Method for stimulating retina ganglion cell regeneration

Abstract

Compositions, nucleic acid molecules, and methods for inducing retinal regeneration and reprogramming Muller glial cells (MGs) into retinal ganglion cells in a subject are described. The development of retina ganglion cell (RGC) transcription factors Pou4f2 and Islet1 increases the MG neurogenesis capability induced by Ascl1. The combination of Ascl1, Pou4f2, and Islet1 stimulates the MG to produce bipolar cells and RGC-like neurons. Likewise, a transcription factor Onecut1, which is expressed in developing retinal cells but not expressed in MG, induces MG to produce RCG-like cells. Other transcription factors that can be used include Irx2, Irx5, Neurod2, Ebf1, and Tcf3. The MG-derived RGC can exhibit action potential in vivo, and exhibit chromatin characteristics similar to those of developing RGC.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 362,361, filed on April 1, 2022, the entire contents of which are incorporated by reference into this application.

[0002] References to sequence listings

[0003] The XML file of the sequence listing named "UW77_seq" is 666kb in size, was created on March 30, 2023, and was submitted electronically with this application, and its contents are incorporated herein by reference in their entirety.

[0004] Thank you for the government's support

[0005] This invention was made with government support under Grant No. R01 EY021482 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Background Art

[0006] Neurodegenerative diseases of the eye can lead to blindness because the mammalian nervous system lacks regenerative capacity. In other vertebrates, such as fish and amphibians, the retina is able to replace lost neurons and restore visual function. Müller glia (MG) are the predominant glial cells in the vertebrate retina and may serve as a source of neurogenic progenitor cells in regenerating species. However, in the mammalian retina, MG respond to retinal damage by undergoing an inflammatory response rather than a regenerative response.

[0007] Many diseases lead to the loss of specific neuronal populations in the retina, resulting in blindness. Therefore, there remains a need to stimulate regeneration of the human retina and develop novel regenerative therapies for patients. Summary of the invention

[0008] Described herein are compositions, nucleic acid molecules, and methods for inducing retinal regeneration in a subject and reprogramming Müller glial cells (MG) into retinal ganglion cells. Examples provided herein show that developmental retinal ganglion cell (RGC) transcription factors Pou4f2 and Islet1 increase the neurogenesis capacity of MG induced by Ascl1. The combination of Ascl1, Pou4f2, and Islet1 stimulates MG to produce bipolar cells and RGC-like neurons. Similarly, Onecut1, a transcription factor expressed in developing retinal cells but not in MG, induces MG to produce RCG-like cells. Other transcription factors that can be used include Irx2, Irx5, Neurod2, Ebf1, and Tcf3. MG-derived RGCs can exhibit action potentials in vivo and display chromatin features similar to those of developing RGCs.

[0009] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor, wherein the RGC transcription factor is selected from the group consisting of Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof. In some embodiments, the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof. In some embodiments, the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof. In some embodiments, the RGC transcription factor is Onecut1. In some embodiments, the RGC transcription factor includes Pou4f2 and / or Islet1. In some embodiments, the RGC transcription factor includes Irx2 and / or Neurod2.

[0010] In some embodiments, the nucleic acid sequence also includes a nucleic acid sequence encoding a proneural basic helix-loop-helix (bHLH) transcription factor. In some embodiments, proneural bHLH transcription factors and RGC transcription factors are expressed in the form of fusion proteins. Representative examples of proneural bHLH transcription factors include, but are not limited to, Ascl1, Atona17 (also known as Math5), Atoh1 (also known as Math1), neurogenin-2, and neuronal differentiation 1 (Neurod1). In one embodiment, the nucleic acid molecule also includes a nucleic acid sequence encoding Ascl1 and Atoh1. In another embodiment, the nucleic acid molecule also includes a nucleic acid sequence encoding Ascl1 and Atoh7. In another embodiment, the nucleic acid molecule also includes a nucleic acid sequence encoding Atoh1 and Atoh7. In another embodiment, the nucleic acid molecule also includes a nucleic acid sequence encoding Ascl1, Atoh1, and Atoh7. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding two, three, four, or all five of Ascl1, Atona17, Atoh1, neurogenin-2, and Neurod1.

[0011] In some embodiments, the nucleic acid comprises Pou4f2 and / or Islet1 and / or Ascl1. In some embodiments, the nucleic acid comprises Pou4f2, Islet1, and Ascl1.

[0012] In some embodiments, the nucleic acid sequence also includes a promoter sequence operably connected to the nucleic acid sequence encoding the RGC transcription factor. In some embodiments, the promoter sequence is not naturally operably connected to the nucleic acid sequence encoding the RGC transcription factor. In some embodiments, the promoter sequence is for genes specifically expressed in glial cells, such as RLBP1 or GLAST. In some embodiments, the promoter sequence is a HES1, RLBP1 or GLAST promoter. In some embodiments, the promoter sequence will be located before the effector gene CRE or tTA that will drive transcription in an inducible manner. In one embodiment, the MG-specific promoter sequence is an Rbpl1 promoter sequence or a portion thereof. In other embodiments, the nucleic acid sequence includes an IRES or 2A self-cleavage site between the sequences encoding the transcription factor, for example, in a polycistronic or polycistronic configuration. In other embodiments, the nucleic acid sequence will include an element that responds to the presence of CRE or tTA so as to trigger gene transcription in an inducible manner. These elements include, but are not limited to, loxP, lox2272, and tetracycline response elements (TRE).

[0013] Also provided are compositions comprising nucleic acid molecules as described herein. In some embodiments, the nucleic acid molecule is mRNA. Such compositions can be formulated for delivery, optionally in the form of a vector. In some embodiments, the vector is a non-viral vector or a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the composition is formulated for administration to the retina. Examples of such administration include, but are not limited to, intravitreal or subretinal injection.

[0014] In some embodiments, the composition further comprises a histone deacetylase (HDAC) inhibitor (HDACi). Representative examples of HDACi include, but are not limited to, trichostatin A (TSA), Istodax TM (also known as (formerly) / romidepsin), Beleodaq TM (also known as (formerly) / belinostat), Farydak TM (also known as (pre) / panobinostat) and Zolinza TM (Also known as (pro) / vorinostat.) Exemplary HDACi peptides include, but are not limited to, 16cyc-HxA, 16lin-HxA, and 16KA (SEQ ID NOs: 58-60).

[0015] Also disclosed herein is a method of inducing retinal regeneration, or a method of stimulating retinal ganglion cell regeneration by administering to cells (e.g., MG cells) a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor (TF). In certain embodiments, the cells are in vitro or ex vivo. Exemplary RGC TFs include Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof. In some embodiments, the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof. In some embodiments, the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof. In some embodiments, the RGC transcription factor is Onecut1. In some embodiments, the RGC transcription factor includes Pou4f2 and / or Islet1. In some embodiments, the RGC transcription factor includes Irx2 and / or Neurod2.

[0016] Also disclosed herein is a method of inducing retinal regeneration, or a method of stimulating the regeneration of retinal ganglion cells in a subject. These methods include administering to the subject's retina a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor. Exemplary RGC TFs include Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3 and combinations thereof. In some embodiments, the transcription factor is selected from Onecut1, Pou4f2, and Islet1 and combinations thereof. In some embodiments, the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3 and combinations thereof. In some embodiments, the RGC transcription factor is Onecut1. In some embodiments, the RGC transcription factor includes Pou4f2 and / or Islet1. In some embodiments, the RGC transcription factor includes Irx2 and / or Neurod2.

[0017] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a proneural basic helix-loop-helix (bHLH) transcription factor, as described herein. In some embodiments, the nucleic acid molecule comprises a first nucleic acid molecule encoding a proneural bHLH transcription factor, and a second nucleic acid molecule encoding an RGC transcription factor. In some embodiments, the nucleic acid molecule is administered in the form of a composition. In some embodiments, the administration comprises administering a composition comprising a first nucleic acid molecule encoding a proneural bHLH transcription factor for the first time, and administering a composition comprising a second nucleic acid molecule encoding an RGC transcription factor selected from the group consisting of: Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3 and combinations thereof at a subsequent time point for the second time. In some embodiments, the transcription factor is selected from Onecut1, Pou4f2, and Islet1 and combinations thereof. In some embodiments, the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3 and combinations thereof. In some embodiments, the RGC transcription factor is Onecut1. In some embodiments, the RGC transcription factor comprises Pou4f2 and / or Islet1. In some embodiments, the RGC transcription factor comprises Irx2 and / or Neurod2. In some embodiments, the proneural bHLH transcription factor and the RGC transcription factor are administered as a fusion protein. In some embodiments, the first and / or second nucleic acid molecule is mRNA.

[0018] In another embodiment, the nucleic acid molecules and methods disclosed herein stimulate the production of functional RGCs from reprogrammed MGs. In another embodiment, the number of functional RGCs derived from MG increases. In another embodiment, the number of functional RGCs increases by 40%. In another embodiment, a retinal disease, retinal damage or degeneration of a subject is treated. In another embodiment, the subject is an adult. In another embodiment, the vector comprises a nucleic acid molecule. In one embodiment, the vector is a non-viral vector or a viral vector, and the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector. In another embodiment, a promoter sequence is operably linked to a nucleic acid encoding a developmental RGC transcription factor. In one embodiment, the promoter is a Cre-inducible or tTA-inducible or MG-specific promoter. In one embodiment, administration to the retina is an intravitreal or subretinal injection.

[0019] Also provided herein is a method of inducing retinal regeneration, the method comprising administering a composition as described herein to a subject. In some embodiments, the method effectively increases the number of RGCs derived from Müller glial cells, induces Müller glial cells to enter the mitotic cell cycle, and / or generates new RGCs. In some embodiments of the method, the number of RGCs increases by at least 40% relative to baseline levels or other reference amounts representing untreated retina. In some embodiments, the number of RGCs increases by 10%, 20%, 25%, 50%, 100%, 150%, 200% or more.

[0020] The subject in the methods disclosed herein is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult. In some embodiments, the subject suffers from a retinal degenerative disease. Examples of such retinal degenerative diseases include, but are not limited to, age-related macular degeneration, glaucoma, ischemia, central retinal artery occlusion, and inherited retinal diseases, such as retinitis pigmentosa or Usher's syndrome.

[0021] The reprogramming of MG and the regeneration of retinal neurons are particularly important for the development of therapeutic products and methods for a range of degenerative eye diseases, such as, but not limited to, retinal degeneration caused by diabetic retinopathy, glaucoma, and age-related macular degeneration. One such retinal degenerative disease is called central retinal artery occlusion (CRAO), in which the blood flow of the central retinal artery is blocked or occluded, usually leading to vision loss. CRAO is caused by thromboembolism, carotid atherosclerosis, giant cell arteritis, aneurysm, or arterial spasm. Current treatment modalities for many such retinal degenerative diseases, especially CRAO, have shown little significant improvement in results. Another indication may be paracentral acute central maculopathy due to venous occlusion (CRVO) or other causes, resulting in thinning of the inner retina and loss of vision. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A-1HPou4f2 and / or Islet1 stimulate regeneration of RGC-like neurons. (1A) Schematic depicting transgenic constructs used to specifically induce Ascl1 and Pou4f2 / Islet1 in MG. Pou4f2 / Islet1 are surrounded by mutually exclusive LoxP-flanked (floxed) sites, resulting in expression of Pou4f2, Islet1, or both in the presence of active Cre. (1B) Experimental paradigm for inducing retinal regeneration in adult mice. Tamoxifen (TMX). (1C) Representative sections of the retina after intravitreal NMDA injury showing transgenic expression of Pou4f2 / Brn3 and / or Islet1 in GFP+ lineage-traced MG. DAPI, 4′,6-diamidino-2-phenylindole. (1D) Quantification of the percentage of transgenic expressing MG expressing Pou4f2, Islet1, or both. (1E and 1F) Representative sections showing MG-derived neurons expressing HuC / D after the regeneration paradigm. (1G) Quantification of the percentage of GFP+ MG-derived neurons expressing HuC / D or Otx2. (1H) Examples of MG-derived neurons expressing Otx2. Significance of differences was determined using an unpaired t-test (asterisk = p < 0.0001); points represent individual animals. Scale bar, 50 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Mouse schematics were created using Biorender.com.

