Method of reprogramming mueller glial cells to induce cell proliferation and neuronal regeneration

Abstract

The application discloses a method for reprogramming Müller glial cells (MG) to induce cell proliferation and neuron regeneration, and belongs to the technical field of biological medicine. The application constructs an FGF2 / Ascl1 / RA combined reprogramming system. The system induces MG to re-enter the cell cycle under the condition of no neurotoxic injury through FGF2 combined with AAV-Ascl1, and promotes the multi-round proliferation of adult and middle-aged and old MG by using FGF2 / MAPK / PI3K-Akt signals, and further combines with RA to induce MG to maintain a regeneration state for a long time and promote neurogenesis. The reprogramming system established by the application successfully solves a plurality of core technical problems in the field of mammalian retina regeneration. This breakthrough not only provides a brand-new strategy for the regenerative medicine intervention of retina degenerative diseases, but also shows great potential and value of the application from basic research to clinical transformation application, so that the application has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration. Background Technology

[0002] Degenerative retinal diseases, including glaucoma, age-related macular degeneration, and hereditary retinal degeneration, can lead to irreversible neuronal loss and permanent vision impairment. Current clinical treatments primarily focus on slowing disease progression, lacking effective regenerative therapies to restore retinal neurons and visual function. Therefore, activating endogenous cell regeneration and achieving retinal neuronal regeneration has become a crucial direction in regenerative medicine and ophthalmology research.

[0003] Müller glial cells (MGs) are the main glial cells in the retina, playing a crucial role in maintaining retinal homeostasis, providing nutritional support, and responding to injury. In zebrafish, MGs possess natural regenerative capabilities, re-entering the cell cycle after injury to form retinal progenitor cells and further differentiating into new retinal neurons. However, mammalian MGs are typically in a quiescent state and rarely spontaneously re-enter the cell cycle, thus their endogenous regenerative capacity is extremely limited.

[0004] In recent years, in vivo glial cell-to-neuron conversion has become an important research direction, mediated by adeno-associated virus (AAV) to deliver neurogenesis transcription factors. Among these, Ascl1 (Achaete-scute homolog 1) is a classic pro-neurogenesis bHLH transcription factor that can promote cell reprogramming towards neurons. Previous studies have found that under retinal injury conditions, Ascl1 can partially induce neuronal differentiation in glial cells; however, neurotoxic damage may lead to the death of a large number of retinal neurons, potentially destroying the original structure and function of the retina and severely hindering subsequent regeneration and repair.

[0005] In addition, fibroblast growth factor 2 (FGF2) and its downstream MAPK / Erk and PI3K / Akt signaling pathways play important roles in cell proliferation, tissue repair and development, but their effects on MG regeneration in adult and middle-aged mammals are poor.

[0006] Therefore, establishing a novel reprogramming system that can efficiently activate MG proliferation and neurogenesis in adult and middle-aged mammals without neurotoxic damage is of great significance for retinal regeneration and visual function recovery.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration. Using this method, Müller glial cell proliferation and neuronal regeneration can be efficiently activated in the retinas of adult and middle-aged mammals without neurotoxic damage.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides a composition for inducing the proliferation and reprogramming of Müller glial cells into neuronal cells; the composition comprises a first component and a second component; The first component contains: an AAV expression vector carrying the Ascl1 gene; The second component contains any one of the following: (a) FGF2; (b) Nucleic acid molecules encoding FGF2; (c) AAV expression vectors carrying constitutively activated forms of MEK1 and AKT1 in the FGF2 signaling pathway.

[0010] In a second aspect, the present invention provides the use of the above-described composition in the preparation of a medicament for improving or treating retinal degenerative diseases.

[0011] Thirdly, the present invention provides the use of the above composition in the preparation of a kit for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration.

[0012] Fourthly, the present invention provides a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration, the method comprising administering an effective amount of the above-described composition to the retina of a subject.

