Composition for regenerating retinal ganglion cells by using endogenous microglial cells and application of composition

Abstract

The invention discloses a composition for regenerating retinal ganglion cells by using endogenous microglial cells and application. In-vivo microglial cell fate lineage tracking finds that a plurality of genes (Brn3b, Sox2, Cbln1 and NP1, called BSCN for short) are expressed in an ectopic manner in microglial cells, so that the microglial cells can be promoted to be converted into retinal ganglion cells. And the regenerated nodule cells project axons to the distant brain, reconstruct an optic nerve circuit and recover the impaired vision of the adult glaucoma mouse. Besides, the regenerated ganglion cells can stably survive as long as one year, and the same regeneration strategy is adopted in old-age glaucoma mice, so that the effectiveness of reprogramming regeneration of the RGCs by the BSCN is proved. Therefore, it is determined that the microglial cells are novel reprogramming seed cells, it is found that the four key genes participate in RGCs regeneration to recover vision, and new therapeutic drugs and means are provided for treatment of glaucoma and related diseases.

Description

Technical Field

[0001] The invention relates to a composition for regenerating retinal ganglion cells by using endogenous microglia and application thereof, belonging to the technical field of biomedicine and retinal nerve cell regeneration. Background Art

[0002] Glaucoma is the most common type of retinal neurodegenerative disease, mainly caused by pathological increase in intraocular pressure. It is an irreversible blinding eye disease characterized by retinal ganglion cell apoptosis, optic nerve atrophy and decreased vision. Ganglion cells are the only output neurons in the retina. They are difficult to regenerate after being damaged by glaucoma, traumatic optic neuropathy, and various genetic, ischemic, and neurological diseases, leading to irreversible vision loss or loss. There are many mechanisms that make optic neuropathy difficult to repair, mainly including: 1) the inhibitory microenvironment of the local optic nerve after injury, the lack of signals to stimulate regeneration after injury, and the apoptosis of ganglion cells themselves after injury. 2) After the optic nerve is injured, retinal microglia and Muller glial cells are activated. The activated glial cells secrete a variety of axon growth inhibitory factors, which promote the formation of glial scars and inhibit the regeneration of retinal ganglion cell axons. 3) The level of oxidative stress in the damaged optic nerve is abnormally upregulated, thereby accelerating the apoptosis of ganglion cell bodies.

[0003] Based on the current situation that retinal ganglion cells are difficult to regenerate, the research focuses on promoting ganglion cell regeneration and repair of optic nerve damage, including drug therapy, cell transplantation and gene therapy. Recent progress in drug therapy mainly promotes ganglion cell axon regeneration 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 retinopathy, especially the generation of induced pluripotent stem cells (iPSCs) provides a new way for cell transplantation. Since iPSCs are directly derived from patients and have no medical ethics and other related issues, they have advantages in the treatment of retinopathy. 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 significant progress in many fields of regenerative medicine in recent years. Given the unique advantages of direct intravitreal injection and immune exemption of the retina, gene therapy has gradually become one of the hot spots in the field of retinal nerve cell regeneration.

[0004] Based on the current status of research at home and abroad and multiple research analyses, it was found that the repair of optic nerve damage mainly includes four steps: inhibiting ganglion cell apoptosis, promoting the regeneration of surviving ganglion cell axons, projecting the regenerated optic nerve to the key nuclei responsible for vision in the brain area, and repairing visual function. Although the regeneration strategy of ganglion cells is already well known, it is still challenging to achieve axon regeneration of retinal ganglion cell cell bodies or promote cell transformation of non-ganglion cell types. Microglia are a type of glial cell that can divide and proliferate in vivo to regulate the retinal immune response. There is no relevant literature reporting whether microglia have the potential to transform into ganglion cells. Therefore, in-depth research on the strategy of reprogramming microglia to regenerate retinal ganglion cells will help promote the treatment of glaucoma and optic neuropathy in the future.

[0005] In addition to mature neural cells such as photoreceptors and ganglion cells, there are also non-neuronal cell types such as Müller glial cells and microglia in the mammalian retina. The multipotency of Müller glial cells is well known (different reprogramming strategies can promote their transformation into photoreceptors and ganglion cells, etc.), but reprogramming strategies will reduce the number of endogenous Müller glial cells to a certain extent, thus affecting the function of the retina to varying degrees. Our previous studies have confirmed that reprogramming amacrine cells can achieve the regeneration of ganglion cells. However, in this process, we also found that as interneurons connecting bipolar cells and ganglion cells, the number of endogenous amacrine cells was significantly reduced after reprogramming, which may induce new retinal dysfunction in the future. Therefore, it is a more ideal cell reprogramming strategy to find endogenous retinal cell types that can divide and proliferate for reprogramming and regenerating ganglion cells.