[0023] Figures 2A-2F MG-derived neurons stimulated by Islet1+Pou4f2+Ascl1(IPA) display complex neuronal morphology. (A) Whole mounts of retina stained for GFP (MG-derived cells) and Brn3. (B to E) Examples of morphology of GFP+MG-derived cells. (F) MG-derived (GFP+) cells with complex neurites co-labeled with Brn3. Scale bar, 50 μm.

[0024] Figures 3A-3G.scRNA-seq analysis of Pou4f2 / Islet1 stimulated neurons reveals molecular signatures of RGCs. (A) UMAP plots of FACS-sorted MG-derived cells after the IPA regeneration paradigm combined with a previous scRNA-seq dataset using Ascl1 alone. (B) The signature map highlights the major clusters of MG (Rlbp1), neurogenic transition (Bhlhe22), bipolar cells (Cabp5), photoreceptors (Rcvrn), and RGC-like cells (Elavl4 and Sox11). (C) Distribution of cells per treatment projected onto the segmented UMAP plots. The ring plots represent the percentage of the dataset that each cluster occupies. (D) Heatmap comparing scRNA-seq datasets treated with Ascl1 alone versus IPA. Selected genes associated with RGCs are depicted. (E) Retinal sections were stained with Satb1 (red) for MG-derived cells (GFP) and the percentage of GFP+ cells that are Satb1+ is quantified. (F) Retinal sections were stained with calretinin (red) for MG-derived cells (GFP), and the percentage of calretinin+GFP+ cells was quantified. (G) Clark et al. integrated UMAP of IPA-derived neurons with scRNA-seq of E14 mouse retinas (21), revealing that IPA neurons cluster similarly to immature RGCs. Scale bar, 50 μm.

[0025] Figures 4A-4M.Islet1 and Pou4f2 co-induce RGC-like neurons from MG in vitro. (A) Schematic representation of transgenic constructs for induction of IPA in all primary MGs by doxycycline in vitro. (B) Paradigm for induction of Ascl1-mediated neurogenesis in vitro. (C to E) Representative images of EdU+ MG-derived neurons expressing neuronal markers. (C) EdU+ (white) MG-derived cells expressing Tuj1 (red). (D) MG-derived neurons expressing EdU (white), neurofilament M (NFM; red), and a GFP transgenic reporter (GFP). (E) MG-derived neurons expressing calcium binding proteins (red) co-labeled with EdU (white), DAPI, and GFP. (F to H) UMAP plots of cultured MG reprogrammed with Ascl1 (F) and IPA (G), integrated with cells from the in vivo IPA regeneration model (H) as reference. (I) Stacked bar graphs show the composition of neuronal clusters in each sample. BC, bipolar cells. (J) Feature map highlights differentially expressed genes in neuronal clusters from either reprogramming strategy. (K) Highlighted cells from the Ascl1 and IPA datasets used for downstream DGE analysis in (L). (M) Heat map of differentially expressed genes under Ascl1 or IPA conditions. WT, untreated cultured MG were included as baseline values. Statistics for differential gene analysis: Wilcoxon Mann-Whitney significance test (P < 0.05). Scale bar, 50 μm.

[0026] Figures 5A-5D Physiological analysis of IPA-induced neurons. (A) Summary of the electrical properties of cells in this study compared to endogenous neurons, endogenous glial cells, and MG-derived neurons in previous regeneration protocols (4, 5, 13). Resting potential and input resistance were estimated from current clamp recordings. (B) Three examples of cell responses to current (left) and voltage (right) steps. (C) Three examples of cells responding to light stimulation. (D) Examples of cells showing action potentials or similar events. The left two graphs are responses to hyperpolarizing and depolarizing current steps from cells that produce clear Na+ spikes. The right two graphs are responses from cells that produce smaller discrete events, likely Ca2+ spikes.

[0027] Figures 6A-6J.scATAC reveals that MG remodels chromatin to an RGC-like state after IPA treatment. (A) UMAP of GFP+ sorted MG and their progeny in an in vivo regeneration paradigm in combination with Ascl1 alone (B) or IPA treatment (C). (D) Overlay of known marker genes used to identify clusters. (E and F) chromVAR scores of Otx2 and Pou4f2 to highlight the differential accessibility of their respective motifs. (G) Scatter plot comparing accessible motifs in E14 RGCs to IPA-induced RGCs. (H) Top “GO biological process” results for E14 RGC-specific peaks compared to IPA-derived RGC-like neurons. (I) Heatmap of chromVAR activity scores of top variable motifs of TFs found in the pseudo-temporal lineage from E14 progenitors to RGCs. (J) Heatmap of the same chromVAR activity scores of E14 motifs plotted over the pseudo-time from MG to RGC-like neurons after IPA treatment. (K) Retinal sections showing GFP+ MG-derived cells co-stained with the MG nuclear marker Sox2 (red) and quantification of GFP+ cells expressing Sox2. Scale bar, 50 μm.

[0028] Figures 7A-7I Addition of Atoh1 to the IPA paradigm facilitates the transition from a progenitor state to differentiated neurons. (A) Schematic representation of transgenic constructs expressing IPA with Atoh1 in MG. (B) Regeneration paradigm in which IPA:Atoh1 expression is induced in MG of injured retina. (C) Representative immunofluorescence images of regenerating neurons in IPA:Atoh1 mice demonstrating that MG-derived neurons (GFP+) are HuC / D+ but not Otx2+. (D) Integrated UMAP of FACS-sorted MG-derived cells following the regeneration paradigm with overexpression of IPA:Atoh1 or IPA alone. Highlighted are RGC-like cells in each dataset that were subsetted for further comparative analysis. (E) Scatter plot highlighting differentially expressed genes between RGC-like cells of the IPA:Atoh1 and IPA alone regeneration paradigms. (F) GO analysis demonstrates that neurodevelopmental terms containing many retinal progenitor genes are downregulated in the IPA:Atoh1 dataset compared to IPA alone. (G) Integrated UMAP of the IPA:Atoh1 data as described above with the previously generated Ascl1:Atoh1 dataset (13). Highlights indicate RGC-like cells in each dataset that were compared in further analysis. (H) Scatter plot highlighting differentially expressed genes between RGC-like cells of the IPA:Atoh1 and Ascl1:Atoh1 regeneration paradigms. (I) Bar graph of GO terms related to neurite outgrowth that are enriched in the IPA:Atoh1 data. Known neuronal projection genes listed are upregulated in IPA:Atoh1 compared to Ascl1:Atoh1.

[0029] Figures 8A-8C .IPA-derived neuronal subsets arise from proliferating MGs. (8A) Experimental paradigm for labeling diving cells during the regeneration experiment described in Figure 1. (8B) Representative sections showing MG-derived cells (GFP+) that have previously undergone cell division (EdU+). (8C) Representative images showing that some MG-derived neurons (GFP+ / HuC / D+, top panel) are the result of proliferating MGs (EdU+, bottom left panel). Scale bar, 50 μm. Abbreviations: ONL, outer nuclear layer, INL, inner nuclear layer, GCL, ganglion cell layer.

[0030] Figures 9A-9E IPA treatment is most effective at reprogramming MG if induced prior to injury. (9A) Experimental paradigm using transgenic mice described in Figure 1 to test whether Islet1 / Pou4f2 / Ascl1 can reprogram MG in the absence of retinal injury. (9B) Representative images of GFP+ MG showing no induction of the ganglionic / amacrine marker HuC / D (upper right) or the bipolar marker Otx2 (lower right). (9C) Experimental paradigm testing whether induction of IPA factors following NMDA injury and TSA application can induce MG neurogenesis. (9D) Representative images of GFP+ MG showing some co-labeling of the neuronal markers HuC / D (upper right) and Otx2 (lower right). (9E) Quantification of the percentage of MG-derived cells expressing HuC / D or Otx2 following IPA induction prior to, following, or without injury. Scale bar 50 μm.

[0031] Figures 10A-10E .scRNA-seq analysis reveals that Pou4f2 biases MG generation toward RGC-like neurons. (10A) Integrated IPA and UMAP of Ascl1-only reprogrammed MG highlighting cells expressing Isl1, Pou4f2, or a combination of both. (10B) Stacked bar graph quantifying the percentage of Islet1-only, Pou4f2-only, or Islet1 / Pou4f2 double-positive cells that ultimately become MG-derived bipolar or RGC-like neurons. (10C) Feature graph highlighting cells expressing Ascl1 that is downregulated upon differentiation into bipolar neurons (Otx2+) or RGC-like neurons (Elav14+). (10D) Heatmap of the top 40 differentially expressed genes between MG and MG-derived neurons found in the GO terms “axon guidance,” “neuritogenesis,” and “axon growth.” (10E) Feature plots of prediction scores from Seurat label transfer using a reference dataset of randomly sampled cells from each major retinal neuron class and MG subsetted from mouse retinal development scRNA-seq data. 23 .

[0032] Figures 11A-11D MG-derived RGCs are a stable population over time. (11A) Combined UMAP of IPA-treated MG at the three and six week endpoints. (11B) Segmented UMAP showing the distribution of cells in the UMAP in (a) at each time point. (11C) Stacked bar graph showing the percentage of each cluster of MG and MG-derived neurons at the three and six week time points. (11D) Analysis of cell death genes found in MG-derived RGC-like neurons. These data indicate that cell death genes are not significantly increased in MG-derived RCSs during this 3 to 6 week survival period.

[0033] Figures 12A-12E MGs from IPA mice express reprogramming factors. (12A) Immunofluorescence of IPA MGs treated with doxycycline for 5 days shows co-labeling of Ascl1-IRES-GFP, Brn3, and Isl1 in most cells. (12B) UMAP of untreated cultured MGs with labeled glial and neuronal cell populations. (12C) Labeling of MGs and various retinal neurons reveals that a small fraction of bipolar and amacrine cells survive dissociation. (12D) UMAP of integrated datasets of in vivo and in vitro IPA overexpression and in vitro Ascl1 overexpression. (12E) Expression of melanopsin (Opn4) in the three datasets from (12D). Scale bar, 50 μm. Abbreviations: MG, Müller glial cells; AC, amacrine cells; BC, bipolar cells; PR, photoreceptor; RGC, retinal ganglion cells.

[0034] Figures 13A-13F .scATAC-seq of E14 embryonic mouse retina. (13A) UMAP plot of scATAC-seq of E14 embryonic mouse retina. Chromvar scores show motif accessibility for identifying clusters of progenitors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). (13F) Pseudotemporal subsets of retinal progenitors transitioning to retinal ganglion cells, further analyzed in Figure 6.

[0035] Figures 14A-14HAddition of Atoh1 to IPA significantly induces MG-derived RGC-like cells and does not require retinal injury. (14A) Transgenic mouse constructs for induction of Ascl1, Atoh1, Pou4f2, and Islet1. (14B) Paradigm used to analyze whether induction of all four transcription factors can induce MG neurogenesis in the absence of retinal injury. (14C) Representative sections of retina after IPAA treatment showing MG-derived (GFP+) cells expressing the neuronal marker HuC / D. (14D) Quantification of the percentage of MG-derived cells expressing the RGC-like marker HuC / D or the bipolar marker Otx2 after IPAA treatment in the absence of injury. (14E-14H) Violin plots of markers used to define clusters and bar graphs showing cluster composition for integrated scRNAseq data from IPA-IPA:Atoh1 (14E-14F) and Ascl1:Atoh1-IPA:Atoh1 (14G-14H).