[0013] The present invention has the following beneficial effects: This invention constructs an FGF2 / Ascl1 / RA combined reprogramming system. This system induces mammalian retinal re-entry into the cell cycle under neurotoxic conditions through the combination of FGF2 and AAV-Ascl1, and promotes multi-round proliferation of adult and middle-aged mammalian retina using FGF2 / MAPK / PI3K-Akt signaling. Furthermore, it combines RA to induce long-term regenerative states in mammalian retinal retina and promote neurogenesis. The reprogramming system established in this invention successfully overcomes several core technical challenges in the field of mammalian retinal regeneration. This breakthrough not only provides a novel strategic framework for regenerative medicine intervention in retinal degenerative diseases but also demonstrates its enormous potential and value in translating basic research into clinical applications. Therefore, this invention has promising application prospects. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram of the structure of the AAV reprogramming carrier of the present invention; Figure 2 This is the result of FGF2 treatment combined with AAV-mediated Ascl1 gene delivery to activate robust cell proliferation in lineage-tracing MG according to Example 1; wherein, 2A is the experimental design for inducing MG proliferation in the retina of adult mice; 2B is a confocal micrograph of AAV-Ascl1 expression in MG lineage tracer mice; 2C is Sun1-GFP + Statistical results of AAV-Ascl1 expression efficiency in lineage-traced MG; 2D shows the integrated analysis results of EdU in the retina during lineage tracking on day 20 after FGF2 / Ascl1 treatment. The yellow arrows in the magnified concave region (lower panel) indicate Sun1-GFP. + Ascl1 + EdU-MG; the pink arrow indicates Sun1-GFP. + EdU + MG, Ascl1 levels decreased; pink arrow indicates Sun1-GFP - Ascl1 - EdU + These are cells that were not traced by lineage (they may be MG cells or not, because the efficiency of Sun1-GFP lineage tracing is about 84%, which cannot reach 100%). 2E represents the integrated analysis results of EdU in lineage-tracking retina 20 days after receiving PBS / Ascl1 treatment; 2F represents the integrated analysis results of EdU in the lineage-tracing retina 20 days after receiving FGF2 / mCherry treatment; 2G is used to quantify EdU from (DF). + Sun1-GFP + Cell number / 100μm retinal slice, data are expressed as SEM mean ±, n=9, p<0.001, one-way ANOVA; 2H describes the analysis of EdU integration in the retina of lineages treated with FGF2 / Ascl1 on day 15 by co-labeling the cell proliferation markers Ki67 (G1, S, G2, and M phases of the cell cycle) or PH3 (M phase of the cell cycle) using immunohistochemistry; yellow arrow: Sun1-GFP. + Ki67 + EdU + or Sun1-GFP + PH3 + EdU + MG; Blue arrow: Sun1-GFP + Ki67 + EdU - MG; Scale size: 40μm; 2I is used to quantify all Sun1-GFP. + Ki67 + or Sun1-GFP + PH3 + The proportion of cells in (H) is expressed as SEM mean ± SEM, n=9; The scale bars in (B) and (DF) are 50 μm; ONL is the outer nuclear layer; INL is the inner nuclear layer; GCL is the ganglion cell layer; Figure 3 To analyze the proliferation time of MG cells stimulated by FGF2 after Ascl1 gene transfer; among which, 3A is the experimental design for MG proliferation time-process analysis. Retinal samples were collected from day 11 to day 16 or day 19 after AAV-Ascl1 gene transfer (i.e. day 1 to day 6 or day 9 after the first injection of FGF2), while EdU was injected 3 hours before sampling (to ensure that it adequately labeled the retinal cells that were undergoing the S phase of the cell cycle at that time). 3B-H is a confocal image showing Sox2 cells undergoing the S phase of the cell cycle at each acquisition point. + Sox9 + MG; Scale size, 50μm; 3I represents the quantitative edU content in retinal slices at each acquisition time point. + Sox2 + Sox9 + Cell number / 300μm; data are expressed as mean ± SEM, n=9; Figure 4 A comprehensive assessment of FGF2 / Ascl1-induced cell cycle reactivation in the mouse retina at different stages; among which, 4A was the experimental design aimed at inducing robust MG proliferation in the retina of C57BL / 6J mice. EdU was injected intraperitoneally (IP) daily on days 11, 13, 14 and 15 after Ascl1 gene transfer, and co-injected intravitreally with FGF2 on day 12 to mark cells in the S phase of the cell cycle. Retinal tissue was collected 3 hours after EdU injection on day 15 for immunohistochemical analysis. 4B is the immunohistochemical analysis of EdU incorporation by co-labeling of MG nuclear antigens Sox2 and Sox9. The arrows indicating enlarged incorporation regions represent EdU after FGF2 / Ascl1 treatment. + Sox2 + Sox9 + Cells, proportional size, 100 μm; 4C represents the co-labeling analysis of the integration of the microglia-specific marker Iba1 with EdU. The arrows indicate EdU that appears after FGF2 / Ascl1 treatment. + Iba1 + Cells, proportional size, 50 μm; 4D represents a co-labeling analysis of the integration of the astrocyte-specific marker S100β with EdU. The arrows indicate the presence of EdU after FGF2 / Ascl1 treatment. + S100β + Cells, proportional size, 50 μm; 4E represents the quantitative measurement of MG cells (EdU) proliferating in 4B-4D. + Sox2 + Sox9 + ), microglia (EdU) + Iba1 + ), astrocytes (EdU + S100β + The proportion of all proliferating cells is expressed as mean ± SEM, n=9; 4F-4H represent the EdU incorporation in whole retinal sections of wild-type mice at 5 months, 9 months, and 13 months of age (under FGF2 / ASCL1 treatment). High-magnification images are represented as (F'-H'), with scale sizes of (FH) and (F'-H') of 500 μm and 100 μm, respectively. 4I represents the quantitative analysis of EdU in the retinas of mice of different ages treated with Ascl1 / FGF2. + Cell number / 100μm, ages 1, 5, 9 and 13 months; data are expressed as mean ± SEM, n = 15; Figure 5 The result of FGF2 / ASCL1 treatment activating MG cells to produce continuous cell division; among which, 5A is an experimental design that demonstrates EdU. + MG and its progeny exhibit radially distributed cell clusters that resemble clonal expansion of retinal progenitor cells during development; FGF2 / Ascl1 treatment and EdU injection were administered in tdTomato lineage-traced mice; retinal samples were collected 20 days after Ascl1 gene transfer for immunohistochemical analysis. Images 5B-5E represent radially distributed MG clusters, suggesting that a MG reactivated during a cell cycle produces multiple daughter cells after multiple cell divisions. The arrows indicate tdTomato. + EdU + Sox2 + or tdTomato + EdU + Sox9 + MG and its descendants; scale size, 20 μm; 5F is used to quantify the number of clones (BE) proliferating MG (which must produce >2 progeny daughter cells) per 330 μm retinal slice; data are expressed as mean ± SEM, n=16; The experiment was designed using 5G to demonstrate the effect of FGF2 / Ascl1 in activating MG to re-enter the cell cycle in Sun1-GFP lineage-traced mice by dual labeling with EdU and BrdU. 5H represents the efficiency analysis of co-labeling with EdU and BrdU, scale size: 50 μm; 5I shows the results of BrdU re-injection 24 hours after EdU injection (i.e., dual labeling with EdU and BrdU). The arrows indicate that EdU and BrdU co-labeled a small subset of MG cells that were reactivated during cell cycle, with a scale size of 50 μm. 5J is EdU (EdU) + BrdU (blue), BrdU (EdU) - BrdU + (red) or both (EdU) + BrdU + MG-derived pluripotent progenitor-like cells (MGPLCs) marked in yellow accounted for the majority of all cell cycle reactivation Sun1-GFP cells. + Percentage of MG cells, data are expressed as mean ± SEM, n=9; Figure 6 This serves as validation of FGF2 activation of the MAPK / Erk and PI3K / Akt signaling pathways in MG (independent of AAV-ASCL1 gene transfer); among which, Experiment 6A was designed to detect the phosphorylation levels of Erk (pErk) and Akt (pKt) in adult mouse MG. ShH10-GFAP-GFP (MG infection marker) and ShH10-GFAP-Ascl1 were co-injected with AAV, and PBS was used as a control injection for FGF2. Wild-type retinas (regardless of whether Ascl1 gene transfer was performed) were collected 4 hours after FGF2 injection for immunohistochemical analysis. 6B-6B'' represents the detection of pErk in the retina after PBS injection; MG is labeled with Sox2 nuclear antigen. 6C-6C'' represents the GFP detected in the retina receiving FGF2 / Ascl1. + Increased pErk levels in MG cell bodies (shown in the enlarged embedding region). 6D-6D'' represents pErk detection in the retina that received Ascl1 gene transfer; 6E-6E'' detection of pErk, Sox2 in the retina of individuals receiving FGF2 injection alone. + Increased pErk levels in MG cell bodies (shown in the enlarged insertion region); 6F is a quantization of Sox2 + or GFP + The relative fluorescence intensity of pErk in MG was derived from (BE). The average pErk level of retinas that received PBS injection was normalized to 1. Data are expressed as mean ± SEM, n=4 retinas, p<0.001, one-way ANOVA. 6G-6G'' represents pAkt assay of the retina injected with PBS; 6H-6H'' is used to detect pAkt and GFP in the retina receiving FGF2 / ASCL1. + Increased pAkt levels in MG cell bodies (shown in the enlarged embedding region); 6I-6I'' represents the detection of pAkt in the retina that received Ascl1 gene transfer; 6J-6J' is used to detect pAkt and Sox2 in the retina of individuals who received FGF2 injections alone. + Increased pAkt levels in MG cell bodies (shown in the enlarged insertion region); 6K is used to quantify Sox2 from (GJ) + or GFP + The relative fluorescence intensity of pAkt in MG, the average pAkt level of retinas receiving PBS injection was normalized to 1, data are expressed as mean ± SEM, n=4 retinas, p<0.001, one-way ANOVA; scale size, 50 μm; Figure 7To verify the results showing that the pro-cell proliferation effect of FGF2 on MG is mediated by the MAPK / Erk and PI3K / Akt signaling pathways; among which, 7A describes the experimental design for drug inhibition of the MAPK / Erk and PI3K / Akt pathways: In Sun1-GFP lineage-traced mice, the drug inhibitor was injected once daily via intravitreal and intraperitoneal injection for 10 to 12 days after Ascl1 gene transfer; FGF2 (0.4 mg / mL) was co-injected with the inhibitor on day 10 at a ratio of 3:1; retinal samples for immunohistochemical analysis were collected 3 hours after the last EdU injection (i.e., day 15 after Ascl1 gene transfer); 7B-7D were used to analyze the effects of U0126 or LY294002 inhibitors on EdU incorporation in FGF2 / Ascl1-treated retina; DMSO was used as a control solvent for both inhibitors; scale size, 50 μm; 7E represents EdU from (BD) quantitative retinal slices. + Sun1-GFP + Cell number / 100μm, data are expressed as mean ± SEM, n=9, **p<0.01, one-way ANOVA; 7F is used to quantify Sun1-GFP from (BD) retinal slices. + Total number of MG and its offspring; data are expressed as mean ± SEM, n=9, *p<0.05, one-way ANOVA; The experimental design for 7G was to analyze the functional gain of AAV in Sun1-GFP lineage-tracing mice. Ascl1 was co-injected with AAV by ShH10-GFAP- and constituent MEK1 and / or AKT1 mutants. EdU was injected once daily on days 8, 10, 12 and 14 after AAV infection. Retinal samples were collected on day 15 for immunohistochemical analysis. 7H-7K represents the integration analysis of EdU after gene transfer to the retina by constituent activating MEK1 (mCherry as reporter) and / or constituent activating AKT1 (mCherry or BFP as reporter) together with Ascl1, scale size, 50 μm; 7L is a quantitative EdU from (HK) + Sun1-GFP + Cell number per 100 μm retinal slice, data are expressed as mean ± SEM, n=9, **p<0.01, ***p<0.001, one-way ANOVA; 7M is used to quantify Sun1-GFP from (HK). + Total number of MG and its progeny in retinal sections, data are expressed as mean ± SEM, n=9, p<0.001, one-way ANOVA; Figure 8 To verify the results of FGF2 / Ascl1 / RA treatment in promoting reprogrammed MG neural differentiation towards photoreceptors and bipolar cell fate; among which, Experiment 8A was designed to investigate MG-derived neurogenesis after FGF2 / Ascl1 / RA (FAR) treatment in Sun1-GFP lineage-traced mice. Retinal samples were collected 90 days after Ascl1 gene delivery for immunohistochemical analysis. ShH10-GFAP-mCherry was used as a viral control for Ascl1. PBS and cottonseed oil were used as controls for FGF2 and RA, respectively. 8B is a confocal image showing lineage tracing of Sun1-GFP. + MG and its progeny, combined with immunofluorescence staining of photoreceptor / bipolar cell marker Otx2 and EdU integration, were all performed under control treatments such as FGF2 / Ascl1 (FA), FAR, or PBS / Ascl1, PBS / Ascl1 / RA; the blue arrow indicates Sun1-GFP. + EdU + Otx2 + MG or Sun1-GFP + Ascl1 + Otx2 + MG; representative Sun1-GFP + EdU + Otx2 + MG-derived cells are shown in the magnified embedding region; scale size, 40 μm; 8C represents Sun1-GFP under different treatments, including (B). + The proportion of MG and its progeny expressing Otx2 was quantified. Data are expressed as mean ± SEM, n = 9, p < 0.001, one-way ANOVA; 8D is a quantitative indicator of Sun1-GFP for cell cycle reactivation. + The proportion of MG and its progeny expressing Otx2 under FA or FAR treatment, data are expressed as mean ± SEM, n=9, **p<0.01; 8E-8G are confocal images demonstrating tdTomato lineage tracing. + MG differentiates into different stages of Otx2-expressing neuron-like cells under FAR treatment, from the early stage (E), i.e., Otx2-derived cells. +MG cells simultaneously express the glial cell marker Sox9 (yellow arrow). By the mid-stage (F), Sox9 expression is significantly reduced (orange arrow), and by the late stage (G), Sox9 expression is undetectable (pink arrow), and the cells exhibit morphological characteristics similar to photoreceptor cells; scale size, 20 μm; 8H-8I are confocal images showing Sun1-GFP cell cycle reactivation. + MG and the photoreceptor / bipolar cell marker Crx were co-labeled under FA or FAR treatment; blue arrow, Sun1-GFP + EdU + Crx + MG; representative Sun1-GFP + EdU + Crx + MG-derived cells are shown in the magnified embedding region; scale size, 40 μm; 8J is used for quantitative analysis of Sun1-GFP. + The proportion of MG and its progeny expressing Crx in HI, data are expressed as mean ± SEM, n=9, **p<0.01; 8K is Sun1-GFP, which is responsible for cell cycle reactivation. + The proportion of Crx expressed by MG and its progeny in (HI) was quantified. Data are expressed as mean ± SEM, n=9, **p<0.01; 8L-8M is a confocal image showing tdTomato cells undergoing cell cycle reactivation. + MG and its progeny co-labeled with the photoreceptor / bipolar cell marker Crx under FA or FAR treatment, blue arrow, tdTomato + EdU + Crx + MG, Orange Arrow, tdTomato + EdU - Crx + MG, proportional size, 40μm; Figure 9 The study investigated the FAR-induced proliferation and neuronal regeneration of MG neurons under NMDA-induced neurotoxic injury; among which, The experimental design in 9A aimed to investigate cell death in the mouse retina under FAR treatment in the Sun1-GFP lineage; intravitreal injection of NMDA (100 mM, 1.5 μL) was used as a positive control, while the uninjected retina was used as a negative control. 9B shows the analysis of TUNEL staining at different acquisition time points under FAR treatment, scale size 50 μm; 9C represents the TUNEL content in retinal slices under different treatments.+ Quantification of cells / 100 μm (B), data are expressed as mean ± SEM, n = 4 retinas; p < 0.001, ns indicate p > 0.05, ANOVA; The experiment was designed as 9D to investigate MG-derived proliferation and neurogenesis in tamoxifen-induced tdTomato lineage-tracing mice after FAR treatment; NMDA (100 mM) and FGF2 (0.4 mg / mL) were co-injected in a 1:1 ratio to induce damage; retinal samples with or without neurotoxin-induced damage (FAR) were collected 90 days after Ascl1 gene delivery for immunohistochemical (IHC) analysis. 9E represents EdU treated with FAR (F) and (I) in the intact (FAR) or damaged (FARN) mouse retina. + tdTomato + Quantitative analysis of cells per 100 μm, data are presented as mean ± SEM, n = 9, p < 0.001; 9F is a confocal image showing cell cycle reactivation and lineage tracing of tdTomato. + MG and its progeny, co-labeled with the photoreceptor / bipolar cell marker Otx2 under FAR or FARN treatment; blue arrow, tdTomato + EdU + Otx2 + MG; Orange arrow, tdTomato + EdU - Otx2 + MG; Scale size, 50μm; 9G is a quantitative tdTomato + The proportion of MG and its progeny expressing Otx2 in ONL, INL, or both layers; data are expressed as mean ± SEM, n=9; **p<0.01, ***p<0.001, ns indicates p>0.05, one-way ANOVA; 9H represents the tdTomato cell cycle reactivation. + The proportion of MG and its progeny expressing Otx2 in ONL, INL or both layers was quantified. Data are expressed as mean ± SEM, n=9, p<0.001, ns indicate p>0.05, and analysis of variance was performed. 9I is a confocal image showing cell cycle reactivation and lineage tracing of tdTomato. + MG and its progeny, co-labeled with the amacrine / receptor ganglion cell (RGC) marker HuC / D under FAR or FARN treatment; blue arrow, tdTomato + EdU+ HuC / D + MG; Scale size, 50μm; 9J represents tdTomato in quantization (I) + The proportion of HuC / D expressed by MG and its progeny; data are expressed as mean ± SEM, n=9, *p<0.05; 9K represents the tdTomato gene for quantifying cell cycle reactivation in (I) + The proportion of HuC / D expressed by MG and its progeny, data are expressed as mean ± SEM, n=9, *p<0.05; Figure 10 These are the results of single-cell transcriptome analysis; among them, The 10A experimental design utilizes scRNA sequencing analysis to examine MG reprogrammed by FAR. FAR treatment was performed in 2-3 month old mice to trace the Sun1-GFP lineage; Sun1-GFP... + MG (Ctrl, without FAR treatment) or reprogrammed MG were purified by fluorescence-activated cell sorting (FACS) at 13, 20 or 90 days after Ascl1 gene transfer for scRNA-seq analysis. 10B-10C are UMAP plots showing MG-derived clusters under different treatment conditions after contamination removal, where n represents the number of lineage-traced MG and its derived cells used for single-cell analysis under each treatment condition. The 10D dot plot shows the expression of cell type-specific marker genes for integrating MG-derived clusters under various treatment conditions in the annotation and definition (EF) section. The 10E UMAP diagram shows 17 MG-derived cell clusters identified by different cell type-specific marker genes; 10F shows the composition of MG-derived cell clusters under different treatment conditions: MG (resting type), cyclic MG, MGPLC (MG-derived retinal progenitor cells), reactive MG, MG-derived neurogenic progenitor cells, MG-derived transitional neurogenic cells, MG-derived rod cells, and biphasic cells. The 10G feature map shows cell cycle-related ( Mki67 , Cdk1 , Mcm6 and Ccnd1 The gene is upregulated in the cellular MG cluster; 10H is a feature map showing neural progenitor / neural progenitor genes (in the cell cycle MG and MGPLC clusters) Btg2 and Gal The adjustment was raised. The 10I dot plot shows the expression of marker genes for different retinal neuron types in transitional neurogenic cell clusters, with particular emphasis on the expression of marker genes for amacrine and cone cells; 10J feature maps show that gene expression of the neuronal marker Otx2 is enriched in transitional neurogenic cells, rod-like and bipolar-like cell clusters; 10K feature maps show that gene expression of the neuronal marker Nrl is enriched in transitional neurogenic cells and rod-like cell clusters; 10L feature maps show that gene expression of the neuronal marker Pcp2 is enriched in transitional neurogenic cells and bipolar-like cell clusters; The 10M-10N feature map shows the absence of long-particle marker genes. Tfap2b and cone photoreceptor marker genes Arr3 It is mainly expressed in transitional neurogenic cell clusters (red dashed lines); 10O validated the MG in FARday90 processed retinal samples. Nrl Upregulated expression demonstrated co-labeling of cell cycle reactivation, a subset of lineage-tracing MG-derived cells, and the rod photoreceptor marker Nrl; the arrows indicate Nrl. + EdU + MG-derived rod cells are localized in the ONL; 10P validated the presence of MG in FARday90-treated retinal samples by displaying a subset of MG-derived cells with cell cycle reactivation and lineage tracing co-labeling with the bipolar cell marker Pcp2. Pcp2 Upregulated expression; blue arrows indicate Pcp2 located in INL. + EdU + MG-derived bipolar-like cells; orange arrows indicate Sun1-GFP that has not been integrated with EdU. + Pcp2 + MG-derived cells; 10Q validated the presence of MG in FARday90-treated retinal samples by demonstrating cell cycle reactivation, lineage tracing of MG-derived cell subsets, and co-labeling with the amacrine cell marker Tfap2b. Tfap2b The upregulation expression; the arrow shows Tfap2b. + EdU + MG-derived amacrine cells are located medially to the INL; 10R validated the MG in FARday90 processed retinal samples. Arr3 Upregulated expression of [a specific substance] indicates a small subset of cell cycle reactivation, lineage-tracing MG, and the cone photoreceptor marker Cone Arrestin (produced by [a specific substance]).Arr3 (Encoding) co-marked; the arrow indicates Arr3 + EdU + MG-derived cone-like cells are located at the top of the ONL; Figure 11 The results are the trajectory analysis results; among them, Figures 11A-11C are UMAP plots, which show two possible fate trajectories of MG after robust cell proliferation; the first direction points to resting MG (red X), and the second points to retinal neurons (blue arrow under FAR treatment); cells in the FAR 13-day sample were set as the starting point for trajectory analysis. 11D is a UMAP diagram showing the pseudo-temporal analysis of the second trajectory, in which the cellular MG under FAR treatment can enter a regenerative state and eventually generate neurogenesis; the cellular MG in the FARday13 group is the starting point, and the MG-derived retinal neurons in the FARday90 group are the ending point. 11E is a heatmap that shows the pseudo-temporal expression of DEGs (differentially expressed genes). The identified DEGs are divided into 5 clusters by k-means clustering. 11F is a dot plot, which shows the representative GO terms enriched in the DEG cluster in the (E) pseudo-time series analysis. All GO terms shown have GeneRatio>0.01 and adjusted p-values<0.0001. 11G is a graphical summary of the progression of MG-related neurogenesis after FAR treatment; Figure 12 The study aimed to verify the effect of microglia depletion on reducing MG proliferation induced by FAR treatment, while enhancing retinal neurogenesis in adult mice after FAR treatment; among which; 12A is a dot plot showing that the expression of inflammatory response genes is upregulated in the cell cycle MG, MGPLC and reactive MG clusters; Experiment 12B was designed to investigate the role of microglia in MG proliferation and neurogenesis after FAR treatment. From 3 days before Ascl1 gene transfer until day 20, gamoxifen-induced Sun1-GFP lineage-tracked mice were fed a plx3397 diet to remove microglia, and MG proliferation was observed. tdTomato lineage-tracked mice were fed a plx3397 diet from day 20 to day 90 to monitor the effect of microglia loss on MG-derived neurogenesis. 12C represents the integration of EdU in the retinas of FAR-treated Sun1-GFP lineage mice under normal (Ctrl-diet) or microglia-deficient (plx3397-diet) conditions by co-labeling the microglia marker Iba1 with EdU via immunohistochemistry. The scale size is 50 μm. 12D-12E represents Iba1 in the retina (12C) of Sun1-GFP mice fed with either the control or plx3397 diet. + Microglia and EdU + Sun1-GFP + Quantitative analysis of cells / 100 μm; data are expressed as mean ± SEM, n=9; *p<0.05, ***p<0.001, unpaired t-test.

