion cells can survive long-term (12 months after reprogramming) in the retina of glaucoma mice and maintain the integrity of the visual circuit using lineage tracing and immunofluorescence techniques.

[0061] (B) Experimental methods

[0062] (1) Construction of AAV expression vector plasmid

[0063] The reverse coding sequences of mouse Brn3b, Sox2, Cbln1, and NP1 were subcloned into the CAG promoter-induced DIO-AAV expression vector (target gene specific expression in CX3CR1-positive microglia cells was achieved through the Cre-LoxP system), and the expression plasmid construction was identified by transformation and plasmid sequencing. The successful construction of DIO-Brn3b plasmid sequence is shown in SEQ ID NO: 1, the DIO-Sox2 plasmid sequence is shown in SEQ ID NO: 2, the DIO-Cbln1 plasmid sequence is shown in SEQ ID NO: 3, and the DIO-NP1 plasmid sequence is shown in SEQ ID NO: 4.

[0064] (2) Extraction of AAV expression vector plasmid

[0065] Take 200 ml of overnight culture and add to a large centrifuge tube, centrifuge at 8000 rpm for 15 min at room temperature to collect the bacteria. Add 10 ml of solution P1 with RNase A to the centrifuge tube containing the bacterial pellet, resuspend the bacterial pellet using a vortex shaker. Add 10 ml of solution P2 to the centrifuge tube, gently invert 10 times to fully lyse the bacteria, and let stand at room temperature for 6 min. Add 10 ml of solution P4 to the centrifuge tube, gently invert 10 times until the solution appears white flocculent precipitate, and let stand at room temperature for 12 min. Centrifuge at 8000 rpm for 20 min, collect the supernatant into a 50 ml sterile centrifuge tube. Add 9 ml of isopropanol to the supernatant, mix well and transfer to the adsorption column. Centrifuge at 8000 rpm for 4 min at room temperature, discard the waste, add 10 ml of rinse solution PW to the adsorption column, centrifuge at 8000 rpm for 4 min, discard the waste, and place the adsorption column back into the collection tube, repeat the above operation once. Add 3 ml of anhydrous ethanol to the adsorption column, centrifuge at 8000 rpm for 2 min at room temperature, remove the waste. Place the adsorption column back into the collection tube, centrifuge at 8000 rpm for 5 min to remove residual waste in the adsorption column. Place the adsorption column in a 50 ml sterile centrifuge tube, add 1.5 ml of elution buffer TB, let stand at room temperature for 8 min, centrifuge at 8000 rpm for 5 min, finally transfer the plasmid eluate to a 1.5 ml sterile centrifuge tube, and store at -20°C.

[0066] (3) Packaging and purification of AAV

[0067] AAV packaging:

[0068] 293T cells were cultured in DMEM + 10% FBS + 1% PS medium, passaged to 10 plates of 15 cm culture dishes, and when the cell confluence reached 80%, the medium was replaced with serum-free and double-antibody-free DMEM medium 1 h before transfection. Prepare the transfection plasmid, for each 15 cm culture dish, Phelper: 20 μg, AAV 7m8 serotype plasmid for packaging AAV: 7 μg, target plasmid: 7 μg. Prepare the transfection reagent, for each 15 cm culture dish, according to the ratio of DNA: PEI = 1:3, PEI 1020 μg is needed, first mix the three plasmids in 17 ml of DMEM, and mix PEI in 23 ml of DMEM. Add the PEI mixture to the plasmid mixture, mix well and let stand for 12 min. Add 4 ml of PEI-plasmid mixture to each 15 cm culture dish, gently shake the dish to mix the medium thoroughly, and replace the medium with serum-containing and double-antibody-containing DMEM medium 24 h after transfection. Harvest 293T cells 72 h after transfection, centrifuge at 2000 rpm for 10 min, wash twice with PBS, and freeze the cell pellet at -80°C or directly purify AAV.

[0069] AAV purification:

[0070] After thawing the cell pellet at -80°C on ice, resuspend in 15 ml Cell Lysis Buffer, freeze-thaw 5 times in liquid nitrogen and 37°C water bath. Add 15 μΐ of 1M MgCl2and 15 μΐ of Benzonase to the resuspended solution, then vortex thoroughly to mix, and digest at 37°C for 45 min. Add the supernatant to a 25 ml grinding jar, grind for 30-40 times, collect the supernatant to a 50 ml centrifuge tube, pre-cool the centrifuge to 4°C, centrifuge at 5500 rpm for 20 min, and aspirate the supernatant to a 50 ml centrifuge tube. Prepare iodixanol gradient centrifugation solution. Add the virus supernatant, 17%, 25%, 40%, and 60% iodixanol gradient centrifugation solution to the sealed tube in order, remove the air bubbles, and seal the tube. This process is performed on ice. Set the speed of the ultracentrifuge to 48000 rpm, the temperature to 4°C, the time to 2 h, the rotor to 70Ti, and the volume to 39.2 ml. After centrifugation, pierce the tube with a needle, carefully aspirate the virus supernatant with a 5 ml syringe, and add it to an ultrafiltration column. Add PBS:F68 (50 ml PBS + 5 μΐ F68) to the ultrafiltration column to the maximum volume, mix by inverting the column, centrifuge at 5500 rpm and 4°C for 5 min, and keep the volume of the virus concentrate at about 100-200 μΐ. Wash the virus concentrate twice with PBS:F68 at 5500 rpm for 5 min. Take the virus supernatant, perform qPCR to determine the titer, and store at -80°C.