[0036] Fig.15 .Using transcription factors specific to developing retinal cells to promote the generation of RGCs. Lentivirus was used to induce the expression of several transcription factors that are expressed in developing retinal cells but not in Müller glial cells. Müller glial cells were grown in cell culture and infected with the virus. The cells were then cultured for 5-7 days and subsequently processed for single-cell RNAseq to determine their fate. Of all the factors tested, Onecut1 (arrow) was able to induce Müller glial cells to generate new cells with expression of genes characteristic of RGCs.

[0037] Fig.16 .Use transcription factors specific to developing retinal cells to promote the generation of RGCs. Onecut1 expression (eg Fig.15 induced Müller glia to generate new cells with expression of genes characteristic of RGCs, including Nefm, Nefl, Ebf1, and Gap43.

[0038] Figures 17A-17C .AAV delivered reprogramming transcription factors can induce neurogenesis. HuC / D+ neuronal lineage was traced from MG using tdTomato. (17A) AAV design using Atoh1. (17B) Lineage tracing scheme. (17C) Immunofluorescence demonstrated successful delivery of reprogramming transcription factors, resulting in reprogramming of HuC / D+ neurons from MG cells.

[0039] Fig.18. Reprogramming efficiency of lineage-traced MG. The bar graph shows that both AAV-delivered Atoh1 and Atoh7 can induce HuC / D+ neurons. The efficiency of Atoh1 and Atoh7 is roughly the same. Under these conditions, the efficiency of AAV delivery of reprogramming factors is much lower than transgene expression.

[0040] Fig.19 .Irx2 and Neurod2 promote axonal growth in IPA-reprogrammed MG.

[0041] Figures 20A-20D .Generation of human Muller glia in vitro from fetal retinas or pluripotent stem cells using retinal spheres and retinal organoids. (20A) Schematic diagram of embryonic stem cell (ESC) differentiation protocol for generating retinal organoids (R0). (20B) Images of MG development over time in retinal organoids labeled with RLBP1 and SOX2. (20C) The upper panel shows a schematic diagram of generating retinal spheres (RS), and the lower panel shows images of retinal spheres made from several fetal retinas and cultured for different times as labeled. (20D) Characterization of MG in RS using RLBP1, VSX2, SOX9, SOX2, and GFAP.

[0042] Figures 21A-21B . Protocol for the generation of dissociated human Muller glial cell cultures in vitro.

[0043] Figures 22A-22E .Ascl1 promotes neurogenesis in human Muller glial cells. (22A) Scheme for delivery of GFP to dissociated cells. (22B-22E) These neurons express some pan-neuronal markers, such as DCX and TUJ1, but are not mature.

[0044] Fig.23 Characterization of the ShH10 capsid and RLBP promoter in NHP-dissociated Muller glial cell cultures.

[0045] Fig.24 .HES1 promoter as an alternative to the RLBP1 promoter. Schematic representation of a lentiviral construct containing the HES1 promoter driving EGFP expression.

[0046] Figures 25A-25D Schematic diagram of the construction of lentivirus containing the HES1 promoter driving the expression of EGFP (control) or driving the expression of proneural factor ASCL1 and EGFP in Muller glial cells (25A). (25B-25D) Testing the specificity of the HES1 promoter for MG expression in human retinal bulbs (D132+77=D 209 of infection date). GFP and HES1 are present in the same cells.

[0047] Fig.26 The HES1 promoter also directs the expression of MG in dissociated cultures of adult NHPs. DETAILED DESCRIPTION

[0048] The molecules, compositions and methods described herein are based on the following surprising discovery: through the targeted overexpression of developmental retinal ganglion cell transcription factors, retinal ganglion-like cells can be regenerated in the damaged adult retina in vivo. As demonstrated herein, AAV vectors can deliver reprogramming transcription factors to Muller glial cells in vivo. Although the neurogenesis rate of Muller glial cells reprogrammed by AAV is much lower than the neurogenesis rate observed by transgenic reprogramming, other RGC transcription factors can increase specific RGC genes, and in some cases, can improve the growth of RGC axons. In addition, studies described herein show that human Muller glial cells can be reprogrammed to produce neurons in isolated cultures, and these human Muller glial cells can be derived from retinal organoids or fetal human retina. This ability to reprogram Muller glial cells to specific types is particularly important for endogenous regeneration strategies, because most blinding diseases are the result of specific neuronal subtype defects. For example, glaucoma is mainly caused by RGC death.

[0049] definition

[0050] Unless otherwise specified, all scientific and technical terms used in this application have the meanings commonly used in the art. As used in this application, the following words or phrases have the designated meanings.

[0051] As used herein, the term "comprising" means that the compositions and methods include the elements described, but do not exclude other elements. As used herein, the transition phrase "consisting essentially of..." (and its grammatical variants) should be interpreted as covering the materials or steps described "and those materials or steps that do not substantially affect the basic and novel characteristics of the described embodiments." Therefore, the term "consisting essentially of..." used in this article should not be interpreted as equivalent to "comprising." "Consisting of..." means excluding other ingredients beyond trace elements and substantial method steps for applying the compositions disclosed herein. The aspects defined by each of these transition terms are within the scope of the disclosure herein.

[0052] As used herein, "retinal neurons" refers to any of the five types of neurons in the retina: photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. In some specific embodiments, the retinal neurons are bipolar neurons, amacrine cells, horizontal cells, and ganglion cells.

[0053] As used herein, the term "nucleic acid sequence" or "polynucleotide" refers to nucleotides of any length, which are deoxynucleotides (i.e., DNA) or derivatives thereof; ribonucleotides (i.e., RNA) or derivatives thereof; or peptide nucleic acids (PNA) or derivatives thereof. The term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, oligonucleotides (oligos), or other natural, synthetic, modified, mutated, or non-natural forms of DNA or RNA.

[0054] MicroRNAs, or "miRNAs" or "miRs," are short, noncoding RNAs that regulate gene expression through post-transcriptional regulation of target genes.

[0055] "Short hairpin RNA" or "shRNA" is a synthetic or non-natural RNA molecule. shRNA refers to RNA with a tight hairpin bend that is used to silence (via RNA interference or RNAi) target gene expression in cells. shRNA is usually delivered via an expression vector (e.g., a DNA plasmid) or via a viral vector.

[0056] The term "vector" refers to, but is not limited to, a recombinant genetic construct or plasmid or expression construct or expression vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the target cell genome. The vector may be derived from or based on a wild-type virus. Aspects of the present disclosure relate to adeno-associated viral vectors, adenoviral vectors, and lentiviral vectors.

[0057] The term "expression control element" as used herein refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). Exemplary expression control elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements can be, but are not limited to, constitutive, inducible, repressive, or tissue-specific. "Promoter" is a control sequence that is a region in a polynucleotide sequence that controls transcription initiation and rate. It may contain genetic elements that regulatory proteins and molecules can bind to, such as RNA polymerase and other transcription factors. In some embodiments, the expression control of a promoter is tissue-specific. "Enhancer" is a region of DNA that can bind to increase the probability or frequency of transcription by activating proteins. Non-limiting exemplary enhancers and post-transcriptional regulatory elements include CMV enhancers and WPRE.

[0058] The term "multicistronic" or "polycistronic" or "bicistronic" or "tricistronic" refers to an mRNA with multiple (i.e., dual or triple) coding regions or exons, and thus will be able to express two or more, three or more, four or more, etc. proteins from a single construct from the mRNA. Polycistronic vectors express two or more independent proteins from the same mRNA at the same time. The two most widely used strategies for constructing a polycistronic configuration are by using 1) an IRES or 2) a 2A self-cleavage site. "IRES" refers to an internal ribosome entry site or portion thereof of a viral, prokaryotic, or eukaryotic origin used in a polycistronic vector construct. In some embodiments, an IRES is an RNA element that allows translation to be initiated in a cap-independent manner. The term "self-cleaving peptide" or "sequence encoding a self-cleaving peptide" or "2A self-cleavage site" refers to a linker sequence used to incorporate a site within a vector construct to promote ribosome jumping and thereby produce two polypeptides from a single promoter, such self-cleaving peptides include but are not limited to T2A and P2A peptides or sequences encoding self-cleaving peptides.

[0059] When used to define amino acid or nucleic acid sequences, the term "substantially complementary" means that a particular sequence (e.g., an oligonucleotide sequence) is substantially complementary to the sequence to which the reference is made. Thus, the sequence is typically highly complementary to the "target" sequence, and the entire sequence will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches. In many cases, it may be desirable for the sequence to be precisely matched, i.e., fully complementary to the sequence to which the nucleic acid specifically binds, and therefore to have zero mismatches along the complementary sequence. Therefore, highly complementary sequences will typically be very specifically bound to the target sequence region, and therefore will very effectively reduce and / or even inhibit the biological activity of the target sequence.

[0060] A substantially complementary nucleic acid sequence will be greater than about 80% complementary (or '% exact match') to the corresponding target sequence to which the nucleic acid specifically binds, and more preferably greater than about 85% complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it is desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the present invention, and in such cases, the nucleic acid sequence will be greater than about 90% complementary to the corresponding target sequence to which the nucleic acid specifically binds, and in certain embodiments, may be greater than about 95% complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including 96%, 97%, 98%, 99% and even 100% exact match complementary to the target to which the designed nucleic acid specifically binds.

[0061] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, or alternatively less than 25% identity with one of the sequences described herein.

[0062] The percent similarity or percent complementarity of any disclosed sequence can be determined, for example, by comparing sequence information using the GAP computer program version 6.0 available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program employs the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix for nucleotides (1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap, with an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for terminal gaps.

[0063] "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide nucleic acid sequences that can be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or synthesized from a series of oligonucleotides to provide a sequence that is capable of specifically binding to a target molecule and acting as an antisense construct to alter, reduce, or inhibit the biological activity of the target.

[0064] As used herein, "directed against" in the context of antisense oligonucleotides means that the antisense oligonucleotide binds to a target miRNA and blocks or inhibits the activity of the target.

[0065] As used herein, the terms "protein", "peptide" and "polypeptide" refer to amino acid subunits, amino acid analogs or peptide mimetics. The subunits can be linked by peptide bonds. On the other hand, the subunits can be linked by other bonds, such as esters, ethers, etc. The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids.

[0066] As used herein, the term "recombinant expression system" or "recombinant expression vector" refers to a genetic construct formed by recombination and used to express certain genetic materials.

[0067] The term "effective amount" or "therapeutically effective amount" or "prophylactically effective amount" refers to an amount of an active agent described herein that is effective to provide a desired / intended result and / or biological activity. Thus, for example, in various embodiments, an effective amount of a composition described herein is an amount effective to cause retinal neuron regeneration and / or improve or alleviate the symptoms of a retinal degenerative disease and / or treat a retinal degenerative disease.

[0068] When the disclosure herein relates to small molecules, polypeptides, proteins, polynucleotides, nucleic acids, oligonucleotides, antisense or miRNA, its equivalent or biological equivalent is intended to be within the scope of the present disclosure. As used herein, when referring to a reference small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense or miRNA, the term "its biological equivalent" is intended to be synonymous with "its equivalent", even those reference molecules with minimum homology while still maintaining desired structure or function. Unless specifically described herein, it is expected that any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide or protein mentioned herein also includes its equivalent. For example, equivalent means at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, or at least about 85%, or at least about 90%, or at least about 95%, or 98% percentage homology or identity and show a biological activity substantially equivalent to a reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, its equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0069] In some embodiments disclosed herein, polypeptide and / or polynucleotide sequences are provided herein for use in the following gene and protein transfer and expression technologies. Such sequences provided herein can be used to provide expression products and substantially identical sequences of proteins having the same biological properties. These "bioequivalent" or "biologically active" or "equivalent" polypeptides are encoded by equivalent polynucleotides described herein. When compared using a sequence identity method run under default conditions, it may have a primary amino acid sequence that is at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to a reference polypeptide. Specific polynucleotide or polypeptide sequences are provided as examples of specific embodiments. Alternative amino acids with similar charges can be used to modify the amino acid sequence. In addition, equivalent polynucleotides are polynucleotides that hybridize with reference polynucleotides or their complements under stringent conditions, or, with respect to polypeptides, are polypeptides encoded by polynucleotides that hybridize with reference coding polynucleotides or their complementary chains under stringent conditions. Alternatively, equivalent polypeptides or proteins are polypeptides or proteins expressed by equivalent polynucleotides.