[0016] 12F represents the analysis of EdU integration in the retinas of FAR-treated tdTomato lineage mice under normal (Ctrl-diet) or microglia-deficient (plx3397-diet) conditions by immunohistochemistry co-labeling Iba1 and EdU; scale size, 50 μm. 12G-12H served as a quantitative control or was used to measure Iba1 in the retinas of tdTomato mice fed a diet of plx3397. + Microglia and EdU + tdTomato + Cells / 100 μm; data are expressed as mean ± SEM, n=9; p<0.001, ns indicate p>0.05, unpaired t-test; 12I is a confocal image showing tdTomato cells undergoing cell cycle reactivation in either the control group (normal feeding) or the microglia-deficient group (plx3397 feeding). + Co-labeling of MG and its progeny with the photoreceptor / bipolar cell marker Otx2; blue arrow, tdTomato + EdU + Otx2 + MG; Orange arrow, tdTomato + EdU - Otx2 + MG; Scale size, 50μm; 12J is tdTomato + The proportion of MG and its progeny expressing Otx2 in (I) was quantified. Data are presented as mean ± SEM, n=9, *p<0.05, unpaired t-test. 12K is the tdTomato gene for cell cycle reactivation. + Proportional quantification of Otx2 expression in MG and its progeny in (I); data are expressed as SEM mean, ± n = 8, *p < 0.05, unpaired t-test; 12L is a confocal image showing tdTomato cells undergoing cell cycle reactivation in either the control group (normal feeding) or the microglia-deficient group (plx3397 feeding). +Co-marking of MG and its progeny with the amacrine / retinocyte (RGC) marker HuC / D; blue arrow, tdTomato + EdU + HuC / D + MG; Scale size, 50μm; 12M is tdTomato + The proportion of HuC / D expressed in MG and its progeny in (L) was quantified. Data are expressed as SEM mean ±, n=9, p<0.001, unpaired t-test; 12N is tdTomato, which is responsible for cell cycle reactivation. + The proportion of HuC / D expressed in MG and its progeny in (L) was quantified. Data are expressed as mean ± SEM, n=9, *p<0.05, unpaired t-test. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] Currently, while there are various methods for inducing Müller glial cell proliferation and neuronal regeneration, NMDA lesioning combined with Ascl1 reprogramming technology can only induce limited neurogenesis in young mice, with significantly reduced efficacy in adult and middle-aged mice. AAV-mediated direct glial cell-to-neuron technology faces lineage controversy, with some so-called "glial-derived neurons" potentially originating from virally infected neurons. YAP / Hippo signaling-activated glial cell proliferation technology primarily promotes glial cell proliferation but struggles to further induce efficient neurogenesis. FGF2-promoted glial cell activation technology has yet to achieve potent glial cell proliferation and sustained neurogenesis under non-invasive conditions.