[0071] (4) CX3CR1 CreERT2-2A-EGFP Construction of Knock In mice and CX3CR1-tdTomato mouse model

[0072] To verify the feasibility of reprogrammed microglia cells to regenerate retinal ganglion cells, we constructed CX3CR1 CreERT2-2A-EGFP Knock In mice to specifically label CX3CR1 positive retinal microglia cells (as shown in Figure 13 The brief process of mouse construction is as follows: a vector (donor vector) containing a CreERT2-2A-EGFP expression frame and homologous recombination arms was constructed by In-Fusion cloning. Cas9 mRNA, gRNA, and donor vector were microinjected into fertilized eggs of C57BL / 6J mice. The injected fertilized eggs were cultured for 3.5 days and then transplanted into pseudopregnant female mice to obtain F0 mice. After long fragment PCR identification, the F0 mice containing correct homologous recombination were mated with C57BL / 6J mice to obtain positive F1 CX3CR1 CreERT2-2A-EGFP mice.

[0073] To trace the fate of CX3CR1-positive microglia, we crossed CX3CR1 CreERT2-2A-EGFP mice with Rosa26-LSL-tdTomato mice to obtain CX3CR1 CreERT2-2A-EGFP ; Rosa26-LSL-tdTomato mice (hereinafter referred to as CX3CR1-tdTomato mice). The strain of mice is activated by tamoxifen (TAM) by gavage, and microglia cells express EGFP and tdTomato at the same time. Since EGFP is regulated by the endogenous promoter, if the microglia cells are completely transformed into ganglion cells after reprogramming, the endogenous EGFP will be turned off, and the regenerated ganglion cells will only express tdTomato and migrate to the retinal ganglion cell layer. Thus, this strategy can effectively trace the fate of microglia cells.

[0074] (5) Construction of a retinal ischemia-reperfusion model of CX3CR1-tdTomato mice

[0075] After the CX3CR1-tdTomato mice were fully anesthetized with sodium pentobarbital, they were given compound tropicamide eye drops for 8 minutes to dilate the pupils, and then local anesthesia was performed with proparacaine hydrochloride eye drops. After the mustache was removed using ophthalmic scissors, the eye was disinfected with medical iodophor. The left eye was used as a blank control group, and the right eye was used as an experimental treatment group. A 30G needle that had been disinfected with alcohol was used to perform anterior chamber puncture on the right eye of the CX3CR1-tdTomato mouse near the corneal limbus, avoiding the lens and iris. The infusion switch was turned on to avoid the generation of bubbles at 110 mm Hg for 1.5 hours. The infusion switch was turned off, the needle was carefully removed, and the CX3CR1-tdTomato mouse was coated with aureomycin hydrochloride eye ointment on the eye surface to prevent secondary infection. After 1 week of ischemia-reperfusion injury, the CX3CR1-tdTomato mouse was euthanized, and the mouse retinal and optic nerve tissues were separated under a dissecting microscope. Cryosections were prepared and the experimental results were analyzed.

[0076] (6) Vitreous cavity AAV injection of CX3CR1-tdTomato mice

[0077] One week after glaucoma surgery, the CX3CR1-tdTomato mice were fully anesthetized with sodium pentobarbital, and were given compound tropicamide and proparacaine hydrochloride eye drops for full mydriasis and topical anesthesia. Iodophor was used to disinfect the eye. A Hamilton micropipette carrying a 32G needle was used to inject 2 μl of mixed 4 AAV (Brn3b+Sox2+Cbln2+NP1) into the vitreous cavity of the CX3CR1-tdTomato mouse from the corneal limbus, avoiding the iris and lens. Each AAV was diluted to ensure a titer of >1x10 12 vg / ml.