[0070] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific means. The complex may comprise two chains forming a double-stranded structure, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction may constitute a step in a broader process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0071] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a washing solution of about 1xSSC, 0.1xSSC or deionized water. Generally speaking, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the washing incubation time is about 1, 2 or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that equivalents of SSC using other buffer systems may be employed.

[0072] As used herein, "treating" or "treatment" of a retinal degenerative disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject susceptible to or not yet showing symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is an approach to obtaining a beneficial or desired result, including a clinical result. With respect to the compositions, combination therapies, nucleic acid molecules, and methods disclosed herein for inducing neurogenesis in MG and / or generating functional neurons from MG, the beneficial or desired result may include, but is not limited to, one or more of the following: alleviation or amelioration of one or more symptoms of retinal degeneration, reduction in the extent of a retinal degenerative condition, including a retinal degenerative disease, stabilization of the state of a retinal degenerative condition, including a disease (i.e., not worsening), delay or slowing of a retinal degenerative condition, including a disease, progression, improvement or alleviation of a retinal degenerative condition, including a disease, a retinal degenerative state, and remission (whether partial or total), whether detectable or undetectable.

[0073] The term "isolated" as used herein refers to naturally occurring DNA fragments, DNA molecules, coding sequences or oligonucleotides removed from their natural environment, or are synthetic molecules or cloned products. Preferably, the DNA fragments, DNA molecules, coding sequences or oligonucleotides are purified, i.e., substantially free of any other DNA fragments, DNA molecules, coding sequences or oligonucleotides and related cell products or other impurities.

[0074] The term "cell" as used herein refers to a prokaryotic cell or a eukaryotic cell, optionally obtained from a subject or a commercially available source. Cells treated, transfected, transformed or otherwise contacted with the compositions and / or nucleic acid molecules disclosed herein include, but are not limited to, cells of humans, non-human animals, mammals or non-human mammals, including, but not limited to, cells of murine, canine or non-human primate species. Cells treated, transfected, transformed or otherwise contacted with the compositions and / or nucleic acid molecules disclosed herein include, but are not limited to, retinal cells, Müller glial cells (MG) and / or retinal neuronal cells, such as retinal neurons, bipolar neurons, amacrine cells, horizontal cells, ganglion cells and / or glial cells. The term "Müller glial cells" or "Müller glial cells" or "MG" refers to cells present in the retina of vertebrates and are supporting cells for neurons. MG is the most common type of glial cell in the retina. Although the MG cell body is located in the inner nuclear layer of the retina, the MG spans the entire retina.

[0075] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.

[0076] As used herein, "a" or "an" means at least one, unless specifically stated otherwise.

[0077] As used herein, "preventing" or "preventing" a condition or disease means hindering, reducing or delaying the onset or progression of the condition or disease.

[0078] When the term "encoding" is applied to a nucleic acid sequence, it means that if the polynucleotide in its native state or when manipulated by methods well known to those skilled in the art can be transcribed and / or translated to produce an effector RNA, mRNA or mRNA that can be used for a polypeptide and / or its fragment, then the polynucleotide is said to "encode" a polypeptide, mRNA or effector RNA. The antisense strand is the complementary strand of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0079] As used herein, the term "expression" or "gene expression" refers to the process of transcribing a polynucleotide into mRNA and / or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample; in addition, the expression levels of multiple genes can be determined to establish an expression profile for a particular sample.

[0080] As used herein, the term "functional" may be applied to the modification of any molecular, biological or cellular material so that it achieves a specific, designated effect.

[0081] As used herein, the term "combination therapy" refers to the delivery of two or more compositions and / or nucleic acid molecules in a combined manner, such as but not limited to sequentially, concurrently, simultaneously and / or stepwise, to achieve a therapeutic effect.

[0082] As used herein, the term "about" when referring to a measurable value (e.g., an amount, level, or concentration), for example, but not limited to, is intended to cover variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of a specified amount, or a fold difference from a quantifiable comparable level of a standard or control or reference material, such as 1-fold, 2-fold, 3-fold, 4-fold... 10-fold, 100-fold, etc., of a specified comparative level.

[0083] In some embodiments, enhancing the expression level of two or more endogenous and / or exogenous proneural bHLH transcription factors refers to an increase in the expression of endogenous and / or exogenous Ascl1, and / or Atoh1, and / or Atoh7 (e.g., but not limited to untreated or single Ascl1 expression) compared to the control sample or explant level. In some embodiments, neurogenesis is increased and / or the generation of functional neurons is increased compared to the control. In some embodiments, the expression level and / or functional neurons are increased by about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 50 times, about 100 times, about 1000 times, or about 10,000 times relative to the control.

[0084] In some embodiments, the terms "reprogramming potentiator" or "reprogramming potentiating agent" used interchangeably herein refer to a small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, miRNA, or its equivalent or bioequivalent, which assists in stimulating and / or promoting the process of neurogenesis from MG in a manner that produces functional neurons from MG. In one embodiment, one or more reprogramming potentiators assist in stimulating neurogenesis and generating functional neurons from MG by inhibiting the HDAC pathway. In another embodiment, one or more reprogramming potentiators assist in stimulating neurogenesis and generating functional neurons from MG by inhibiting the Jak / STAT pathway. In another embodiment, one or more reprogramming potentiators assist in stimulating neurogenesis and generating functional neurons from MG by inhibiting the HDAC+Jak / STAT pathway. In another embodiment, one or more reprogramming potentiators assist in stimulating neurogenesis and generating functional neurons from MG by enhancing and / or increasing endogenous and / or exogenous Ascl1 expression levels. See also our previous work in WO2019 / 210320 and WO2020 / 223308, each of which is incorporated herein by reference in its entirety.

[0085] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc. disclosed herein means that the ingredient, range, dosage form, etc. is suitable for the disclosed purpose.

[0086] The term "adeno-associated virus" or "AAV" as used herein refers to members of the class of viruses associated with this name and belonging to the Parvoviridae family, the genus Dependent Parvovirus. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells of various tissue types. At least 11 or 12 serotypes numbered in sequence are disclosed in the prior art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, such as AAV2, AAV5 and AAV8, or engineered serotypes, such as AAV-SHH10 and AAV-7m8. AAV structural particles are composed of 60 protein molecules, which are composed of VP1, VP2 and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins, arranged in an icosahedral structure.

[0087] Nucleic acid molecules and compositions

[0088] Provided are compositions and / or nucleic acid molecules for retinal regeneration, enhancing retinal regeneration, stimulating retinal ganglion cell regeneration, restoring vision, and treating retinal degenerative diseases, injuries, or damage.

[0089] Such nucleic acid molecules can be delivered by viral or non-viral means. An example of viral delivery is adeno-associated virus (AAV). Other examples include retrovirus and lentivirus delivery. An example of non-viral delivery method is cell penetrating peptide (CPP). Polynucleotide constructs can also be modified, for example, by chemical modification, to improve their stability and / or delivery suitability. In some embodiments, oligonucleotides are modified by locked nucleic acid and / or thiophosphate bonds. In some embodiments, a delivery system is selected to improve bioavailability, such as pegylated liposomes, lipidoids or biodegradable polymers.

[0090] In some embodiments, the composition further comprises one or more additional synergists or therapeutic agents, including, for example, reprogramming synergists. In some embodiments, the composition does not contain reprogramming synergists. Optionally, a composition comprising one or more small molecule reprogramming synergists can be administered sequentially or simultaneously with the nucleic acid molecules disclosed herein. In another embodiment, one or more proteins / peptides or miR-based reprogramming synergists can be incorporated into the nucleic acid molecules disclosed herein. Such one or more reprogramming synergists are selected from HDACi, STATi, Jak / STATi and RNAi-based Ascl1 activators. Please also refer to our previous work in WO2019 / 210320 and WO2020 / 223308, each of which is incorporated herein by reference in its entirety.

[0091] In one embodiment, the HDAC signaling pathway inhibitor is selected from the group consisting of peptidomimetics, small molecule inhibitors, oligonucleotides, peptides and proteins. Representative examples of small molecule HDACi include, but are not limited to, trichostatin A (TSA); Istodax TM , also known as (formerly) / romidepsin; Beleodaq TM , also known as (formerly) / belinostat; Farydak TM , also known as (pre) / panobinostat; and Zolinza TM, also known as (pre) / vorinostat, Quisinostat, Abexinostat, Givinostat, Resminostat, Phenylbutyric Acid, Valproic Acid, Depsipeptide, Entinostat, Mocetinostat and Tubastatin A. Exemplary HDACi peptides include, but are not limited to, 16cyc-HxA, 16lin-HxA and 16KA.

[0092] In some embodiments, the inhibitor, mimetic, activator or antagomir is an oligonucleotide or nucleotide sequence. Accordingly, the invention provides nucleotide constructs for use in the compositions or combination therapies or nucleic acid molecules and methods described herein.

[0093] In some embodiments, the reprogramming potentiator is selected from one or more STAT signaling pathway inhibitors; and one or more Ascl activators, such as but not limited to miR-25 and / or miR-124; and one or more let-7 family inhibitors.

[0094] Optionally, a composition is provided herein, comprising any one or more of the following: an RNAi-based Ascl1 activator and / or a combination therapy of HDACi+STATi, and / or a nucleic acid sequence encoding a developmental RGC transcription factor or a vector comprising a nucleic acid sequence disclosed herein, and a carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0095] An exemplary nucleic acid sequence encoding human Onecut1 can be found in NCBI Reference Sequence No. NC_000015.10 (SEQ ID NO: 1). An exemplary nucleic acid sequence encoding human Pou4f2 can be found in NCBI Reference Sequence No. NC_000004.12 (SEQ ID NO: 3). An exemplary nucleic acid sequence encoding human Islet1 can be found in NCBI Reference Sequence No. NC_000005.10 (SEQ ID NO: 5). An exemplary nucleic acid sequence encoding human Irx2 can be found in NCBI Reference Sequence No. NC_000005.10 (SEQ ID NO: 8). The mouse version of Irx2 used in the Examples herein can be found in NCBI Reference Sequence: NM_010574.4 (SEQ ID NO: 64; encoding amino acid sequence SEQ ID NO: 65). An exemplary nucleic acid sequence encoding human Irx5 can be found in NCBI Reference Sequence No. NC_000016.10 (SEQ ID NO: 13). The mouse version of Irx5 used in the examples herein can be found in NCBI Reference Sequence: NM_018826.2 (SEQ ID NO: 66; encoding amino acid sequence SEQ ID NO: 67). An exemplary nucleic acid sequence encoding Neurod2 can be found in NCBI Reference Sequence No. NC_000017.11 (SEQ ID NO: 17). An exemplary nucleic acid sequence encoding human Ebf1 can be found in NCBI Reference Sequence No. NC_000005.10 (SEQ ID NO: 19). The mouse version of Ebf1 used in the examples herein can be found in NCBI Reference Sequence: NM_001290709.1 (SEQ ID NO: 68; encoding amino acid sequence SEQ ID NO: 69). An exemplary nucleic acid sequence encoding human Tcf3 can be found in NCBI Reference Sequence No. NC_000019.10 (SEQ ID NO: 48). The mouse version of Tcf3 used in the examples herein can be found in the NCBI reference sequence: NM_001164147.2 (SEQ ID NO: 70; encoding amino acid sequence SEQ ID NO: 71). This mouse sequence can be used to guide the selection of the corresponding human or humanized Tcf3.

[0096] An exemplary nucleic acid sequence encoding human Ascl1 can be found in NCBI reference sequence number NG_008950.1 (SEQ ID NO: 50). In another embodiment disclosed herein is a nucleic acid sequence encoding the human Ascl1 amino acid sequence of UniProtKB / Swiss-Prot: P50553.2 (SEQ ID NO: 51) or a portion thereof. Ascl1 homologs derived from species such as murine, canine, equine, etc. are included herein but are not limited thereto.