[0019] To address the aforementioned issues, this invention proposes a method combining AAV-Ascl1 and FGF2. AAV-mediated Ascl1 expression, coupled with enhanced FGF2 signaling, induces resting MG to re-enter the cell cycle. Furthermore, MAPK / Erk-dominated proliferation signaling promotes multiple rounds of cell division to form MG-derived progenitor-like cells. Further, RA is combined to maintain MG regeneration and promote neurogenesis, thereby achieving reprogramming and neurogenesis-inducing MG regeneration in the retina of adult and middle-aged mammals under conditions free from neurotoxic damage.

[0020] Based on the above research results, the present invention can provide a composition for inducing the proliferation of Müller glial cells and reprogramming them into neuronal cells; wherein the first component and the second component; The first component contains: an AAV expression vector carrying the Ascl1 gene; The second component contains any one of the following: (a) FGF2; (b) Nucleic acid molecules encoding FGF2; (c) AAV expression vectors carrying constitutively activated forms of MEK1 and AKT1 in the FGF2 signaling pathway.

[0021] MEK1 and AKT1 are key downstream signaling molecules that initiate cellular responses with FGF2. Co-infection with AAV expression vectors carrying constitutively activated forms of MEK1 and AKT1, along with AAV expression vectors carrying the Ascl1 gene, can activate Muller cell proliferation. Specifically, the serotypes of the aforementioned AAV expression vectors are ShH10, 7m8, or AAV9.

[0022] In some embodiments, the AAV expression vector comprises a nucleotide sequence encoding a target gene, operably linked to a glial cell-specific promoter. More preferably, the glial cell-specific promoter includes the GFAP promoter. The structure of the AAV expression vector described above can be found in [reference needed]. Figure 1 .

[0023] It should be noted that the sequences of the Ascl1 gene and GFAP promoter have been published. Both the Ascl1 gene and GFAP promoter used in this invention can be found in conventional databases in the field. For example, the Ascl1 gene can be referenced in NM_008553.4, and the GFAP promoter can be referenced in the sequence shown in SEQ ID NO.1. In addition, other sequences obtainable by those skilled in the art can also be used, and this invention does not specifically limit these. The MEK1 and AKT1 genes are constitutively activated MEK1 and AKT1. Constitutively activated MEK1 and AKT1 are commercially available reagents, which can be obtained by those skilled in the art through purchase or by referring to published techniques for preparation. The constitutively activated form is a protein form that maintains its activity independently of upstream signals, generated through artificial modification or natural mutation. In optional schemes, the mutation methods for MEK1 include S218E, S222D, or R4F; in optional schemes, a myristylation signal is added to the N-terminus of the AKT1 protein to form constitutively activated AKT1, or the serine residue at position 473 is mutated to aspartic acid.

[0024] More preferably, the above composition further includes a third component, which is a neural differentiation promoting factor, specifically retinal acid (RA). When AAV-Ascl1, FGF2 and RA are combined to reprogram Müller glial cells, it can maintain the regenerative state of MG cells and promote neurogenesis while promoting MG cell proliferation, thereby achieving the effect of reprogramming and inducing neurogenesis in the retina of adult and middle-aged mammals without neurotoxic damage.