[0078] (7) Immunofluorescence detection of the distribution and localization of regenerative ganglion cells RBPMS, Brn3a

[0079] Whole mount immunofluorescence: The eyeballs of CX3CR1-tdTomato mice were separated under a surgical microscope, the cornea and lens were removed, and the eye cup containing the retina was fixed in 4% PFA for 8 h. The retina was separated under a body microscope and placed in TD-buffer for washing 3 times, 10 min each time. 2% BSA prepared with PBS was added for 1 h of room temperature blocking. The blocking solution was aspirated, and the primary antibody diluted with 1% BSA was added dropwise, and incubated at 4°C overnight. The primary antibody was aspirated, and the tissue was placed in TD-buffer for washing 3 times, 10 min each time. Under light-avoiding conditions, the second antibody labeled with FITC or Alexa Fluo594 was added dropwise, and incubated at 4°C for 4 h. Subsequently, under light-avoiding conditions, TD-buffer was washed 3 times, 10 min each time. Hoechst was diluted with PBS at 1:100, and incubated for 10 min, and washed with PBS 3 times. Colorless mounting medium H-1000 was used for mounting, and the results were observed under a Zeiss laser confocal microscope.

[0080] Tissue section immunofluorescence: The CX3CR1-tdTomato mouse retina was frozen sectioned, fixed with 4% PFA for 20 min, and washed with PBS 3 times, 5 min each time. 0.2% Triton was used to permeabilize for 10 min, and PBS was washed 3 times, 5 min each time. 2% BSA was added for 1 h of blocking. The primary antibody was prepared with 1% BSA, and incubated at 4°C overnight. The primary antibody was washed, and PBS was washed 3 times, 10 min each time. After wiping dry, the secondary antibody was added, and incubated for 1 h in the dark. The secondary antibody was washed, and PBS was washed 3 times, 10 min each time, with attention to avoid light. DAPI (H-1200) was used for mounting, and the experimental results were observed under a ZEISS 900 laser confocal microscope.

[0081] (8) Pattern Electroretinogram (PERG) evaluation of the electrical physiological function of retinal ganglion cells after microglial BSCN reprogramming

[0082] The CX3CR1-tdTomato mice were taken in natural light adaptation state, and after full anesthesia, they were fixed on the movable experimental table. After the test eye was given artificial tears, the PERG recording electrode was fixed at the corneal margin, the reference electrode and the ground electrode were placed in the subcutaneous tissue of the cheek and the tail of the test eye, respectively, and the position of the mouse was adjusted so that the visual axis of the test eye was perpendicular to the stimulation screen. Using the Chongqing Guote visual electrophysiology detection system, the stimulation mode was set to chessboard, the distance from the corneal vertex to the center of the stimulation field was 15 cm, and the resistance was maintained below 10 kΩ during recording. The stimulation frequency was 1.0 Hz, superimposed 128 times, the spatial frequency was set to 0.5 cpd, the contrast was 98.6%, and the passband (0.3-100 Hz). The N1-P wave amplitude and peak time of mouse PERG were recorded using the above experimental parameters, and the improvement of the electrical physiological function of retinal ganglion cells after BSCN treatment in glaucoma animals was analyzed.

[0083] (III) Experimental results

[0084] (1) BSCN reprograms microglia to regenerate retinal ganglion cells in normal mouse retina

[0085] After 1 month of BSCN (Brn3b, Sox2, Cbln1 and NP1) treatment, we detected a large number of tdTomato-positive ganglion cells in the retinal ganglion cell layer of normal mice, confirming that microglia can be efficiently converted into ganglion cells by BSCN reprogramming Figure 1 ), and the axons of regenerated ganglion cells can establish synaptic connections with the optic chiasm (OPC), optic tract (OPT), lateral geniculate nucleus (dLGN) and superior colliculus (SC) in the lower brain area, indicating that regenerated ganglion cells can integrate into the visual neural circuit Figure 2 ).

[0086] (2) BSCN reprograms microglia to regenerate retinal ganglion cells in glaucoma mouse retina

[0087] In order to verify whether BSCN microglia reprogramming strategy can be implemented to regenerate retinal ganglion cells in glaucoma animal models, we established an acute glaucoma model induced by retinal ischemia-reperfusion injury. The experimental results confirmed that after 2 months of BSCN reprogramming, a large number of regenerated ganglion cells were detected in the ganglion cell layer, and the regenerated ganglion cells had normal electrical physiological function Figure 3 ). At the same time, we also found that the axons of regenerated ganglion cells can establish complete visual neural circuit with key nuclei in the lower brain area Figure 4 ).