[0006] Microglia originate from the embryonic yolk sac and, as immune cells, play different roles in a variety of retinal diseases. Current research focuses on the role of microglia in regulating retinal immune responses. However, as a cell type that can stably proliferate, its multipotency has not been reported in the literature. If microglia are targeted for reprogramming, promoting their in vivo transformation into stable surviving retinal ganglion cells and rebuilding the visual neural circuit from the eye to the brain, it will help promote the treatment of clinical glaucoma in the future. Summary of the invention

[0007] Purpose of the present invention: In view of the shortcomings of the existing retinal nerve cell regeneration technology, the present invention provides a composition and application of regenerating retinal ganglion cells using endogenous microglia.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides an application of a composition, wherein the composition comprises an agent for promoting the expression or biological activity function of Brn3b, Sox2, Cbln1 and NP1 genes in microglia, and the application of the composition is selected from any one of the following:

[0010] a) A product for preparing retinal ganglion cell regeneration;

[0011] b) for preparing a product for inducing microglia to reprogram into ganglion cells;

[0012] b) for preparing a drug for treating diseases related to retinal ganglion cell apoptosis;

[0013] c) Used for preparing drugs for treating retinal neurodegenerative diseases.

[0014] Preferably, the agent is a nucleotide or nucleic acid construct that targets microglial cells to express Brn3b, Sox2, Cbln1 and NP1 genes or increases their biological activities.

[0015] Preferably, the nucleic acid construct comprises the coding sequences of Brn3b, Sox2, Cbln1 and NP1, and a promoter associated with each coding sequence, wherein the promoter is used to drive the exogenous gene to achieve efficient and stable expression in mammalian cells.

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

[0017] Preferably, the DIO-AAV expression plasmid sequences of the Brn3b, Sox2, Cbln1 and NP1 genes are shown as SEQ ID NOs: 1 to 4, respectively.

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

[0019] Preferably, the retinal neurodegenerative disease comprises glaucoma.

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

[0021] The present invention also provides a method for inducing microglia to reprogram into ganglion cells, which at least comprises: transfecting nucleotides or nucleic acid constructs encoding Brn3b, Sox2, Cbln1 and NP1 genes into microglia to convert them into retinal ganglion cells.

[0022] The present invention also provides a recombinant cell, which is obtained by 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 present invention has the following beneficial effects:

[0024] (1) This invention is the first to confirm that microglia can be transformed into ganglion cells, and to identify four key genes, Brn3b, Sox2, Cbln1 and NP1, that exert the effect, providing a reliable theoretical basis for the study of retinal ganglion cell regeneration;

[0025] (2) Cell gene reprogramming can promote the regeneration of other types of cells, but it is unclear whether the number of seed cells will drop sharply, thereby inducing new retinal dysfunction. The present invention clarifies a new gene therapy strategy for BSCN reprogramming microglia to regenerate retinal ganglion cells, and further explains the maintenance of the inherent immune homeostasis of microglia after transformation, providing a safety guarantee for the new strategy of ganglion cell regeneration.

[0026] (3) Maintaining the stability of the visual neural circuit plays an important role in promoting the restoration of vision in animals with glaucoma. However, it is still unclear whether the ganglion cells regenerated by microglia can survive stably for a long time and rebuild the visual circuit. The present invention fully clarifies the feasibility of maintaining the stability of retinal ganglion cell regeneration and visual circuit reconstruction through lineage tracing technology, and the present invention fully clarifies the new gene therapy strategy of reprogramming microglia to regenerate retinal ganglion cells through in vivo experiments, which will help provide new methods and theoretical basis for the treatment of clinical glaucoma and optic neuropathy in the future, and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : BSCN reprograms microglia to regenerate retinal ganglion cells; wherein, a. Construction strategy of AAV plasmid expression vector: Brn3b, Sox2, Cbln1 and NP1 were subcloned into DIO vector respectively; b. Detailed experimental strategy for regenerating retinal ganglion cells by reprogramming microglia in vivo; cd. Representative images of co-localization of RBPMS with EGFP and tdTomato in the retina of the control group (c) and BSCN-treated group (d); ef. Representative images of co-localization of Brn3a with EGFP and tdTomato in the retina of the control group (e) and BSCN-treated group (f); gh. tdTomato in the retinal GCL (ganglion cell layer) of the control group (g) and BSCN-treated group (h) + or tdTomato + RBPMS +Quantitative analysis of cell number; ij. tdTomato in the retinal GCL of the control group (i) and BSCN-treated group (j) + or tdTomato + Brn3a + Quantification of cell numbers; arrow heads indicate regenerating retinal ganglion cells in the GCL; (cf) scale bars = 50 μm.