[0025] The present invention provides for the use of the above composition in any of the following: (1) To prepare drugs for improving or treating retinal degenerative diseases; (2) Prepare a kit for inducing cell proliferation and neuronal regeneration in reprogrammed Müller glial cells; (3) Reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration; (4) Improve or treat retinal degenerative diseases.

[0026] However, it should be noted that while reprogramming Muller cells to regenerate nerve cells may slow the progression of hereditary diseases and even improve visual function for a period of time, it cannot completely cure hereditary gene defects in the long run.

[0027] In some embodiments, the aforementioned retinal degenerative diseases include glaucoma, age-related macular degeneration, and hereditary retinal degeneration.

[0028] Based on the above, the present invention can also provide a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration, the method comprising: administering an effective amount of the above composition to the retina of a subject.

[0029] In this invention, the proliferation of subjects or Müller glial cells can be induced without neurotoxic damage such as NMDA.

[0030] In some embodiments, the subjects include adult or middle-aged and elderly mammals. Experiments have demonstrated that the method of the present invention is not only applicable to young mammals, but also efficiently activates MG regeneration capacity in the retinas of adult, middle-aged, and elderly mammals, overcoming the shortcomings of existing NMDA lesion combined with Ascl1 reprogramming techniques.

[0031] In this invention, the subject refers to any animal (e.g., mammals, birds, reptiles, amphibians, fish), including but not limited to humans, non-human primates, rodents, etc., which becomes the recipient of the specific treatment.

[0032] In this invention, an effective amount refers to the amount required to produce the desired therapeutic outcome. For example, an effective amount is an effective level of delaying or improving the symptoms of a disease for which the composition or drug is administered. The determination of an effective amount for a specific therapeutic target will depend on a variety of factors, including the disease being treated and its severity and / or stage of development / progress; bioavailability and the activity of the composition or drug used; the route or method of administration and the site of introduction on the subject; the clearance rate and other pharmacokinetic properties of the composition or drug; the duration of treatment; the infection regimen; drugs used in combination with or concurrently with the composition or drug; the age, weight, sex, diet, physiological and general health status of the subject; and similar factors well known to those skilled in the art. Depending on the individual being treated, some necessary variations in dosage may occur, and in any case, the appropriate dosage for an individual patient will be determined by a physician or other individual administering the treatment.

[0033] In this invention, "application" refers to delivering a therapeutically effective amount of the composition to the subject via intravitreal, intraocular, ocular, subretinal, intraperitoneal injection, oral administration, or topical administration. Specifically, the first and second components of the composition are administered via intravitreal injection; the third component is administered via intraperitoneal injection, and may also be administered via direct oral administration of a drug containing retinoic acid or its precursor, or via topical administration of ophthalmic preparations (including eye drops, injections, ointments, and other dosage forms).

[0034] The compositions of the present invention can be administered alone, but may also be administered together with other compounds, excipients, fillers, binders, carriers, or other delivery agents, depending on the chosen route of administration and standard pharmaceutical practice. Administration may be by means of a carrier or delivery agent, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; sustained-release gels, etc.

[0035] In some embodiments, FGF2 can be prepared as a sustained-release formulation, including sustained-release gels and microspheres; it can be administered via eye drops, intravitreal injection, etc., and by controlling the degradation rate of the polymeric material, the drug can maintain a stable and effective blood concentration in the eye.

[0036] The above method can also be applied to in vitro treatment. This method includes: contacting an effective amount of the above composition with Müller glial cells in an in vitro culture system, and culturing the treated cells under conditions suitable for cell survival and transdifferentiation.

[0037] In in vitro processing methods, the amount of the above-mentioned composition can be adjusted and optimized according to the experimental subjects, and the present invention does not limit this. Any reprogramming of Müller glial cells to induce cell proliferation and neuronal regeneration using the technical concept of the present invention, whether in vivo or in vitro, falls within the protection scope of the present invention.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] The experimental animals involved in the embodiments of the present invention: Wild-type mice (strain C57BL / 6J, JAX#000664), transgenic mouse strain Glast-CreERT (JAX#012586), and reporter gene mouse strains Rosa-CAG-LSL-Sun1-GFP (JAX#021039) and Rosa-CAG-LSL-tdTomato (Ai9, JAX#007909) were all purchased from Jackson Laboratories.

[0040] Heterozygous Glast-CreERT mice and homozygous reporter mice were mated. Following standard operating procedures published by the Jackson laboratory, PCR genotyping was performed on Glast-CreERT;Rosa-CAG-LSL-Sun1-GFP and Glast-CreERT;Rosa-CAG-LSL-tdTomato MG lineage-tracing mice for validation.

[0041] All mice were housed in a temperature-controlled environment, following a 12-hour light / dark cycle, and had free access to water and food. Male and female mice were randomly assigned to groups during the experiment. All animal experiments were conducted in accordance with the ethical guidelines and procedures for animal use established by the American Academy of Visual and Ophthalmology (ARVO).

[0042] The cell lines involved in the embodiments of the present invention are as follows: AAVpro 293T cell line (632273, RRID: CVCL_B0XW) was purchased from Takara Bio USA, Inc. and cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37°C under 5% CO2.

[0043] Example 1 This embodiment describes a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration, as detailed below: S1. Constructing the AAV reprogramming carrier The structure of the AAV reprogramming carrier is as follows: Figure 1As shown: the Ascl1 coding sequence, GFP, mCherry and BFP with P2A self-cleavage site were PCR-based subclones inserted into the AAV vector backbone, and expression was driven by the mini GFAP (SEQ ID NO.1) promoter.

[0044] A single adeno-associated virus (AAV) was generated in the AAVpro 293T cell line by co-transfection with the ShH10 and Helper plasmids. AAV production and purification were performed using discontinuous iodixanol gradient ultracentrifugation. The purified AAV was washed with PBS and concentrated using an Amicon Ultra-15 filter unit (Millipore). Viral titers (copies per milliliter of genome) were determined by real-time PCR. The obtained AAV reprogramming vector was designated ShH10-GFAP-Ascl1.

[0045] S2.AAV reprogramming vector and FGF2 intravitreal injection In 2-3 month old tamoxifen-induced Sun1-GFP MG lineage-tracing mice, ShH10-GFAP-Ascl1 was injected intravitreally, followed by two intravitreal injections of FGF2 on days 10 and 12 post-Ascl1 gene transfer. EdU was injected intraperitoneally twice daily (IP) from days 11 to 15 post-Ascl1 gene transfer, and on day 12, it was co-injected intravitreally with FGF2 (INV) to label cells in S phase of the cell cycle. GFAP-mCherry and PBS served as controls for GFAP-Ascl1 and FGF2, respectively.

[0046] The method for inducing Sun1-GFP MG lineage tracking mice with tamoxifen was as follows: tamoxifen (SigmaT5648) was dissolved in corn oil at a concentration of 20 mg / mL, and injected at a dose of 100 mg / body weight daily for five consecutive days.

[0047] The intravitreal injection was administered as follows: Mice were anesthetized via intraperitoneal injection with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg). The injection was then performed using a microsyringe equipped with a 33-gauge needle: the needle tip penetrated the sclera, near the dorsal margin of the eye, and entered the vitreous cavity. The injection volume of AAV, FGF2 (R&D SYSTEMS, 233-FB, with a final concentration of 0.2 μg / μL in PBS when injected alone or co-injected with EdU), and EdU (with a final concentration of 2 mg / mL in PBS when co-injected with FGF2) was 1.5 μL per eye. Intravitreal injection of antibiotic eye ointment was also given to prevent infection.

[0048] EdU (Invitrogen, E10187) and BrdU (Invitrogen, B23151) were both prepared into 10 mg / mL solutions using PBS. Mice were injected with either EdU solution at a dose of 2 mL / kg or 5 mL / kg body weight.

[0049] S3. Lineage Tracing and Neural Development Analysis Retinal samples were collected 10, 15, 20, or 90 days after Ascl1 gene transfer for immunohistochemical analysis, EdU integration analysis and BrdU detection, Ki67 detection, single-cell RNA sequencing, UMAP cluster analysis, and Slingshot trajectory analysis.

[0050] The results are as follows: (1) Following AAV-mediated Ascl1 gene delivery, FGF2 treatment activated strong MG proliferation in adult and middle-aged mice without excitotoxic damage.