[0097] The protein atonal homolog 1 (Atoh1) is a proneural member of the bHLH transcription factor family. The protein activates an E-box that is different from the Ascl1 gene. An exemplary nucleic acid sequence encoding human Atoh1 can be found in NCBI reference sequence number NM_005172.1 (SEQ ID NO: 52). In another embodiment disclosed herein is a nucleic acid sequence encoding the human Atoh1 amino acid sequence of NP_005163.1 (SEQ ID NO: 53) or a portion thereof. Included herein are, but not limited to, Atoh1 homologs, orthologs and / or paralogs derived from species such as murine, canine, equine, etc.

[0098] The Atoh7 family bHLH transcription factor 7 (Atoh7) gene encodes a proneural member of the basic helix-loop-helix (BHLH) transcription factor family. An exemplary nucleic acid sequence encoding human Atoh7 can be found in NCBI reference sequence number NM_008553.4 (SEQ ID NO: 54). In another embodiment disclosed herein is a nucleic acid sequence encoding the human Atoh7 amino acid sequence of NP_660161.1 (SEQ ID NO: 55) or a portion thereof. Included herein are, but are not limited to, Atoh7 homologs, orthologs and / or paralogs derived from species such as murines, canines, equines, and the like. An exemplary nucleic acid sequence encoding neurogenin-2 (also known as NEUROG2 and NGN-2) can be found in NCBI reference sequence number NM_024019. An exemplary nucleic acid sequence encoding Neurod1 can be found in NCBI reference sequence number KR709666.

[0099] Exemplary nucleic acid sequences of Ascl1, Atoh1 and / or Atoh7 or other proneural bHLH transcription factors used herein include, but are not limited to, portions of the corresponding sequences of Ascl1, Atoh1 and / or Atoh7 for configuring polycistronic, bicistronic and / or tricistronic constructs, plasmids and / or expression vectors. It should be understood that portions of the sequences cited herein may be selected for use, wherein the selected portions are sufficient to encode the transcription factors and / or other elements.

[0100] Exemplary sources of promoter sequences can be found in HES1 (SEQ ID NO:56), RLBP1 (SEQ ID NO:57), or GLAST (also known as SL1A3; SEQ ID NOs:61-63; see NCBI Reference Sequence No. NM_004172).

[0101] Viral vectors

[0102] In some embodiments, the vector disclosed herein is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenovirus / retroviral chimera vector, a herpes simplex virus I or II vector, a parvovirus vector, a reticuloendotheliosis virus vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector, or any hybrid or chimeric vector combining the advantages of two or more viral vectors. In some embodiments, the vector also includes one or more expression control elements operably connected to the polynucleotide. In some embodiments, the vector also includes one or more selection markers.

[0103] In some embodiments, the vector disclosed herein is a low toxicity AAV vector. In some embodiments, the AAV vector is not incorporated into the host genome, so the possibility of causing insertional mutagenesis is low. In some embodiments, the AAV vector can encode a series of total polynucleotides of 4.5kb to 4.75kb. In some embodiments, exemplary AAV vectors that can be used in any of the compositions, systems, methods, and kits described herein can include AAV1 vectors, modified AAV1 vectors, AAV2 vectors, modified AAV2 vectors, AAV3 vectors, modified AAV3 vectors, AAV4 vectors, modified AAV4 vectors, AAV5 vectors, modified AAV5 vectors, AAV6 vectors, modified AAV6 vectors, AAV7 vectors, modified AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV.rh10 vectors, modified AAV.rh10 vectors, AAV.rh32 / 33 vectors, modified AAV.rh32 / 33 vectors, AAV.rh43 vectors, modified AAV.rh43 vectors, AAV.rh64R1 vectors, and modified AAV.rh64R1 vectors, and any combinations or equivalents thereof.

[0104] In some embodiments, the vector disclosed herein is a lentiviral vector. In one embodiment, the lentiviral vector is an integrase competent lentiviral vector (ICLV). In some embodiments, a lentiviral vector may refer to a transgenic plasmid vector, and a transgenic plasmid vector combined with a related plasmid (e.g., a packaging plasmid, a rev expression plasmid, an envelope plasmid), and a lentiviral-based particle capable of introducing exogenous nucleic acids into cells through a viral or virus-like entry mechanism. Lentiviral vectors are well known in the art. In some embodiments, exemplary lentiviral vectors may be used with any of the compositions, nucleic acid molecules, and / or methods described herein, and may include human immunodeficiency virus (HIV) 1 vectors, modified human immunodeficiency virus (HIV) 1 vectors, human immunodeficiency virus (HIV) 2 vectors, modified human immunodeficiency virus (HIV) 2 vectors, sooty mangabey immunodeficiency virus (SIV SM ) vector, modified sooty mangabey immunodeficiency virus (SIV SM ) vector, African green monkey immunodeficiency virus (SIV AGM ) vector, modified African green monkey immunodeficiency virus (SIV AGM ) vector, equine infectious anemia virus (EIAV) vector, modified equine infectious anemia virus (EIAV) vector, feline immunodeficiency virus (FIV) vector, modified feline immunodeficiency virus (FIV) vector, Vesna / Maedi virus (VNV / VMV) vector, modified Vesna / Maedi virus (VNV / VMV) vector, caprine arthritis-encephalitis virus (CAEV) vector, modified caprine arthritis-encephalitis virus (CAEV) vector, bovine immunodeficiency virus (BIV) or modified bovine immunodeficiency virus (BIV).

[0105] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the vector of the present disclosure is a viral vector. In some embodiments, the viral vector comprises a sequence separated from or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence separated from or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence separated from or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence separated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication-defective. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0106] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the viral vector comprises a sequence separated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises a reverse terminal repeat or capsid sequence of an AAV separated from or derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or a vector and / or component derived from a synthetic AAV serotype, such as, but not limited to, Anc80AAV (ancestor of AAV 1, 2, 6, 8 and 9). In some embodiments, the viral vector is replication-deficient. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).

[0107] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or a lipoplex, a polymer vesicle, a polymer complex, or a dendrimer.

[0108] In some embodiments, the expression vectors or viral vectors disclosed herein are used to transfect, transform or contact cells that are eukaryotic cells. In some embodiments, the cells are animal cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are bovine, mouse, cat, horse, pig, dog, monkey or human cells. In specific embodiments, the cells are retinal neurons or MGs of animals or mammals.

[0109] In some embodiments, the cell is a packaging cell or a producer cell for producing viral particles.

[0110] In some embodiments, provided herein are viral particles comprising, consisting of, or consisting essentially of a vector comprising, consisting of, or consisting essentially of a polynucleotide sequence encoding a developmental RGC transcription factor and, optionally, an Ascl1 protein.

[0111] In general, methods for packaging genetic material (e.g., RNA or DNA) into one or more vectors are well known in the art. For example, packaging vectors and cell lines can be used to package genetic material and introduced by traditional recombinant methods.

[0112] In some embodiments, packaging vectors may include but are not limited to retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors. Packaging vectors contain elements and sequences that help deliver genetic material to cells. For example, a retroviral construct is a packaging plasmid that includes at least one retroviral auxiliary DNA sequence derived from a replication-defective retroviral genome, and the sequence trans-encodes all virion proteins required for packaging replication-defective retroviral vectors, and is used to produce virion proteins that can package replication-defective retroviral vectors in high titers without producing replication-competent helper viruses. The retroviral DNA sequence lacks the region of the natural enhancer and / or promoter of the viral 5'LTR of the encoding virus, and lacks the psi functional sequence responsible for packaging the auxiliary genome and 3'LTR, but encodes an external polyadenylation site, such as an SV40 polyadenylation site, and an external enhancer and / or promoter that guides effective transcription in the cell type where the virus needs to be produced. The retrovirus is a leukemia virus, such as Moloney murine leukemia virus (MMLV), human immunodeficiency virus (HIV) or gibbon ape leukemia virus (GALV). The foreign enhancer and promoter can be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the enhancer and promoter (U3 region) of Moloney murine sarcoma virus (MMSV), the U3 region of Rous sarcoma virus (RSV), the U3 region of spleen focus forming virus (SFFV), or the HCMV IE enhancer joined to the natural Moloney murine leukemia virus (MMLV) promoter.

[0113] The retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector, for example, wherein the first helper sequence contains cDNAs encoding the gag and pol proteins of the ecotropic MMLV or GALV, and the second helper sequence contains cDNAs encoding the env protein. The Env gene that determines the host range may be derived from genes encoding heterotropic, amphoteric, ecotropic, polytropic (mink lesion formation) or 10A1 murine leukemia virus env proteins, or the gibbon ape leukemia virus (GALV) env protein, human immunodeficiency virus env (gp160) protein, vesicular stomatitis virus (VSV) G protein, human T-cell leukemia (HTLV) type I and II env gene products, chimeric envelope genes derived from a combination of one or more of the above env genes, or chimeric envelope genes encoding the cytoplasmic and transmembrane of the above env gene products, and monoclonal antibodies against specific surface molecules on the desired target cells. Similar vector-based systems may employ other vectors, such as the Sleeping Beauty vector or transposon elements.

[0114] The resulting packaged expression system can then be introduced via an appropriate route of administration, as discussed in detail with respect to the method aspects disclosed herein.

[0115] Pharmaceutical composition

[0116] The pharmaceutical compositions disclosed herein include one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The compositions disclosed herein may be formulated for intraocular administration.

[0117] cell

[0118] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the cells of the present disclosure are retinal cells, such as Müller glial (MG) cells, or rod or cone photoreceptor cells. In some embodiments, the cells are neuronal cells. In some embodiments, the neuronal cells of the present disclosure are neurons of the retina. In some embodiments, the neuronal cells of the present disclosure are neurons of the optic nerve. In some embodiments, the neuronal cells of the present disclosure are glial cells or glial cells. In some embodiments, the cells are bipolar neurons, horizontal cells, ganglion cells or amacrine cells. In some embodiments, the cells of the present disclosure are astrocytes. In some embodiments, the cells of the present disclosure are macroglial cells or microglial cells or glial cells.

[0119] In some embodiments of the disclosed compositions and methods, the disclosed cells are cultured cells.

[0120] In some embodiments of the present disclosure, the cells are in vivo, in vitro, ex vivo or in situ. In some embodiments, the cells are modified ex vivo and transplanted and / or administered into the retina of a subject in need thereof.

[0121] In some embodiments, the cells of the disclosure are autologous or allogeneic and used for transplantation.

[0122] In some embodiments, the cells of the present disclosure are stem cell-derived or embryonic stem cell-derived retinal cells. In some embodiments, the cells are derived from retinal cells derived from induced pluripotent stem cells (iPS cells). In some embodiments, the cells are derived from retinal organoids.

[0123] method

[0124] Methods of stimulating retinal ganglion cell regeneration and / or inducing retinal regeneration in a subject are described herein. Methods of enhancing retinal regeneration, improving retinal neurogenesis, enhancing retinal regeneration, restoring vision, and treating retinal degenerative diseases, injuries, damage, or blindness are also provided.

[0125] Also provided herein is a method of inducing retinal regeneration, the method comprising administering to a subject a composition as described herein. In some embodiments, the method effectively increases the number of retinal ganglion cells derived from Müller glial cells, induces Müller glial cells (MG) to enter the mitotic cell cycle, and / or generates new retinal neurons, including generating new ganglion cells. In some embodiments of the method, the number of retinal neurons increases by at least 25% relative to baseline levels or other reference amounts representing untreated retina. In other embodiments, the number of retinal neurons increases by at least 40%. In some embodiments, the number of retinal neurons increases by 10%, 20%, 50%, 100%, 150%, 200% or more.