[0028] Figure 2 : Transformed retinal ganglion cells establish synaptic connections with lower brain regions through axons; ab. Distribution of tdTomato in microglia in the optic nerve of the Vehicle group; cd. Regenerative projection of tdTomato in the optic nerve of the AAV-treated group; e. Regenerative projection of tdTomato in the OPC, OPT, dLGN and SC regions; (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 the retina of glaucoma mice; wherein, a. Experimental process of retinal ganglion cell (RGC) regeneration in glaucoma mouse model; bc. Representative images of co-localization of RBPMS with EGFP and tdTomato in I / R retina of control group (b) and BSCN-treated group (c); de. Representative images of co-localization of Brn3a with EGFP and tdTomato in I / R retina of control group (d) and BSCN-treated group (e); fg. Representative images of tdTomato in I / R retina of control group and BSCN-treated group (g) + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell numbers; arrows indicate ganglion cells regenerating in the GCL; hi. tdTomato in control and BSCN-treated I / R retinas + (h) or tdTomato + Brn3a + (i) Quantitative analysis of cell number; jl. Representative PERG waveforms in response to inverted contrast checkerboard stimulation in the retinas of normal control (j), I / R control (k) and BSCN-treated I / R mice (l); (be) Scale bar = 50 μm.

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

[0031] Figure 5 :Retinal ganglion cell regeneration is time-dependent; Among them, a. Detailed experimental strategy for regenerating retinal ganglion cells in vivo; b. Representative images of RBPMS immunostaining in the retina of the normal control group; c. Representative images of RBPMS immunostaining in the retina 2 weeks after BSCN reprogramming; d. Representative images of RBPMS whole-piece immunostaining in the control group retina; e. Representative images of RBPMS whole-piece immunostaining in the retina 2 weeks after BSCN reprogramming; fg. tdTomato in the GCL (ganglion cell layer) of the retina of the control group + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell number; tdTomato in retinal GCL after 2 weeks of hi.BSCN reprogramming + (h) or tdTomato + RBPMS + (i) Quantitative analysis of cell number; (ad) Scale bar = 50 μm.

[0032] Figure 6 :The axonal projection of regenerated ganglion cells showed time dependence; a. tdTomato expression in microglial BSCNs at 2 weeks of reprogramming + Labeled regenerating optic nerve; b. EGFP expression in microglia BSCN after 2 weeks of reprogramming - tdTomato + Labeled regenerated optic nerve; c. The regenerated optic nerve has not yet established synaptic connections with LGN and SC through OPC and OPT.

[0033] Figure 7:Regenerated retinal ganglion cells and axonal projections show time dependence; Among them, a. Detailed experimental strategy for regenerating retinal ganglion cells in vivo; b. Representative images of RBPMS immunostaining in the retina of the control group; c. Representative images of RBPMS immunostaining in the retina 2 weeks after BSCN reprogramming; d. Representative images of RBPMS whole-piece immunostaining in the retina of the control group; e. Representative images of RBPMS whole-piece immunostaining in the retina 2 weeks after BSCN reprogramming; fg. tdTomato in the GCL of the retina of the control group + (f) or tdTomato + RBPMS + (g) Quantitative analysis of cell number; tdTomato in retinal GCL after 2 weeks of hi.BSCN reprogramming + (h) or tdTomato + RBPMS + (i) Quantitative analysis of cell number; (ad) Scale bar = 50 μm.

[0034] Figure 8 :Regeneration of retinal ganglion cells in aged glaucoma mice; ab. Immunostaining images of RBPMS in the retina of aged glaucoma mice treated with EGFP; cd. Representative immunostaining images of RBPMS in the retina of aged mice treated with BSCN; ef. tdTomato in the GCL (ganglion cell layer) of the retina of aged mice treated with control and BSCN + (e) or tdTomato + RBPMS + (f) Quantitative analysis of cell numbers; arrows indicate regenerating RGC-like cells in the GCL, and arrow heads indicate retinal ganglion cells migrating from the INL (inner nuclear layer) to the GCL; (ad) Scale bar = 50 μm.

[0035] Fig. 9 : The regenerated ganglion cells of aged glaucomatous mice establish visual circuits with lower brain regions; ab. The distribution of tdTomato-labeled microglia in the ZI (zona incerta), dLGN and SC in the retina of aged glaucomatous mice treated with EGFP; cd. In the brain of BSCN-treated mice, tdTomato-labeled ganglion cell axons project to OPC, ZI, dLGN and SC; (a, c) Scale bar = 50 μm; (b, d) Scale bar = 50 μm.