[0051] like Figure 2 As shown, Part A presents the experimental design for inducing MG proliferation in the retina of adult mice; Part B, with its confocal micrographs, shows the detection of Sun1-GFP expression and AAV-mediated Ascl1 gene transfer by immunohistochemistry on day 10 before FGF2 injection; Part C reveals that approximately 88.2% of the Sun1-GFP-tracked MGs were immunoreactive to Ascl1 10 days after AAV infection, indicating that Ascl1 is efficiently transferred to lineage-tracked adult mouse MGs; Part D shows the Sun1-GFP tracked in the retina treated with FGF2 / Ascl1. + A large amount of EdU was detected in MG. + Cells, in comparison, contain very little EdU + Cells were injected with PBS (as a control for FGF2 injection) after AAV gene delivery of Ascl1 (see [link to PBS]). Figure 2 Detected in the retina of E), and in the retina of AAV gene-delivered mCherry (as a control for Ascl1 gene transfer) after receiving FGF2 injection, see [link to relevant documentation]. Figure 2 F and Figure 2 G.

[0052] Since EdU only marks circulating cells in the S phase of the cell cycle, co-immunostained staining experiments were performed 3 hours after the last EdU injection using two cell cycle markers, Ki67 or PH3, which are expressed at different stages of the cell cycle. The tracked Sun1-GFP subset (~23.9%) of MG was immunoreactive to Ki67, marking active cell cycle phases (G1, S, G2, and M); while a small subset of Sun1-GFP MG (~2.8%) was immunoreactive to PH3, specifically marking M-phase cells (see...). Figure 2 I and 2H). To characterize the time course of MG proliferation, we performed daily EdU injections starting one day after the first FGF2 injection (see I and 2H). Figure 3 A), and retinal samples were collected 3 hours after each daily EdU injection for EdU integration assay. EdU was first detected in Sox2. + Sox9 + The time of detection in MG was 2 days after the first FGF2 injection (see...). Figure 3 B and 3C). The peak of MG proliferation occurred 1 day after the second FGF2 injection, corresponding to day 13 after AAV injection (see B and 3C). Figure 3 D). The proliferative response gradually weakened over the following 3-6 days, until EdU signal was undetectable 7 days after the second injection (equivalent to day 19 after AAV injection) (see...). Figure 3 These results indicate that MG expressing Ascl1 rapidly re-enters the cell cycle upon FGF2 stimulation.

[0053] Next, we examined whether a small subset of proliferating cells in the wild-type retina originated from a non-MG cell population (see...). Figure 4 A). The vast majority of EdU + Cells (~91.5%) were Sox2 + Sox9 + MG, of which ~3.7% EdU + Cells showed an immunoreactivity to the microglia-specific marker Iba1, with ~3.6% of EdU... + Cells exhibit an immunoreactivity to the astrocyte-specific marker S100β. Figure 4 B-4E).

[0054] Next, we examined whether FGF2 / Ascl1 could induce sustained MG proliferation in mice aged 1, 5, 9, and 13 months during aging. Surprisingly, MG maintained robust proliferation in the retinas treated with Ascl1 / FGF2 across all age groups. Figure 4 F-4H). Pooled EdU testing showed similar levels of proliferative response across age groups (see...). Figure 4I). These results indicate that the FGF2 / Ascl1 treatment paradigm is sufficient to restore the proliferative response of MG in middle-aged and older mice, overcome age barriers, and restore the ability of MG to self-renew to a retinal stem cell state.

[0055] (2) FGF2 / Ascl1 stimulation of continuous multi-round MG splitting The EdU incorporation assay only labeled a subset of MGs that were reactivated during the S phase of the cell cycle at the time of EdU injection, while the total number of MGs re-entering the cell cycle could be far greater than the number of EdU-labeled cells. Since MG cell body migration to the ONL is a marker of MG proliferation activation, we did observe a large number of tracked Sun1-GFP lineages or Sox2... + Sox9 + MG migrated to ONL, but was not flagged by EdU (see...) Figure 2 (D and 4B). Given the strong proliferative response in the retina treated with FGF2 / Ascl1, we next investigated whether this strong MG proliferation was caused by MG undergoing several successive rounds of cell division. Indeed, we frequently observed EdU cells aligning radially along the tdTomato lineage 20 days after FGF2 / Ascl1 treatment. + Sox2 + Or EdU + Sok9 + Cell population tracking MG ( Figure 5 This phenomenon (A-5F) is reminiscent of the clonal expansion of a single retinal progenitor cell to produce multiple daughter cells after several rounds of cell division during retinal development.

[0056] To determine whether MG undergoes continuous cell division after FGF2 / Ascl1 treatment, we employed an EdU / BrdU dual-labeling procedure, used to analyze whether MG undergoes multiple rounds of cell division in the adult mouse retina. One day after FGF2 injection, MGs traced to the Sun1-GFP lineage were first labeled with EdU, and circulating MGs were labeled with BrdU 24 hours later to examine the EdU levels. + Sun1-GFP + Does the cell enter a second S phase through another cell division to incorporate BrdU? Figure 5 G). First, the labeling efficiency when EdU and BrdU were administered simultaneously was tested. The results showed that the vast majority of MG tracked by Sun1-GFP (~93.8%) was co-labeled by EdU and BrdU (G). Figure 5H and 5J), indicating that proliferating MG showed similar labeling efficiency for EdU and BrdU during the S phase of the cell cycle. Notably, the EdU / BrdU dual labeling experiment showed that approximately ~7.4% of Sun1-GFP cells that had previously entered the S phase of the cell cycle were labeled with EdU and BrdU. + MG underwent at least two rounds of cell division when tagged with EdU and BrdU (see...) Figure 5 (I-5J). In summary, our results indicate that FGF2 / Ascl1 treatment can stimulate continuous cell division in the MG retina of adult mice, leading to their massive proliferation.

[0057] (3) FGF2 activates the MAPK / Erk and PI3K / Akt signaling pathways in MG. FGF2 signaling is a key factor in regulating stem cell pluripotency by activating the MAPK / Erk and PI3K / Akt signaling pathways. To investigate the roles of MAPK / Erk and PI3K / Akt signaling in regulating MG proliferation in the adult mouse retina, we first examined the phosphorylation levels of Erk and Akt, which reflect the activation of these two pathways in FGF2 / Ascl1-treated MG (see...). Figure 6 A). Although pErk levels are almost undetectable after PBS injection into the retina (see A). Figure 6 B), but after 4 hours of treatment with FGF2 in retina that received Ascl1 gene transfer, pErk in MG (tagged with ShH10-GFAP-GFP) was significantly increased (see B). Figure 6 C, 6F). To determine whether Erk activation was caused by Ascl1 gene transfer or by the therapeutic effect of FGF2, we examined the retina of individuals who received Ascl1 gene transplantation but did not receive FGF2 (PBS as a control for FGF2) and observed no increase in Erk phosphorylation in MG (see [link to relevant documentation]). Figure 6 D, 6F). Interestingly, the Sox2 in the retina received only FGF2 injections + MG has similar Erk activation levels (see) Figure 6 These results indicate that FGF2 independently activates MAPK / Erk signaling in MG, independent of Ascl1 gene transfer.

[0058] Then, the activation of Akt in the FGF2 / Ascl1-treated retina was further examined. The baseline level of Akt phosphorylation was observed in the PBS-injected outer retinal retina (OPL) (see...). Figure 6 G). Although not as strongly activating as Erk phosphorylation, Akt phosphorylation in the MG was slightly increased 4 hours after FGF2 injection in FGF2 / Ascl1-treated retina. Figure 6H, 6K quantification). Sox2 cells that received FGF2 injection but not Ascl1 gene transfer... + A similar increase was also observed in pAkt in MG (see Figure 6 Following FGF2 injection, the phosphorylation level downstream of S6 (pS6) in MG continued to increase, and this was not related to Ascl1 gene transfer. Taken together, these results indicate that FGF2 strongly activates the MAPK / Erk signaling pathway in adult MG and to a lesser extent activates the PI3K / Akt signaling pathway, regardless of whether the Ascl1 gene has been transferred.

[0059] (4) MAPK / Erk and PI3K / Akt signaling regulate MG proliferation in adult mice.