[0088] (3) Regeneration of retinal ganglion cells and axon projection show time dependence

[0089] To verify whether the conversion of microglia into retinal ganglion cells is time-dependent, we analyzed the regeneration and axonal projection of retinal ganglion cells at different time points after the genetic reprogramming of microglia by lineage tracing technology. The experimental results show that the BSCN-mediated gene therapy can detect tdTomato + RBPMS + regenerated ganglion cells, but the ganglion cell axons have not yet established synaptic connections with the inferior brain area Figures 5-6 ). Three weeks after reprogramming, the regenerated ganglion cell axons cross the optic chiasm (OPC) and the optic tract (OPT), but axonal projections are still not detected in the lateral geniculate nucleus (dLGN) and the superior colliculus (SC) Figure 7 ). After one month of reprogramming, axonal projections of the newly generated ganglion cells can be detected in the OPC, OPT, dLGN, and SC Figures 1-2 . The experimental results suggest that the BSCN reprogrammed microglia can be converted into retinal ganglion cells that can integrate into the visual neural circuit.

[0090] (4) Regeneration of retinal ganglion cells and reconstruction of the visual neural circuit in aged glaucoma mice

[0091] The regeneration potential of aged mice is poor. To verify whether the BSCN reprogramming strategy can achieve the regeneration of ganglion cells in aged mice, we performed lineage tracing of microglia fate in aged mice. The experimental results reveal that the same genetic reprogramming strategy in aged mice can still promote the conversion of microglia into retinal ganglion cells and establish synaptic connections with the inferior brain area Figures 8-9 . The experimental results suggest that the BSCN microglia reprogramming strategy can achieve the regeneration of ganglion cells in adult and aged glaucoma models.

[0092] (5) Regenerated retinal ganglion cells can long-term stable survival and maintain the integrity of the visual circuit

[0093] To verify whether the regenerated retinal ganglion cells can long-term stable survival and maintain the integrity of the visual neural circuit, we analyzed the retinal and brain sections again after 12 months of ganglion cell regeneration. The experimental results confirm that the tdTomato-labeled regenerated ganglion cells can still be detected in the GCL layer Figure 10 , and the ganglion cell axons can still establish synaptic connections with the key nuclei responsible for vision in the inferior brain area Figure 11 . The experimental results suggest that the reprogrammed microglia can regenerate retinal ganglion cells that can long-term stable survival and maintain the integrity of the visual neural circuit.

[0094] (6) BSCN microglia reprogramming strategy is an ideal method for retinal ganglion cell regeneration

[0095] To analyze whether the BSCN reprogramming strategy of microglia can reduce the number of endogenous microglia, which may induce retinal dysfunction in the future. Therefore, we analyzed the retinal flat mount of CX3CR1-tdtomato mice after 1 month of BSCN reprogramming. The experimental results show that the number of microglia in the BSCN reprogramming experimental group has no significant difference compared with the control group Figure 12 ), suggesting that the microglia reprogramming strategy is a relatively safe method for retinal ganglion cell regeneration.

[0096] The above experimental results suggest that: (1) The BSCN reprogramming of microglia mediated by Brn3b, Sox2, Cbln1 and NP1 can achieve the regeneration of retinal ganglion cells, and the projection of the regenerated ganglion cell axons in the brain area shows time dependence. (2) The BSCN reprogramming strategy does not cause the number of endogenous microglia to decrease, and it is a relatively safe and ideal method for retinal ganglion cell regeneration. This new strategy of microglia reprogramming to regenerate ganglion cells will help to provide new methods and theoretical basis for the treatment of glaucoma patients in the future.

[0097] The above is only the preferred embodiment of the present application, and is not any form and substantial limitation of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. Use of a composition, characterized in that, The composition comprises an agent for promoting expression of Brn3b, Sox2, Cbln1 and NP1 genes in microglial cells, and the use of the composition is selected from any one of the following: a) for preparing a product for inducing reprogramming of microglial cells into ganglion cells; b) for preparing a drug for treating glaucoma; The agent is a nucleotide or nucleic acid construct targeting Brn3b, Sox2, Cbln1 and NP1 genes expressed by microglial cells.

2. Use according to claim 1, characterized in that, The nucleic acid construct comprises coding sequences of Brn3b, Sox2, Cbln1 and NP1, and a promoter combined with each coding sequence, which is used to drive efficient and stable expression of exogenous genes in mammalian cells.

3. Use according to claim 2, characterized in that, The nucleic acid construct comprises a DIO-AAV expression plasmid of Brn3b, Sox2, Cbln1 and NP1 genes or an AAV viral vector thereof.

4. Use according to claim 3, characterized in that, The DIO-AAV expression plasmid sequences of Brn3b, Sox2, Cbln1 and NP1 genes are shown in SEQ ID NO: 1-4, respectively.

5. The use according to any one of claims 1 to 4, characterized in that, The product is an agent.

6. A recombinant cell, characterized in that, It is obtained after introducing a nucleotide or nucleic acid construct capable of expressing Brn3b, Sox2, Cbln1 and NP1 genes in microglial cells.

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