[0036] Fig.10:Regenerated retinal ganglion cells can survive stably in the retina for a long time; ab. Staining images of RBPMS in the retina of glaucoma mice in the control group (a) and BSCN-treated (b) after 12 months; cd. Staining images of Brn3a in the retina of glaucoma mice in the control group (c) and BSCN-treated (d) after 12 months; ef. tdTomato in the retina of glaucoma mice and the GCL (ganglion cell layer) of glaucoma mice treated with BSCN + (e) or tdTomato + RBPMS + (f) Quantitative analysis of the number of cells; gh. tdTomato in the GCL of the retina of glaucomatous mice and the retina of glaucomatous mice treated with BSCN + (g) or tdTomato + Brn3a + (h) Quantitative analysis of cell number; (ad) Scale bar = 50 μm.

[0037] Fig.11 : Regenerating retinal ganglion cell axons can maintain the integrity of the visual circuit for a long time; a. tdTomato marks the regenerated axons in the optic nerve of mice after BSCN treatment for 12 months; b. tdTomato marks the endogenous microglia in the optic nerve of the retina of glaucomatous mice; c. Representative images of regenerated ganglion cell axons projecting through the OPC nerve to the target brain areas (OPT, ZI and dLGN); d. Magnified images of the OPC, OPT, ZI and dLGN areas in the brains of BSCN-treated glaucomatous mice; e. Magnified images of the OPC, OPT, ZI and dLGN areas in the brains of glaucomatous mice in the control group; (ab) scale bar = 400μm, (c) scale bar = 400μm, (de) scale bar = 100μm.

[0038] Fig.12 :BSCN reprogramming strategy does not reduce the number of endogenous microglia; a. In normal retina, EGFP + tdTomato + Labeled microglia; b. EGFP at 1m of BscN reprogramming of microglia + tdTomato + Labeled microglia; c. Normal retina and BscN reprogrammed retina with EGFP + tdTomato + Statistical analysis of microglial cell numbers; (ab) scale bars = 50 μm.

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

[0040] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

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

[0042] Example 1

[0043] Sox2, Oct4, Klf4 and c-Myc are four Yamanaka factors that can effectively induce fibroblasts to reverse to dedifferentiated pluripotent stem cells (iPSCs), and therefore have strong translational prospects in the field of regenerative medicine. Lu et al. found that in a mouse optic nerve injury model, combined OSK (OCT4; Sox2; Klf4) treatment can restore aging ganglion cells to a young DNA methylation pattern and promote axon regeneration of ganglion cell cell bodies. Thomas et al. found that specifically initiating the gene expression of OSKM (OCT4; Sox2; Klf4; c-Myc) can promote myocardial cell regeneration in mice with myocardial infarction model. In order to explore the potential role of Yamanaka factors in promoting the transformation of microglia into retinal ganglion cells, we used the Cre-dependent DIO (Direction inverted open reading frame) system (which can achieve specific expression of the target gene in microglia) to construct the above four expression plasmids ( Figure 1 a) and packaged them into AAV viruses, injected into the vitreous cavity of CX3CR1-tdTomato mice, and analyzed whether microglia could be transformed into ganglion cells. The results showed that 2 months after AAV injection, no tdTomato-positive regenerated ganglion cells were detected in the retinal ganglion cell layer, indicating that Yamanaka factors could not effectively promote the transformation of microglia into retinal ganglion cells, and therefore the microglia reprogramming strategy needed to be redesigned.

[0044] Sox2 is a member of the SoxB1-HMG box transcription factor family and one of the core transcription factors for the formation of induced pluripotent stem cells (iPSCs). It and Brn3b play a key role in regulating the development of retinal ganglion cells, the maintenance of neural progenitor cells, and the determination of cell fate. Therefore, we explored the possibility of combining Sox2 and Brn3b to reprogram microglia into retinal ganglion cells. The results showed that after combined Sox2 and Brn3b gene therapy, tdTomato appeared in the retinal ganglion cell layer. + RBPMS +However, when we analyzed the visual neural circuit, we found that the ganglion cells converted from microglia could not transmit signals through axons through the optic chiasm to the distant brain to establish synaptic connections. These results suggest that there is still a need to optimize the method of retinal ganglion cell regeneration.

[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 injection of Cbln1 and NP1 into the hippocampus of Alzheimer's mice induced a large number of excitatory synapses and significantly improved the spatial memory of mice. In order to achieve the reprogramming of microglia to regenerate retinal ganglion cells and rebuild the synaptic connection between the retina and the brain, we constructed Brn3b, Sox2, Cbln1 and NP1 into the DIO plasmid expression vector ( Figure 1 a), and packaged into AAV virus. The specific experimental design, experimental methods and results are as follows:

[0046] 1. Experimental design, grouping and testing indicators

[0047] Healthy 8-week-old adult CX3CR1-tdTomato mice (half male and half female) were randomly selected and housed in the Experimental Animal Center of Shanghai Health Medical College. All animal experimental procedures followed the relevant regulations of the American Association for Research in Vision and Ophthalmology (ARVO) and were approved by the Experimental Animal Ethics Committee.