[0060] Building upon the aforementioned studies, this embodiment also involved drug intervention in mice: the MAPK / Erk signaling pathway inhibitor U0126 (Tocris Bioscience, 1144) and the PI3K / Akt signaling pathway inhibitor LY294002 (TocrisBioscience, 1130) were administered via intraperitoneal and intravitreal routes. For the intraperitoneal route, the inhibitors were dissolved in DMSO and then diluted with PBS at a 2:3 ratio, resulting in final doses of 15 mg / kg (U0126) and 15 mg / kg (LY294002), respectively. For the intravitreal route, they were similarly dissolved in DMSO at concentrations of 5 mg / mL and 4 mg / mL, respectively. Each inhibitor was subsequently injected once daily for 10 to 12 days following AAV-mediated Ascl1 gene delivery. On day 10, at the first injection, the inhibitor-containing solution was diluted with FGF2 (0.4 μg / μL, PBS) at a 1:3 ratio. DMSO served as a control for all inhibitors.

[0061] Figure 7 A-7F uses U0126 and LY294002 inhibitors to inhibit the MAPK / Erk and PI3K / Akt signaling pathways, respectively. Figure 7 G-7M utilizes co-transfection of AAV with constituent activating MEK1 and / or AKT mutants (MEK1S218E / S222D, MEK1R4F, caAKT1) and Ascl1 for gene delivery. These results indicate that to maximize the robust proliferation of MGs in the adult mouse retina using FGF2 treatment, simultaneous activation of MAPK / Erk and PI3K / Akt signaling is necessary. MAPK / Erk signaling plays a crucial role in mediating FGF2 / Ascl1-stimulated MG proliferation in the adult mouse retina, while PI3K / Akt plays a synergistic and supportive role in this process.

[0062] Example 2 This embodiment describes a method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration. The difference from Embodiment 1 is that, in this embodiment, mice were intraperitoneally injected with all-trans retinoic acid after or simultaneously with S3, as detailed below: All-trans retinoic acid (RA, Thermo Scientific, 207340010) was dissolved in cottonseed oil at a concentration of 5 mg / mL. RA was administered to mice via intraperitoneal injection at a daily dose of 25 mg / body weight for three consecutive days.

[0063] The methods for constructing the AAV reprogramming vector, intravitreal injection of FGF2, lineage tracing, and neurogenesis analysis are the same as in Example 1.

[0064] The results are as follows: (1) FGF2 / Ascl1 / RA treatment will reprogram MG into a regenerative state, thereby enabling subsequent neurogenesis.

[0065] This embodiment verifies whether MG treated with FGF2 / Ascl1 (denoted as FA) enters a regenerative state for further neurogenesis after exiting the cell cycle. Therefore, 90 days after Ascl1 gene transfer, immunohistochemical analysis was performed on FA-treated samples using a set of retinal neuron markers (see [link to study]). Figure 8 A), FA treatment induced Sun1-GFP. + A portion of MG (~7.7%) expresses Otx2 (a marker for photoreceptor cells and bipolar neurons). Notably, these Sun1-GFPs... + Otx2 + The majority (~4.9%) of cells also contain EdU. + This indicates that they originate from MG (metastatic neuropathy) during cell cycle reactivation. Figure 8 B-8D). In contrast, MG showed very little Otx2 expression under various control treatments, including Ascl1 gene transfer, FGF2 injection, or vector injection (see B-8D). Figure 8 We also observed a small subset of Sun1-GFP. + MG cells (~2.1%) expressed HuC / D (a marker of amacrine cells and retinal ganglion cells (RGCs)) only under FA treatment, not under control treatment; these Sun1-GFP cells were present in the control group. + HuC / D + Approximately 1.3% of the MG cells were also co-labeled with EdU, indicating that they originated from proliferative MG. These results suggest that FA-treated MG cells can enter a regenerative state after exiting the cell cycle, thereby undergoing neurogenesis.

[0066] To evaluate the role of RA in regulating MG proliferation and MG-derived neurogenesis in the adult mouse retina, RA was administered in combination with FGF2 / Ascl1 treatment. MG proliferation and neurogenesis induced by the FGF2 / Ascl1 / RA (FAR) treatment modality were assessed at 20 and 90 days post-Ascl1 gene transfer (see [link to FAR]). Figure 8 A). The results showed that RA did not affect MG proliferation in the retina of adult mice, whether used in combination with FGF2, Ascl1 or both.

[0067] Notably, 90 days after Ascl1 gene transfer, FAR treatment resulted in Sun1-GFP. + The percentage of Otx2 expression in the MG group (approximately 13.5%) was significantly higher than that in the FA group (approximately 7.7%). Figure 8 B, Figure 8 C). Of these, approximately 8.6% (approximately 4.9% in the FA group) also co-marked with EdU, indicating its origin in proliferative MG (see [link to relevant data]). Figure 8 D). Next, we applied FAR treatment to tdTomato lineage-tracing mice to visualize the morphology of MG-derived cells. Interestingly, some of these Otx2 cells... + tdTomato + MG-derived cells, located in the ONL, exhibit a photoreceptor-like morphology after losing the typical Müller glial cell morphology that spans the three layers of the retina. Figure 8 EG). These Otx2 + tdTomato + MG-derived cells also showed reduced expression of the MG marker Sox9 (see...) Figure 8 E and 8F) or below the detection level (see E and 8F) or below the detection level (see Figure 8 These findings suggest that FAR-reprogrammed MGs, while losing their original MG identity, undergo a process of differentiation into photoreceptor and bipolar cell fate. Next, we performed immunohistochemistry on Crx in the retinas of FA and FAR-treated lineage-tracing mice, another marker of photoreceptors and bipolar cells (see G). Figure 8 HM). Sun1-GFP expressing Crx + The proportion of MG cells increased from ~4.7% in the FA group to ~8.3% in the FAR group. Furthermore, Crx cells undergoing cell cycle reactivation... + EdU + Sun1-GFP + The proportion of MG-derived cells also increased from ~3.4% under FA treatment to ~6.4% after FAR treatment (see...). Figure 8JK). On the other hand, the percentage of HuC / D or NeuN expression in lineage-traced MG cells, i.e., markers of amacrine protocysts and rGCs, did not show a significant difference between the FA and FAR groups (regardless of whether the proportion of cell cycle reactivation was counted). Interestingly, some HuC / D cells re-entered the cell cycle... + or NeuN + MG cells reside in the lower part of the INL, exhibiting an amacrine-like morphology and lacking the typical MG morphology. Overall, our results indicate that adding RA to FA treatment enhances MG-derived cell photoreceptor and bipolar cell fate neurogenesis, but does not alter the degree of MG proliferation.

[0068] To assess whether FAR treatment induced cell death, we performed TUNEL assays on retina samples collected on days 13, 20, and 90 of FAR. As expected, NMDA-induced neurotoxicity resulted in significant cell death; however, TUNEL was rarely detected in FAR-treated samples. + Cells, and their levels are comparable to those of an undamaged, intact retina (see...). Figure 9 This indicates that FAR treatment does not lead to significant short-term or long-term cell death.

[0069] (2) MG proliferation and neurogenesis stimulated by FAR treatment under NMDA-induced neurotoxic injury.

[0070] We further investigated whether NMDA-induced neurotoxicity affected MG proliferation and neurogenesis, again at 90 days after FAR treatment (see [link]). Figure 9 NMDA (Sigma #454575, prepared as 100 mM in PBS) and FGF2 (0.4 μg / μL) were co-injected intravitreally on day 10 following Ascl1 gene delivery via AAV at a 1:1 ratio. Surprisingly, MG proliferation was moderately suppressed in the retina treated with FGF2 / Ascl1 / RA / NMDA (denoted as FARN) (see...). Figure 9 Next, we examined MG-derived neurogenesis following FARN treatment. The results showed that NMDA-induced neurotoxicity shifted the fate of FAR-stimulated neurogenesis towards intraretinal neurons (i.e., those without long processes and optic ganglion cells), while moderately inhibiting MG proliferation (see...). Figure 9 ).

[0071] (3) Single-cell sequencing analysis revealed different cellular states during the reprogramming of MG to neurons after FAR treatment.