[0126] Optionally, the methods disclosed herein may utilize a combination therapy composition comprising one or more, or two or more small molecule reprogramming synergists. The agent may be administered sequentially or simultaneously with the nucleic acid molecules disclosed herein. In another embodiment, one or more proteins / peptides or miR-based reprogramming synergists may be incorporated into the nucleic acid molecules used in the methods disclosed herein. In some embodiments, the method is performed in the absence of such reprogramming synergists.

[0127] The subject is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult. In some embodiments, the subject suffers from a retinal degenerative disease. Examples of such retinal degenerative diseases include, but are not limited to, age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), central retinal artery occlusion (CRAO), vitreoretinopathy, and glaucoma.

[0128] Administration and Dosage

[0129] The compositions and / or nucleic acid molecules disclosed herein are administered in any suitable manner, usually with a pharmaceutically acceptable carrier. Suitable methods for administering compositions, compounds, molecules, nucleic acids and vectors in the context of the present invention to the eye or retina of a subject are available, and although more than one approach can be used to administer a particular composition, a particular approach can generally provide a more direct and more effective response than another approach. For the treatment of the retina, intraocular injections, such as but not limited to intravitreal injections and subretinal injections are the most common approaches to deliver to the retina. However, in some embodiments, periocular, suprachoroidal, systemic or topical administration is more suitable for efficacy and safety of delivery.

[0130] In the context disclosed herein, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time, or to inhibit disease progression. Thus, a composition is administered to a subject in an amount sufficient to induce an effective response and / or alleviate, mitigate, cure, or at least partially inhibit the symptoms and / or complications of a retinal disease or injury. An amount sufficient to achieve this is defined as a "therapeutically effective dose".

[0131] The route of administration, sequence and / or frequency and dosage of the therapeutic compositions disclosed herein will vary from individual to individual and can be readily determined using standard techniques. In general, an appropriate dosage and treatment regimen provides an amount of active compound sufficient to provide therapeutic and / or preventive benefit. This response can be monitored by determining an improvement in clinical outcomes in treated patients compared to untreated patients.

[0132] Example

[0133] The following examples are provided to illustrate the invention and to aid those of ordinary skill in making and using the invention. The examples are not intended to otherwise limit the scope of the invention in any way.

[0134] Example 1: Reprogramming Müller glial cells to regenerate ganglion-like cells using developmental transcription factors

[0135] Many neurodegenerative diseases lead to the degeneration of specific types of neurons. For example, glaucoma leads to the death of retinal ganglion cells, while other neurons remain intact. Neurons in the central nervous system of adult mammals do not regenerate. However, in non-mammalian vertebrates, glial cells spontaneously reprogram into neural progenitor cells and replace neurons after injury. We recently developed a strategy to stimulate functional neuronal regeneration in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glial cells. In this example, we tested the ability of additional transcription factors (TFs) to guide the regeneration of specific types of retinal neurons. We engineered mice to express different TF combinations in MG, including Ascl1, Pou4f2, Islet1, and Atoh1. Using IHC, scRNA-seq, scATAC-seq, and electrophysiology, we found that retinal ganglion-like cells can regenerate in vivo in damaged adult mouse retinas and target overexpression of developmental RGC transcription factors.

[0136] Over the past several years, our group and others have developed strategies to stimulate adult mouse MG to behave like fish and generate neurons (4–9). We screened multiple transcription factors (TFs) that are differentially expressed between mouse MG and retinal progenitor cells for their ability to stimulate MG neurogenesis in vitro (10). One factor identified in this screen was the proneural basic helix-loop-helix (bHLH) TF Ascl1, which is able to induce MG neurogenesis in puppies (11) and in adult mice when co-treated with the histone deacetylase trichostatin A (TSA) (4). MG expressing Ascl1 adopt a molecular phenotype similar to that of developing retinal progenitor cells, and a subset of these cells undergo mitosis (5, 12). Some of these newly generated cells go on to differentiate into retinal neurons that connect to endogenous circuits (4, 5). Stimulating MG neurogenesis with Ascl1 results in the majority of MG-derived neurons adopting a bipolar cell fate, with a minority resembling amacrine cells (4, 12). Recently, we reported that the efficiency and range of neuronal cell types generated by MG reprogramming can be significantly improved by adding additional atonal class (Atoh1 / 7) bHLH TFs. With this combination, up to 80% of MGs expressing Ascl1 and Atoh1 will become neurogenic precursors and ultimately neurons (13).

[0137] Our previous results showed that combining two TFs of the same type (bHLH proneural) significantly increased the efficiency of MG reprogramming in vivo, but we were unable to control the neuronal types generated by MGs in this paradigm. This is somewhat analogous to the regeneration process in zebrafish, where injury triggers the generation of all neuronal types, regardless of whether the injury is widespread or targeted to a specific cell type (14, 15). However, specific retinal diseases are often the result of defects in specific neural classes. For example, in glaucoma, loss of retinal ganglion cells (RGCs) leads to blindness (16). Therefore, there is an impetus to direct endogenous regeneration to the specific affected type of neurons for cell replacement strategies.

[0138] Here, we tested whether combining Ascl1 with other classes of developmentally important TFs could more precisely direct MG-derived retinal progenitors to specific retinal cell fates. Specifically, we explored the effects of combining Ascl1 overexpression with two other distinct classes of TFs, Islet1, LIN, Islet1, MEC3 (LIM) homeodomain TF and Pou4f2, class IV, Pit-Oct-Unc (POU) homeodomain TF. Both factors have been shown to be important for cell fate determination in the developing retina, with Pou4f2 being required for RGC differentiation and Islet1 being important for the development of several retinal neurons, including RGCs (17, 18).

[0139] We found that in adult mouse MG, expression of Pou4f2 and Islet1, along with Ascl1, directs a subset of MG-derived neurons toward a cell fate resembling that of RGCs. MG-derived RGC-like neurons (i) can be immunolabeled with markers of normal RGCs; (ii) have a transcriptome similar to that of developing RGCs based on single-cell RNA sequencing (scRNA-seq); (iii) have a broader range of electrophysiological features, such as action potentials, than neurons generated by Ascl1 alone; and (iv) display chromatin accessibility patterns similar to those of developing RGCs. In summary, our results suggest that neurogenesis in MG can be directed to specific cell types using combinations of developmentally relevant TFs.

[0140] Materials and methods

[0141] Primary cell culture

[0142] Retinas of tetO-Ascl1-GFP or tetO-Pou4f2-Islet1-Ascl1-GFP mice of both sexes were harvested at postnatal day 12 (P12) for MG culture. For dissociation, the retinas were incubated in a solution of papain and deoxyribonuclease (DNase) (Worthington) at 37°C for 10 minutes and then wet-grinded. To stop the reaction, an equal volume of ovomucoid (Worthington) was added. The cells were then spun at 300g for 10 minutes at 4°C and resuspended in growth medium consisting of Neurobasal (Gibco), 10% fetal bovine serum (FBS) (Clontech), N2 (Invitrogen), 1mM L-glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and mouse epidermal growth factor (100ng / ml) (R&D Systems). The cells were seeded in six-well plates at a density of two retinas per 10cm2 and incubated at 37°C. The medium was changed every 2 days until confluence (about 7 days). At confluence, cells were passaged with TrypLE (Gibco); resuspended in freezing medium consisting of 50% growth medium, 40% FBS and 10% dimethyl sulfoxide (DMSO); and stored in liquid NO2 for at least 1 day for at least 24 hours. After thawing, cells were grown in growth medium without FBS (1%) and treated with doxycycline to induce tetO-mediated genes.

[0143] animal

[0144] All animals were treated and maintained according to protocols approved by the Institutional Animal Care and Use Committee of the University of Washington. (i) Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP, (ii) Glast-CreER:LNL-tTA:tetO-P&I:tetO-mAscl1-ires-GFP, (iii) Glast-CreER:LNL-tTA:tetO-Atoh1:tetO-P&I:tetO-mAscl1-ires-GFP mice, (iv) rtTa:tetO-Ascl1-ires-GFP, and (v) rtTa:tet0-P7I:tetO-Ascl1-ires-GFP were from a mixed background of C57BL / 6 and B6SJF1. Glast-CreER, LNLtTA, and rtTa mice were from the Jackson Laboratory. The tetO-mAscl1-GFP mice were a gift from M. Nakafuku (University of Cincinnati), the tetO-Atoh1 mice were a gift from P. Chen (Emory University), and the tetO-P&I mice were a gift from X. Mu (University of Buffalo). Both males and females were used in the experiments at the same frequency. All in vivo experiments were performed on adult mice older than 40 days.

[0145] Immunohistochemistry

[0146] After CO2-mediated euthanasia, the cornea was removed and the eyeball was fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 30 minutes. The fixed eyes were then incubated overnight in 30% sucrose at 4°C. The retina was then frozen in the optimal cutting temperature compound (OTC) and frozen sections were made at 18 μm. For immunostaining, the sections were washed twice in PBS for 10 minutes each, and then incubated overnight in 0.5% Triton X-10 and PBS containing the primary antibody. The slides were then washed in PBS and incubated in the secondary antibody for 1 hour. Finally, the slides were washed again in PBS and coverslipped with Fluoromount-G (SouthernBiotech). Antibody and concentration information is shown in Table 1.

[0147] Cultured MGs were plated on glass coverslips containing FBS-reduced growth medium (as described above). To activate the transgene, doxycycline was added at a concentration of 3 μg / ml every 24 hours for 5 days. The coverslips were washed with PBS and then fixed with 4% PFA for 10 minutes at room temperature (RT), followed by 3×5 minute washes with PBS. Primary antibody incubations were performed overnight at 4° C. in PBS containing 0.5% Triton X-10 and 10% normal horse serum (NHS). Coverslips were washed in PBS and then labeled with EdU using click chemistry (Click-iT EdU Assay, Invitrogen), followed by incubation with secondary antibodies and 4′,6-diamidino-2-phenylindole for 1 hour at room temperature. After staining, coverslips were washed three more times and mounted on slides with Fluoromount-G (SouthernBiotech).

[0148] Table 1: List of antibodies used in the study. Each column identifies the antibody used for immunohistochemistry, source, catalog number, RRID identifier and concentration.

[0149]

[0150]

[0151] Fluorescence activated cell sorting

[0152] After euthanasia, retinas were dissociated into single cells as described for cell culture; after centrifugation at 300 g at 4°C, cells were resuspended in Neurobasal solution and passed through a 35 μm filter. FACS was performed on GFP+ cells using a BD FACSAria III cell sorter (BD Bioscience).

[0153] injection

[0154] Intravitreal injections were performed on mice anesthetized with isoflurane using a 32-G Hamilton syringe. NMDA was injected in a volume of 1 μl at a concentration of 100 mM in PBS. TSA (Sigma-Aldrich) was administered in DMSO by intravitreal injection at a concentration of 1 μg / μl. Tamoxifen (1.5 mg per 100 μl corn oil) was injected intraperitoneally into adult mice for four consecutive days to induce the expression of tetO-mAscl1-ires-GFP, tetO-P&I, and tetO-Atoh1 genes.

[0155] Microscopy / cell counting

[0156] Images were taken on a Zeiss LSM880 confocal microscope. To quantify cell number, at least four images were taken for each retina at the same magnification using a 20x objective.

[0157] Electrophysiology

[0158] Recordings were the same as in our previous reports (4, 5). Mice were adapted to a dark environment before recording. After euthanasia, retinas were cut into 200 μm slices for recording. Tissue recordings were performed at 32°C in Ames medium and oxygenated with 95% O2 / 5% CO2. GFP+ cells were recorded using infrared light and video differential interference contrast of a confocal microscope. Light responses were measured under infrared conditions, and the tissue was exposed to full-field illumination by blue and green light-emitting diodes. Pulled glass pipettes were used for recording and filled with a solution containing the following solution: 123 mM K-aspartate, 10 mM Hepes, 1 mM MgCl2, 10 mM KCl, 1 mM CaCl2, 2 mM EGTA, 0.5 mM guanosine triphosphate, 4 mM MG-adenosine triphosphate, and 0.1 mM AlexaFluor 695 hydrazide.