[0048] The specific design is as follows: After CX3CR1-tdTomato mice were fully anesthetized and mydriasis, retinal ischemia-reperfusion surgery was performed, the left eye was used as the blank control group, and the right eye was used as the 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 ganglion cell regeneration phenotype, visual neural circuit reconstruction, and physiological function of regenerated ganglion cells were analyzed at different time points after treatment (for details on the construction of the retinal ischemia-reperfusion model, see the experimental method). The experimental process is as follows Figure 1 As shown in b.

[0049] Experimental groups:

[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 indicators:

[0055] 1) Lineage tracing and immunofluorescence (RBPMS, Brn3a) were used to evaluate the damage of ischemia-reperfusion to retinal ganglion cells and optic nerves in CX3CR1-tdTomato mice.

[0056] 2) Use immunofluorescence and lineage tracing to analyze the distribution and localization of regenerating ganglion cells in the CX3CR1-tdTomato mouse glaucoma model after gene therapy;

[0057] 3) Immunofluorescence and lineage tracing techniques were used to analyze the reconstruction of the visual neural circuit in the CX3CR1-tdTomato mouse glaucoma model after gene therapy.

[0058] 4) Pattern ERG was used to evaluate the physiological function of regenerated retinal ganglion cells in the CX3CR1-tdTomato mouse glaucoma model.

[0059] 5) Use immunofluorescence and lineage tracing techniques to analyze whether the regeneration of retinal ganglion cells and the reconstruction of visual circuits are time-dependent at different time points (2 weeks, 3 weeks, 1 month, and 2 months) after microglia reprogramming.

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

[0061] (II) 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 DIO-AAV expression vector induced by the CAG promoter (specific expression of the target gene in CX3CR1-positive microglia was achieved through the Cre-LoxP system), and the successful construction of the expression plasmid was confirmed by transformation and plasmid sequencing. The sequence of the successfully constructed DIO-Brn3b plasmid is shown in SEQ ID NO: 1, the sequence of the DIO-Sox2 plasmid is shown in SEQ ID NO: 2, the sequence of the DIO-Cbln1 plasmid is shown in SEQ ID NO: 3, and the sequence of the DIO-NP1 plasmid is shown in SEQ ID NO: 4.

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

[0065] Take 200ml of overnight cultured bacterial solution and add it to a large centrifuge tube. Centrifuge at 8000rpm for 15min at room temperature to collect bacteria. Add 10ml of solution P1 with RNase A to the centrifuge tube with bacterial pellet, and use a vortex oscillator to resuspend the bacterial pellet. Add 10ml of solution P2 to the centrifuge tube, gently flip 10 times to fully lyse the bacteria, and let it stand at room temperature for 6min. Add 10ml of solution P4 to the centrifuge tube, gently flip 10 times until the solution appears white flocculent precipitation, and let it stand at room temperature for 12min. Centrifuge at 8000rpm for 20min, and collect the supernatant into a 50ml sterile centrifuge tube. Add 9ml of isopropanol to the supernatant, mix well, and transfer to the adsorption column. Centrifuge at 8000rpm for 4min at room temperature, discard the waste liquid, add 10ml of rinsing solution PW to the adsorption column, centrifuge at 8000rpm for 4min, discard the waste liquid, put the adsorption column back into the collection tube, and 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, and remove the waste liquid. Put the adsorption column back into the collection tube and centrifuge at 8000 rpm for 5 min to remove the residual waste liquid 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, and finally transfer the plasmid eluate to a 1.5 ml sterile centrifuge tube and store at -20 °C.

[0066] (3) AAV packaging and purification

[0067] AAV packaging:

[0068] 293T cells were cultured in DMEM+10% FBS+1% PS medium, and subcultured to 10 15 cm culture dishes. When the cell confluency reached 80%, the medium was replaced with DMEM medium with serum and no dual antibody 1 hour before transfection. Prepare the transfection plasmid. For each 15 cm culture dish, Phelper: 20 μg, AAV 7m8 (Serotype plasmid for packaging AAV): 7μg, target plasmid: 7μg. Prepare transfection reagent. For each 15cm culture dish, according to the ratio of DNA:PEI=1:3, PEI 1020μg is required. First, mix 3 plasmids in 17ml DMEM, and PEI in 23ml DMEM. Add the PEI mixture to the plasmid mixture, mix well and let stand for 12min. Add 4ml PEI-plasmid mixture to each 15cm culture dish, gently shake the culture dish to fully mix the culture medium, and replace it with DMEM medium with serum and double antibodies 24h after transfection. Harvest 293T cells 72h after transfection, centrifuge at 2000rpm for 10min, wash twice with PBS, freeze the cell pellet at -80℃ or purify AAV directly.