[0072] To analyze the transcriptome of reprogrammed MG, we performed single-cell RNA sequencing (scRNA-seq) analysis on Sun1-GFP lineage-traced MG purified by fluorescence activated cell sorting (FACS) after FAR treatment. To characterize the progression of the reprogramming process, we selected three time points for transcriptome analysis: an early time point (day 13), corresponding to the peak of MG proliferation; a mid-term time point (day 20), marking the cessation of MG proliferation; and a late time point (day 90), analyzing the long-term status of the reprogrammed MG (see [link to relevant documentation]). Figure 10 A). The dataset was merged, normalized, and analyzed using R and Seurat to create a UMAP. To exclude Sun1-GFP... + A small number of contaminating non-MG cell populations generated during MG purification were re-clustered at high resolution. Vascular / endothelial cells (VE), microglia, astrocytes, and resident endogenous neuronal types shared across all samples were considered contaminants and removed from subsequent analyses. After projecting the filtered cells onto a single UMAP plot, FARday13 (6,977 cells), FARday20 (13,462 cells), and FARday90 (7,099 cells) were obtained compared to the untreated control group (Ctrl, 8,224 cells) (see [link to UMAP]). Figure 10 (B and 10C), we identified 17 cell clusters and annotated them based on the expression of unique cell type-specific markers for each cluster, resulting in 2 MG clusters (MG-1 and MG-2) in the resting state, 3 cyclic MG clusters, 6 MG-derived progenitor-like cell (MGPLC) clusters, 2 reactive MG clusters, 1 neurogenic progenitor cluster, 1 transient neurogenic cell cluster, 1 rod-like cell cluster, and 1 bipolar-like cell (BC-like) cluster. Figure 10 D-10E). In the vast majority (~97.6%) of untreated control samples, purified MG was projected into two resting MG clusters (MG-1 and MG-2), which contain glial cell genes such as... Rlbp1 , Slc1a3 and Glul High expression, but reactive genes such as Gfap , Stat1 and Irf1 The expression is extremely low.

[0073] In contrast, purified MG in FARday13 and FARday20 samples rapidly altered the molecular characteristics of their glial cells, generating three cell cycle MG clusters and multiple MGPLC clusters (see...). Figure 10D and 10E). Cyclic MG clusters comprised ~25.4% of FARday13 cells, but their proportion in FARday20 cells decreased dramatically to only ~2.0% (see D and 10E). Figure 10 This is consistent with our time analysis results, showing that MG proliferation almost ceased on FAR19. Cell cycle status analysis further confirmed that cells with high G2 / M or S phase scores were confined to the cellular MG cluster, which showed cell cycle regulators such as Cdk1 and Mcm6 (see FAR1). Figure 10 D, 10G) and proliferation-related genes such as Mki67 (Markers of all active cell cycle phases) and Ccnd1 Strong enrichment (see) Figure 10 (D, 10G). Simultaneously, glial cell genes were transiently downregulated in the cyclic MG cluster, indicating that MG undergoes dedifferentiation during proliferation. Results from 10D-10H show that between day 13 and day 20, FAR treatment reprogrammed cyclic MG into a progenitor-like cell state, ultimately entering the neurogenic phase; Figure 10 I-10N further reflects the intermediate state of transitional neuronal cell clusters that are transforming into neurons but have not yet fully determined their specific neuronal fate. Finally Figure 10 O-10R validated specific neuronal gene expression in rod-like, bipolar-like, and transitional neurogenic cell clusters identified in FARday90 samples: Sun1-GFP for cell cycle reactivation. + or tdTomato + A subset of MGs expressed Nrl (rod cell marker) in the ONL and Pcp2 (bipolar cell marker) in the NINL. Furthermore, a small subset of lineage-traced MGs expressed Tfap2b (amacrine-agnostic cell marker) in the lower INL and Arr3 (cone cell marker) in the apex of the ONL.

[0074] (4) MG treated with FAR differentiates into retinal neurons rather than returning to a quiescent state.

[0075] To further analyze the trajectory of MG-derived regeneration, we used the single-cell lineage and pseudo-time inference tool Slingshot to analyze gene expression profiles. Starting from the cyclical MG in the FARday13 sample, two different post-division fates emerged: differentiation into retinal neurons or reversion to the resting MG state (see...). Figure 11 A). Trajectory analysis showed that after leaving the cell cycle, cyclic MGs transitioned via MGPLCs in the FARday20 sample and progressed into the neuronal lineage in FARday90, rather than returning to a quiescent state (see [reference]). Figure 11B and 11C). Pseudo-temporal analysis along neuronal trajectories also revealed that reprogrammed MG differentiated into vision-related retinal neuron fates (e.g., B and 11C). Figure 11 D-11F).

[0076] Combining immunohistochemical and morphological analyses of the FARday90 samples, our single-cell transcriptomic and trajectory analyses revealed that a significant proportion of MG cells undergoing cell cycle reactivation first pass through a progenitor-like regeneration state (primarily present in the FARday20 samples) before further differentiating toward retinal neuron fate (see [link to study]). Figure 11 G).

[0077] (5) Microglia promote the proliferation of MG induced by FAR treatment but inhibit neuronal regeneration.

[0078] The inflammatory immune response following retinal injury is a crucial factor regulating MG proliferation and retinal regeneration. Microglia play a key role in this process, serving as a major source of inflammatory cytokines in the central nervous system. These cytokines can either support or inhibit the regenerative response depending on the specific context. Our preliminary results indicate that a small number of proliferating cells (~3.7%) in the retinas of adult mice treated with FGF2 / Ascl1 are actually microglia. Figure 4 C and 4E).

[0079] Figure 12 Further analysis of scRNA-seq data from A revealed the expression of a set of inflammatory response genes, including Stat1 , Bst2 , Isg15 These findings were significantly upregulated in cyclic MG, MGPLC, and reactive MG clusters. Together, these findings suggest that microglia may play a regulatory role in FAR-stimulated MG proliferation and neurogenesis.

[0080] To determine whether microglia regulate FAR-stimulated MG proliferation, we administered PLX3397 (a potent CSF1R inhibitor widely used to deplete microglia) to Sun1-GFP lineage-tracing mice via diet three days prior to Ascl1 gene transfer, and collected FAR-treated retinas on day 20. Figure 12 B). Our analysis indicates that with the reduction of microglia, MG proliferation in the retina is moderately reduced (B). Figure 12 C-12E).

[0081] Next, we investigated whether microglia affect neurogenesis in MG. To separate MG proliferation from its subsequent neuronal regeneration, we tracked mice in the FAR-treated tdTomato lineage by feeding them a diet containing PLX3397 starting on day 20 post-Ascl1 gene transfer (when MG proliferation was largely complete), and collected retinas on day 90. We first confirmed that the number of FAR-induced MG and its derived cells was not affected by microglia depletion after cell division (see [link to relevant documentation]). Figure 12 F-12H). It is worth noting that tdTomato + The proportion of MG expressing Otx2 increases after microglia depletion (see...). Figure 12 I-12K). Similarly, tdTomato + The ratio of MG expressing HuC / D also increases further after microglia depletion (see...). Figure 12 L-12N). The above results indicate that microglia have an inhibitory effect on MG-derived neurogenesis in the retina of adult mice after FAR treatment.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, The composition is used to induce the proliferation of Müller glial cells and reprogram them into neuronal cells; The composition comprises a first component and a second component; The first component comprises: an AAV expression vector carrying the Ascl1 gene; The second component comprises any one of the following: (a) FGF2; (b) Nucleic acid molecules encoding FGF2; (c) AAV expression vectors carrying constitutively activated forms of MEK1 and AKT1 in the FGF2 signaling pathway.

2. The composition according to claim 1, characterized in that, The serotype of the AAV expression vector is ShH10, 7m8 or AAV9; Preferably, the AAV expression vector contains a nucleotide sequence encoding a target gene, which is operatively linked to a glial cell-specific promoter; Preferably, the glial cell-specific promoter includes the GFAP promoter.

3. The composition according to claim 1, characterized in that, The composition also includes a third component, which is a neural differentiation promoting factor; Preferably, the neural differentiation promoting factor includes retinoic acid.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for improving or treating retinal degenerative diseases.

5. The application according to claim 4, characterized in that, The degenerative retinal diseases mentioned include glaucoma, age-related macular degeneration, and hereditary retinal degeneration.

6. Use of the composition according to any one of claims 1 to 3 in the preparation of a kit for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration.

7. A method for reprogramming Müller glial cells to induce cell proliferation and neuronal regeneration, characterized in that, The reprogramming is performed in vivo; the method includes administering an effective amount of the composition according to any one of claims 1 to 3 to the retina of the subject.

8. The method according to claim 7, characterized in that, The reprogramming also includes being performed in vitro; the method includes contacting the Müller glial cells with an effective amount of the composition according to any one of claims 1 to 3 in an in vitro culture system, and culturing the treated cells under conditions suitable for cell survival and transdifferentiation.

9. The method according to claim 8, characterized in that, Neither the subject nor the Müller glial cells had undergone neurotoxic damage; Preferably, the subjects include adult or middle-aged and elderly mammals.

10. The method according to claim 7, characterized in that, The composition may be administered by injection; Preferably, the first and second components of the composition are administered via intravitreal injection; the third component is administered via intraperitoneal injection.

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