[0159] Single-cell RNA library construction

[0160] For the in vivo dataset, after FACS purification of GFP+MG, cells were centrifuged at 300 g at 4°C and resuspended at a concentration of 1000 cells / μl. Library construction was performed using the 10x Genomics 3′ Single Cell RNA V3 or V3.1 Sequencing Kit according to the manufacturer's instructions. Cells were encapsulated in gel beads and given unique barcodes using the 10x Chromium controller and 10x Genomics G-chip. Libraries were multiplexed using single index and dual index kits (10x Genomics).

[0161] For in vitro experiments, MG cultures from three to five mice were used. After 5 days of doxycycline treatment, cells were dissociated for 2 minutes using a mixture of Accutase (Sigma-Aldrich, Saint-Louis, MO) and DNAse (Worhington, Lakewood, NJ) and then spun at 400 relative centrifugal force for 7 minutes at 4°C. The pellet was resuspended in culture medium to reach a target concentration of 1000 cells / μl. Following the protocol of Chromium Single Cell 3′Reagents Kits v3.1 (10x Genomics, Pleasanton, CA), cells were passed through a filter and loaded into a 10x Genomics Chromium Single Cell Chip G.

[0162] Single-cell RNA sequencing, mapping, and data analysis

[0163] Libraries were sequenced using an Illumina NextSeq 500, in most cases using multiplexed libraries with the High Output 150 kit. Data were demultiplexed and aligned to the mm10 genome using CellRanger version 3.0. Filtered output files were further analyzed in R using Seurat version ≥3.0, ggPlot2, data.table, dplyr, tidyr, and other commonly used R packages. Low-quality cells (determined as having low read depth or high mitochondrial content; >10%) were removed from the dataset. Microglia and any astrocytes or endothelial cells present were identified and removed from the data prior to downstream analysis. Additionally, gene expression data were normalized and scaled prior to analysis, and cells were clustered using principal component analysis and UMAP using tools provided in the Seurat R package version ≥3.0. Comparisons between datasets were done by canonical correlation analysis as described in the Satija lab vignette ( 46 ) ( https: / / satijalab.org / seurat / archive / v3.0 / integration.html ).

[0164] Integration with developmental data

[0165] To compare with the developmental retina, data were first downloaded from the Gene Expression Omnibus (GEO) (21). Label transfer was performed in Seurat using a reference dataset consisting of 432 randomly sampled cells from each major cell class in the developmental retina dataset (rods, cones, bipolar cells, amacrine cells, horizontal cells, retinal ganglion cells, and Müller glia), for a total of 3024 cells identified by canonical markers. Reads were downsampled to a common mean depth prior to analysis. In addition, IPA neurons were directly integrated with a subset consisting only of E14 cells from the developmental dataset.

[0166] Single-cell ATAC sequencing

[0167] The Cellranger ATAC pipeline (2.0.0) was used to preprocess the data generated by sequencing (47). First, the BCL files were converted to fastq and the reads were demultiplexed using "cellranger-atac mkfastq". Next, "cellranger-atac count" was run to map the Tn5 locus to mm10 (mouse genome), remove duplicate reads, and remove background cells. This returns a peak by cell matrix and barcode fragment files, which are loaded into Signac (48) (R (4.0.4) (R Core Team, 2021) package). Macs2 was then run on the Signac object and barcode fragment files to call peaks using Signac's "CallPeaks" function (49). Fragments were mapped to peaks called by Macs2 and assigned to cells using Signac's "FeatureMatrix" function. Further quality control (QC) metrics were measured in Signac using the "NucleosomeSignal" and "TSSEnrichment" functions. Cells with outliers in the QC metric categories were removed following Signac’s standard processing guidelines. Latent semantic indexing (LSI) was performed in Signac using the “RunTFIDF” and “RunSVD” functions. Signac’s “DepthCor” was used to identify LSI sizes that were highly correlated with read depth; these LSI sizes were excluded from downstream analysis. Signac / Seurat’s “RunUMAP” function was run to calculate UMAP embeddings. To identify clusters, Signac / Seurat’s “FindClusters” was then run at different resolutions. Clusters were assigned to known retinal cell types by examining Tn5 insertions within 100kb of known marker genes using Signac’s “CoveragePlot” and further supported using chromVAR scores for known lineage-specific TFs. Clusters of the same type were grouped for visualization purposes. Vertebrate motifs were obtained from the Jaspar 2020 database. Signac’s “AddMotifs” function was used to map these motifs to peaks within the Signac object. The “RunChromvar” function of Signac was used to calculate the motif accessibility z-scores for all cells.

[0168] Dataset Integration

[0169] Before integrating the Signac objects, we first ran all previous computational steps independently on each sample. Next, we created a shared peak set for all objects to be integrated using BEDOPS(-m)(50). Signac's FeatureMatrix function was run on each sample and the merged peaks were set to place all samples in the same feature space. The samples were next downsampled to the same average read depth using the DropletUtils "downsampleMatrix" function. The samples were merged using the "Merge" function of Signac / Seurat, and standard Signac normalization, dimensionality reduction, clustering, and visualization were performed on the merged objects as described above. The objects were then split by sample and the samples were integrated together. Anchors between samples were computed using "FindIntegrationAnchors" and the integrated embedding space was then computed using Signac's "IntegrateEmbeddings" function. UMAP and clustering of the integrated objects were performed as previously described.

[0170] Pseudo-time analysis

[0171] To calculate pseudotime and identify trajectory branches, samples were loaded into Monocle 3 using the “as.cell_data_set” function of SeuratWrapper (51). Clustering was performed and partitions were defined using Monocle 3’s “cluster_cells”. Monocle 3’s “learn_graph” was then run to define the main trajectory graph. Pseudotime roots were placed in progenitor-like or MG-like clusters using Monocle 3’s “order_cells”. Branches were selected using Monocle 3’s “choose_graph_segments”. Pseudotime and branch data were transferred back to the Seurat object for subsequent analysis.

[0172] Scatter plot

[0173] To create scatter plots, the “FindMarkers” function of Signac / Seurat was run between the cell groups of interest using the top 25% most accessible peaks in these cell groups. Scatter plots were then drawn showing the percentage of cells in each group that could reach each peak. Peaks with a mean log base-two change greater than 0.05 were selected for each group. For GO analysis, these peak sets were then loaded into GREAT (Genomic Region Enrichment Annotation Tool) (52) or analyzed for scRNA-seq data using the gprofiler2 package in R.

[0174] Cascading heatmap

[0175] To construct the Cascade heatmap, motif names were converted to mouse gene names using R and biomart’s “getLDS” function. The pseudo-temporal lineage branches of RGCs were subsetted from RNA and ATAC data from the E14 sample. Variable motifs were identified by running chromVAR’s “addGCBias”, “getBackgroundPeaks”, “computeDeviations”, and “computeVariability” functions. Peaks with speech scores greater than 1.2 were retained for further analysis. This list of motifs was then subsetted again; only motifs corresponding to TFs expressed by more than 10% of cells in the RGC branch were retained. Finally, conjoined motifs were removed. Motif enrichment scores for selected motifs were pseudo-temporally ordered in the E14 RGC branch of the scATAC-seq data by fitting their ChomVAR scores to a third-order polynomial function and ordering motifs by the maximum value of this function within our pseudo-time. To create the heatmap, chromVAR motif accessibility z-scores were plotted within the RGC lineage in the order previously derived over pseudo-time. The RNA heatmap was created by plotting genes whose binding sites were in the final motif list in the same order in RNA objects within the RGC lineage. A cascade heatmap of reprogrammed cells was created by plotting motifs and factors identified in the developmental data.

[0176] result

[0177] Pou4f2 and Islet1 increase neuronal diversity in Ascl1-reprogrammed MG

[0178] We previously developed a mouse in which Ascl1 is specifically induced in the MG (Glast-CreER:LNL-tTA:teto-mAscl1-GFP:ccGFP) by application of tamoxifen. After Ascl1 induction, retinal injury [N-methyl-D-aspartate (NMDA)] followed by injection of a histone deacetylase inhibitor (TSA) induces the MG to express genes associated with developing retinal progenitors and generate new neurons in vivo in the adult mouse retina. The majority of these newly generated neurons adopt a bipolar cell fate (4, 5, 12). To examine whether expression of additional TFs (Pou4f2 or Islet1) can direct MG-derived progenitors toward alternative neuronal fates, we crossed Glast-CreER:LNL-tTA:teto-mAscl1-GFP mice with the tetO-Pou4f2-tetO-Islet1 transgenic mouse line (tetO-IPA; Figure 1A). This construct encodes Pou4f2 and Islet1 separated by two different loxP variants (19). When exposed to Cre recombinase, the cassette allows the expression of Pou4f2, Islet1, or sometimes both.

[0179] The tetO-IPA mouse line allowed us to test whether treatment with Pou4f2+Ascl1, Islet1+Ascl1, or Islet1+Pou4f2+Ascl1 (hereafter referred to as IPA) enhances MG reprogramming. After intraperitoneal administration of tamoxifen to induce TF in MG, we induced retinal injury by intravitreal injection of NMDA followed by TSA ( Figure 1B ). Mice were euthanized 3 weeks later and immunofluorescence analysis was performed to assess the fate of recombinant MG (Fig. 1, C to E). Fig. 1 (C and D) shows that this protocol induced transgenes [green fluorescent protein positive (GFP+)] in MG and neurons derived therefrom. We found that GFP+ MG-derived cells expressed Brn3(Pou4f2) (17%), Islet1 (33%), or both TFs (7%) (Fig. 1, C and D).

[0180] Additional immunohistochemistry (IHC) analysis demonstrated that the IPA combination effectively promoted neurogenesis in MG. Most glial-derived cells acquired neuronal morphology 3 weeks after injury ( Figure 1E Quantification of MG-derived cells in retinal sections of IPA-treated mice confirmed that MG-derived GFP+ neuron-like cells expressed the ganglion / amacrine cell marker HuC / D (Figure 1, F and G) or the bipolar marker Otx2 (Figure 1, G and H). IPA expression significantly enhanced MG neurogenesis in HuC / D neurons compared with Ascl1 alone ( Figure 1G ). Consistent with our previous report, a subset of MG-derived neurons was derived from EdU+, proliferating MG ( Figures 8A-8C Together, these data suggest that addition of the TFs Pou4f2 and / or Islet1 enhances neuritogenic capacity and expands the cell fate of Ascl1-MG in vivo.

[0181] We previously showed that the Ascl1:Atoh1 combination stimulates neurogenesis in MG in the absence of retinal injury (13). In contrast, IPA induction in the intact retina was insufficient to stimulate neurogenesis (Fig. 9, A, B, and E). We also tested whether MGs are capable of neurogenic reprogramming if we activate the IPA factor in MGs after NMDA injury and TSA rather than before NMDA and TSA (as in Fig. 1). We found that induction of IPA after injury stimulated a small amount of neurogenesis, but much less than in our initial paradigm (Fig. 9, C to E).

[0182] Because a large fraction of newborn neurons in the injured retina express the ganglion / amarine cell marker HuC / D, we performed whole-mount imaging of IPA-derived neurons to better assess their neuronal morphology. This revealed cells with large, branching dendritic arbors reminiscent of RGCs or broad-field amacrine cells; cells with this morphology had not been seen previously using Ascl1 alone (Fig. 2, A to F).

[0183] IPA induces neurons with an RGC-like transcriptome

[0184] Next, we used scRNA-seq to analyze how Islet1 and Pou4f2 alter the phenotype of Ascl1-mediated MG reprogramming. Three weeks after initiating the IPA regeneration protocol, MG cells and their progeny were purified by fluorescence-activated cell sorting (FACS) and processed for scRNA-seq as previously described (5, 12). To directly compare IPA-induced cell fate changes with those caused by Ascl1 expression alone, we used Seurat to integrate the IPA-treated data with the previously obtained Ascl1-only reprogramming library (4) and clustered the cells ( Figure 3A The combined data from the IPA experiments and the previous Ascl1 dataset were projected onto a single Uniform Manifold Approximation and Projection (UMAP) map, and cell type clusters were identified using known marker genes ( Figure 3B ).