[0069] AAV purification:

[0070] Thaw the -80℃ cell pellet on ice, resuspend in 15ml Cell Lysis Buffer, and freeze-thaw repeatedly in liquid nitrogen and 37℃ water bath for 5 times. Add 15μl 1M MgCl 2 and 15μl Benzonase, then vortex vigorously to mix thoroughly, and digest in a 37℃ water bath for 45min. Add the supernatant to a 25ml grinding bottle, grind 30-40 times, collect the supernatant into a 50ml centrifuge tube, precool the centrifuge to 4℃, centrifuge at 5500rpm for 20min, and aspirate the supernatant into a 50ml centrifuge tube. Prepare iodixanol gradient centrifuge. Add the virus supernatant, 17%, 25%, 40% and 60% iodixanol gradient centrifuge to the sealed tube in sequence, remove bubbles and seal the sealed tube. This process is performed on ice. The speed of the ultracentrifuge was adjusted to 48000rpm, the temperature was set to 4℃, the time was set to 2h, the rotor was selected as 70Ti, and the volume was selected as 39.2ml. After the centrifugation, several holes were pierced around the tube mouth. After the virus supernatant was carefully sucked out with a 5ml syringe, it was added to the ultrafiltration column, and PBS:F68 (50ml PBS plus 5μl F68) was added to the maximum volume of the ultrafiltration column. After mixing upside down, centrifuged at 5500rpm and 4℃ for 5min, the volume of the virus concentrate was kept at about 100μl-200μl. The virus concentrate was washed twice with PBS:F68, 5500rpm-5min. The virus supernatant was removed, the titer was tested by qPCR and stored for a long time at -80℃.

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

[0072] To verify the feasibility of reprogramming microglia to regenerate retinal ganglion cells, we constructed the CX3CR1 gene using the CRISPR-Cas9 system. CreERT2-2A-EGFP Knock In mice to specifically label CX3CR1-positive retinal microglia (e.g. Fig.13 The brief process of mouse construction is as follows: A vector (donor vector) containing the CreERT2-2A-EGFP expression frame and homologous recombination arms was constructed by the In-Fusion cloning method. Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL / 6J mice. After 3.5 days of culture, the injected fertilized eggs were transplanted into pseudo-pregnant female mice to obtain F0 generation mice. After long-fragment PCR identification, the F0 generation mice containing correct homologous recombination were mated with C57BL / 6J mice to obtain positive F1 generation CX3CR1 CreERT2-2A-EGFP Mouse.

[0073] To perform lineage tracing of the fate of CX3CR1-positive microglia, we isolated CX3CR1 CreERT2-2A-EGFP Mice were crossed with Rosa26-LSL-tdTomato mice to obtain CX3CR1 CreERT2-2A-EGFP ; Rosa26-LSL-tdTomato mice (hereinafter referred to as CX3CR1-tdTomato mice). This strain of mice was gavaged with tamoxifen (TAM) to activate Cre, and microglia expressed both EGFP and tdTomato. Since EGFP is regulated by the endogenous promoter, if microglia 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. Therefore, this strategy can be used to effectively trace the fate of microglia.

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

[0075] After CX3CR1-tdTomato mice were fully anesthetized with sodium pentobarbital, compound tropicamide eye drops were given to dilate the pupil for 8 minutes. When the pupil was dilated to the limbus, proparacaine hydrochloride eye drops were used for local anesthesia. After the whiskers were removed with ophthalmic scissors, medical iodine was used to disinfect the periorbital area. The left eye was used as the blank control group, and the right eye was used as the experimental treatment group. The anterior chamber was punctured near the limbus of the right eye of CX3CR1-tdTomato mice with a 30G needle disinfected with alcohol, avoiding the lens and iris. The infusion switch was turned on to avoid bubbles and maintained at 110 mm Hg for 1.5 h. The infusion switch was turned off, the needle was carefully pulled out, and chloramphenicol hydrochloride eye ointment was applied to the ocular surface of CX3CR1-tdTomato mice to prevent secondary infection. One week after ischemia-reperfusion injury, CX3CR1-tdTomato mice were killed by anesthesia, and the mouse retina and optic nerve tissue were separated under a dissecting microscope, frozen sections were made, and relevant experimental results were analyzed.

[0076] (6) Intravitreal AAV injection in CX3CR1-tdTomato mice

[0077] One week after glaucoma surgery, CX3CR1-tdTomato mice were fully anesthetized with sodium pentobarbital. After full mydriasis and topical anesthesia with compound tropicamide and proparacaine hydrochloride eye drops, iodine was used to disinfect the eye area. A Hamilton microinjector with a 32G needle was used to insert the needle from the limbus of the CX3CR1-tdTomato mouse cornea, avoiding the iris and lens, and slowly injecting 2μl of mixed 4AAV (Brn3b+Sox2+Cbln2+NP1) into the vitreous cavity. Each AAV was diluted to ensure that the titer was >1x1012 vg / ml.

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

[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 cups containing the retina were fixed in 4% PFA for 8 hours. The retina was separated under a stereo microscope and washed in TD-buffer 3 times, 10 minutes each time. 2% BSA prepared in PBS was added to block at room temperature for 1 hour, 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 washed in TD-buffer 3 times, 10 minutes each time. Under light-proof conditions, FITC or Alexa Fluo594-labeled secondary antibodies were added dropwise and incubated at 4°C for 4 hours. Subsequently, TD-buffer was washed 3 times, 10 minutes each time, under light-proof conditions. Hoechst was diluted 1:100 with PBS, incubated for 10 minutes, and washed 3 times with PBS. The slides were mounted with colorless mounting medium H-1000 and observed under a Zeiss laser confocal microscope.

[0080] Tissue section immunofluorescence: Cryosections of CX3CR1-tdTomato mouse retina were fixed with 4% PFA for 20 minutes and washed with PBS three times, 5 minutes each time. 0.2% Triton permeabilization for 10 minutes, washed with PBS three times, 5 minutes each time, and blocked with 2% BSA for 1 hour. Primary antibody was prepared with 1% BSA and incubated at 4°C overnight. Primary antibody was washed and washed with PBS three times, 10 minutes each time. Dry, add secondary antibody, and incubate in dark for 1 hour. Secondary antibody was washed and washed with PBS three times, 10 minutes each time, and be careful to avoid light. DAPI (H-1200) was used to seal the slides and the experimental results were observed under a ZEISS 900 laser confocal microscope.

[0081] (8) Pattern electroretinogram (PERG) to evaluate the electrophysiological function of retinal ganglion cells after microglial BSCN reprogramming

[0082] CX3CR1-tdTomato mice were taken in a natural light adaptation state, and they were fixed on a movable experimental table after being fully anesthetized. After artificial tears were given to the test eye, the PERG recording electrode was fixed at the corneal limbus, and the reference electrode and the ground electrode were placed under the cheek and tail of the ipsilateral side of the test eye, respectively. The body position of the mouse was adjusted so that the visual axis of the test eye was perpendicular to the stimulation screen. The Chongqing Guote visual electrophysiological detection system was used, and the stimulation mode was set to a checkerboard. The corneal vertex was 15 cm away from the center of the stimulation field, 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 was (0.3-100 Hz). The above experimental parameters were used to record the amplitude and peak time of the N1-P wave of the mouse PERG, and the improvement of the electrophysiological function of retinal ganglion cells in glaucoma animals after BSCN treatment was analyzed.

[0083] (III) Experimental results

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

[0085] In this example, after one month of combined treatment with Brn3b, Sox2, Cbln1, and NP1 (BSCN for short), we detected a large number of tdTomato-positive ganglion cells in the ganglion cell layer of the retina of normal mice, confirming that microglia can be efficiently converted into ganglion cells through BSCN reprogramming ( Figure 1 ), the regenerated ganglion cell axons establish synaptic connections with the lateral geniculate nucleus (dLGN) and superior colliculus (SC) of the lower brain regions through the optic chiasm (OPC) and optic tract (OPT), suggesting that the regenerated ganglion cells can be integrated into the visual neural circuit ( Figure 2 ).

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

[0087] In order to verify whether the BSCN microglia reprogramming strategy can be implemented in glaucoma animal models to regenerate retinal ganglion cells, 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 electrophysiological functions ( Figure 3 At the same time, we also found that the regenerated ganglion cell axons can establish a complete visual neural circuit with the key nuclei in the lower brain regions ( Figure 4 ).

[0088] (3) Retinal ganglion cell regeneration and axonal projection are time-dependent

[0089] In order to verify whether the conversion of microglia into retinal ganglion cells is time-dependent, we used lineage tracing technology to analyze the regeneration and axonal projection of retinal ganglion cells at different time points after microglia gene reprogramming. The experimental results showed that BSCN-mediated gene therapy could detect tdTomato at two weeks after microglia reprogramming. + RBPMS + Regenerated ganglion cells, however, the ganglion cell axons have not yet established synaptic connections with lower brain areas ( Figure 5-Figure 6 ). At three weeks of reprogramming, regenerated ganglion cell axons crossed the optic chiasm (OPC) and optic tract (OPT), but no axonal projections were detected in the lateral geniculate nucleus (dLGN) and superior colliculus (SC) ( Figure 7 ). One month after reprogramming, axonal projections of newborn ganglion cells were detected in OPCs, OPTs, dLGNs, and SCs ( Figure 1-Figure 2 ). The experimental results suggest that retinal ganglion cells transformed by microglia BSCN reprogramming can be integrated into the visual neural circuit.

[0090] (4) Regeneration of retinal ganglion cells and reconstruction of visual neural circuits in aged glaucomatous mice

[0091] Aged mice have poor regenerative potential. To verify whether the BSCN reprogramming strategy can achieve ganglion cell regeneration in aged mice, we performed lineage tracing of microglia fate in aged mice. The experimental results revealed that the same gene reprogramming strategy in aged mice can still promote the transformation of microglia into retinal ganglion cells and establish synaptic connections with lower brain regions ( Figure 8-Figure 9 ). The experimental results suggest that the BSCN microglia reprogramming strategy can achieve ganglion cell regeneration in adult and elderly glaucoma models.

[0092] (5) Regenerated retinal ganglion cells can survive stably for a long time and maintain the integrity of the visual circuit

[0093] In order to verify whether the regenerated retinal ganglion cells can survive stably for a long time and maintain the integrity of the visual neural circuit, we analyzed the retina and brain slices again 12 months after ganglion cell regeneration. The experimental results confirmed that the regenerated ganglion cells labeled with tdTomato can still be detected in the GCL layer ( Fig.10 ), while the ganglion cell axons can still establish synaptic connections with the key nuclei responsible for vision in the lower brain areas ( Fig.11 ). The experimental results suggest that retinal ganglion cells reprogrammed by microglia can survive stably for a long time 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 microglial BSCN reprogramming strategy would reduce the number of endogenous microglia, which could potentially induce retinal dysfunction in the future, we analyzed retinal flat mounts of CX3CR1-tdtomato mice one month after BSCN reprogramming. The experimental results showed that there was no significant difference in the number of microglia in the BSCN reprogramming experimental group compared with the control group ( Fig.12 ), suggesting that microglial reprogramming strategy is a safer method for retinal ganglion cell regeneration.

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

[0097] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. An application of a composition, characterized in that: The composition comprises an agent for promoting the expression or biological activity function of Brn3b, Sox2, Cbln1 and NP1 genes in microglia, and the application of the composition is selected from any one of the following: a) A product for preparing retinal ganglion cell regeneration; b) for preparing a product for inducing microglia to reprogram into ganglion cells; c) for preparing a drug for treating diseases related to retinal ganglion cell apoptosis; d) Used for preparing medicine for treating retinal neurodegenerative diseases.

2. The use according to claim 1, characterized in that: The reagent is a nucleotide or nucleic acid construct that targets microglial cells to express Brn3b, Sox2, Cbln1 and NP1 genes or increases their biological activities.

3. The use according to claim 2, characterized in that: The nucleic acid construct comprises the coding sequences of Brn3b, Sox2, Cbln1 and NP1, and a promoter combined with each coding sequence, and the promoter is used for driving the exogenous gene to realize efficient and stable expression in mammalian cells.

4. The use according to claim 3, characterized in that: The nucleic acid construct includes a DIO-AAV expression plasmid or an AAV virus vector thereof of Brn3b, Sox2, Cbln1 and NP1 genes.

5. The use according to claim 4, characterized in that: The DIO-AAV expression plasmid sequences of the Brn3b, Sox2, Cbln1 and NP1 genes are shown in SEQ ID NOs: 1 to 4, respectively.

6. The use according to any one of claims 1 to 5, characterized in that: The diseases associated with retinal ganglion cell apoptosis include glaucoma and traumatic optic neuropathy.

7. The use according to claims 1 to 5, characterized in that: The retinal neurodegenerative diseases include glaucoma.

8. The use according to claims 1 to 5, characterized in that: The product is a kit, an injection or a medicine.

9. A method for inducing microglia to reprogram into ganglion cells, characterized in that: At least: The nucleotides or nucleic acid constructs encoding Brn3b, Sox2, Cbln1 and NP1 genes are transfected into microglia to convert them into retinal ganglion cells.

10. A recombinant cell, characterized in that The method is obtained by introducing nucleotides or nucleic acid constructs capable of expressing Brn3b, Sox2, Cbln1 and NP1 genes into microglia.

Images