[0185] The combined UMAP plot of Ascl1 alone and IPA treatment contains the cell type clusters (e.g., MG, progenitor, and bipolar cells) that we have previously observed during Ascl1-mediated reprogramming ( Figure 3A This analysis revealed two additional phenotypes unique to the IPA condition. First, IPA increased the efficiency of neurogenesis in MG by more than two-fold compared to Ascl1 alone ( Figure 3C ), which is consistent with our IHC data and suggests that the combination of these three TFs can effectively stimulate neurogenesis in adult MG. Second, a new cluster that was not present in the Ascl1-only condition appeared after IPA treatment ( Figure 3C While cells expressing Islet1 but not Pou4f2 were primarily found in bipolar clusters, cells expressing Pou4f2 (with or without detectable Islet1) were primarily found in novel RGC-like clusters ( Figures 10A to 10C ).

[0186] IPA-induced new cell clusters showed high expression of RGC characteristic genes, such as Elavl4 and Sox11 ( Figure 3B)(20). In addition, we found that these IPA neurons expressed many genes found in the Gene Ontology (GO) terms “axon guidance,” “axon growth,” and “axonogenesis” ( Fig. 10D ). Since Islet1 and Pou4f2 are located upstream of the RGC fate-inducing regulatory network, we analyzed whether this factor combination could induce multiple RGC genes in MG-derived neuronal clusters. We used Seurat's label transfer function to extensively compare the transcriptome of IPA neurons with reference datasets of all major retinal neuron classes (21, 22). The new cluster was classified as an RGC cluster (mean prediction score = 0.74), indicating overall transcriptome similarity to native RGCs ( Fig. 10E Compared with Ascl1-induced neurons, a large number of RGC-related genes were expressed in IPA-induced neurons ( Figure 3D ). These genes include Sox4 and Sox11, which are redundantly essential genes for RGC fate acquisition (23, 24), and the axon growth-related gene Gap43, which is highly expressed in developing RGCs (25). We found that both Satb1 and Cntn5 are expressed in IPA-induced neuronal subpopulations. Satb1 is highly expressed in the ON-OFF direction-selective subtype of RGCs, where it controls Cntn5 expression (26). Using IHC, we were able to confirm that IPA-induced neurons express Satb1 protein ( Figure 3E In addition, the RGC and amacrine marker calretinin (Calb2) was also detected at the RNA and protein levels (Fig. 3, C and F).

[0187] Although MG-derived RGC-like neurons expressed a large number of RGC genes, these cells failed to express some typical RGC markers, such as Pou4f1, Sncg, Rbpms, and Nefm ( Figure 3D ). This suggests that the newly born neurons have not yet fully differentiated into mature RGCs. Consistent with this observation, when we integrated the scRNA-seq data of IPA-induced neurons with a dataset from E14 (embryonic day 14) embryonic mouse retina (21), we found that MG-derived RGC-like neurons were most similar to immature RGCs ( Figure 3G We compared IPA-induced neurons at the 3-week endpoint with neurons at a longer survival time point (6 weeks) and found that the regenerating RGC-like neurons were a stable population and showed no evidence of increased cell death or stress during this period ( Figures 11A-11D ).

[0188] Simultaneous expression of Islet1-Pou4f2 and Ascl1 induces more uniform RGC states in MG in vitro

[0189] Because the tetO-Pou4f2 and Islet1 transgenic constructs contain mutually exclusive loxP sites, most MGs expressing Cre in vivo express Ascl1 and either Pou4f2 or Islet1. Although a few cells were labeled with both Pou4f2 and Islet1 in vivo (Fig. 1, C and D), to better assess the effects of uniform overexpression of all three transgenes, we bred mice containing the tetO-Pou4f2-Islet1 and tetO-Ascl1-GFP cassettes into the germline Rosa26-rtTA line and then performed in vitro MG reprogramming experiments (Fig. 4, A and B). As previously described, MGs from postnatal day 1111 mice were cultured for 7 days before passaging (10). The cultures obtained in this way were primarily composed of MGs, although some surviving neurons were observed (10). 5-Ethynyl-2′-deoxyuridine (EdU) was added to the culture medium to determine which cells were derived from proliferating MGs and which cells were likely neurons that survived the initial isolation. After passaging, doxycycline was added to the culture medium to induce transgene expression, and the cells were then analyzed by immunofluorescence and scRNA-seq ( Figure 4B ).

[0190] Analysis of cultures 5 days after treatment confirmed that the cells expressed the transgene. Immunolabeling of the transgene showed that the cells co-expressed Pou4f2, Islet1, and Ascl1 ( Fig. 12A Most of the cells adopted neuronal morphology, and immunolabeling showed that EdU+ cells and the neuronal marker Tuj1 ( Figure 4C ), neurofilaments ( Figure 4D ) and calcium binding protein ( Figure 4E ) are marked together.

[0191] To determine how overexpression of IPA differed from Ascl1 alone, we repeated the reprogramming experiments as described above and studied sister MG cultures from tetO-Ascl1;Rosa26-rtTA mice with or without doxycycline treatment to activate transgene expression. We performed scRNA-seq as described above for each sample. Untreated MGs were largely homogeneous, with one glial cluster and one small cluster containing only a few surviving neurons (Fig. 12, B and C). To compare cluster composition between treatment conditions as well as the in vivo IPA dataset, we integrated all three (Fig. 4, F to I, and Fig.12D). We identified cells in both in vitro Ascl1-only and in vitro IPA-reprogrammed MGs that mapped to neuronal clusters in the IPA in vivo dataset. However, each treatment stimulated a major neuronal cluster from the MG; in Ascl1 cultures, neurons most resembled bipolar cells ( Fig. 4, F and I ), whereas in IPA cultures, neurons acquired an RGC-like fate ( Fig. 4, G and I ), with a minority of neurons differentiating into bipolar cells. This differs from our observations in vivo, where the proportion of bipolar clusters was similar to that in Ascl1-only samples ( Figure 1D and 4, F and H).

[0192] We next compared gene expression profiles of induced neurons in the IPA and Ascl1-only in vitro datasets. We observed that bipolar genes, such as Cabp5, were largely localized to the Ascl1-only neuronal clusters. RGC genes, such as Grin2a and Calb1, were found only in IPA neurons ( Figure 4J To identify unique marker genes expressed in IPA neurons, we made subsets of all neuronal populations in the IPA and Ascl1-only integrated datasets ( Figure 4K ), and differential gene expression (DGE) analysis was performed ( Figure 4L ). We identified many RGC genes enriched in IPA neurons, while bipolar genes were enriched in Ascl1 neurons. In addition, we found that some typical markers of the RGC lineage were not induced in vivo, such as Sncg, Nefm, and Pou4f1, which were induced in vitro ( Figure 4M We also failed to detect cells expressing the intrinsically photosensitive RGC marker Opn4 ( Fig.12E Together, these results suggest that simultaneous overexpression of all three IPA factors biases MG-derived neurons toward an RGC-like fate and induces a more homogeneous RGC gene expression.

[0193] IPA induces neurons with different electrical properties

[0194] We previously showed that Ascl1 stimulates MG-derived neurons with physiological features of endogenous retinal neurons, particularly bipolar cells (4, 13). Because IPA treatment results in a distinct molecular and morphological neuronal phenotype compared with Ascl1 alone, we characterized the light responses and electrical properties of these cells. We performed patch clamp electrophysiology analysis of GFP+ cells in retinal slices and whole mounts following the IPA in vivo regeneration paradigm. We measured responses to current and voltage steps as well as responses to light stimulation. We plotted the membrane resistance and resting potential ( ) of IPA-induced neurons compared to GFP-MG, endogenous neurons, and neurons in our previous regeneration strategies using Ascl1 or Ascl1:Atoh1. Figure 5A )(4, 5, 13). Glial cells have low membrane resistance, hyperpolarized resting potential, and few voltage-activated conductances. Neurons have higher resistance, have less negative resting potential, and express voltage-activated conductances.

[0195] Most IPA-induced neurons showed resting potential and membrane resistance characteristics similar to those of endogenous neurons; however, some of them still had glial-like hyperpolarized membrane potentials ( Figure 5A This is consistent with our IHC and scRNA-seq analyses, in which a subset of glial cells do not reprogram after IPA treatment (see Figures 1 and 3). Figure 5B Examples of responses to a family of current or voltage steps recorded from IPA-treated GFP+ cells are shown. These cells exhibit a range of characteristics. Some have a neuronal phenotype and appear to express voltage-activated K+ conductances that limit the degree of depolarization of the current steps, while others retain features of glial cells ( Figure 5B ). Six of the 16 cells recorded responded to brief flashes of light, indicating that they had established synaptic connections with other components of the retinal circuitry ( Figure 5C Some cells respond to the current step by generating action potentials, whereas others generate spiking activity that may represent Ca2+ spikes ( Figure 5D ). This diversity in physiological properties is consistent with the phenotypes observed in Figures 1 and 3. Of note, we did not observe action potentials (a prominent feature of RGCs) in our previous strategy for stimulating MG-derived neurons (4, 5, 13). Thus, IPA increases the diversity of electrical properties of MG-derived neurons, including the generation of some cells that can generate Na+ and / or Ca2+ action potentials.

[0196] IPA expression remodels MG chromatin toward an imperfect RGC-like fate

[0197] We next performed single-cell analysis of transposase-accessible chromatin sequencing (scATAC-seq) for Ascl1 versus IPA-reprogrammed MG to better understand the mechanistic differences between these two reprogramming strategies. Mice were treated with the same in vivo retinal regeneration paradigm described in Figure 3, but were processed using scATAC-seq rather than scRNA-seq. Nuclei from Ascl1 alone (1692) and 2451 from IPA treatment passed our quality control metrics (see Materials and Methods). Single cells from both treatments were then integrated and plotted as UMAPs to identify cell types (Figure 6, A to C). Cell type clusters were identified by accessible chromatin patterns near genes identified for specific retinal cell types. The overlay shows representative peaks for the groups we identified: MG (R1bp1+), neurogenic transition (Islet1+), MG-derived bipolar cells (Crx+), induced RGC-like cells (Pou4f2+), and photoreceptors (Arr3+) ( Fig.6D When UMAP was split between the two treatment groups, it was apparent that both Ascl1 and IPA conditions resulted in MG-derived bipolar neurons, although a large cluster of cells retained the MG phenotype (Fig. 6, B and C). However, IPA treatment induced a distinct cluster of cells with accessible chromatin that was most similar to RGCs ( Figure 6C ). Consistent with the accessibility pattern, Otx2 motifs were enriched in bipolar clusters, whereas Pou4f2 motifs were highly represented in accessible chromatin in IPA-unique clusters ( Fig. 6F ). This is consistent with our scRNA-seq findings and suggests that these MG-derived neurons have a pattern of cis-regulatory regions consistent with their transcriptome identity.

[0198] To determine how similar the chromatin of MG-derived RGCs is to that of normally developing RGCs, we generated a scATAC library from E14 developing mouse retinas for comparison with MG-derived RGCs ( Figures 13A-13F ). Figure 6G The scatter plot in Figure 2 shows a significant correlation between the accessibility peaks in E14 RGCs and MG-derived neurons; however, there are clearly many areas where accessibility differs between cell types ( Figure 6G ). When we analyzed GO enrichment to understand the types of genes that were more highly represented in nearby accessible chromatin in E14 RGCs than in MG-derived RGCs, many of the top terms were related to axonal growth ( Figure 6H and Table 2). This is consistent with the fact that many MG-derived RGCs do not extend long axons.

[0199] Current models of retinal development suggest that a series of TF cascades are activated sequentially as cells transition from multipotent progenitors to neurogenic precursors and ultimately to a final neural cell fate (27). We therefore sought to examine whether induced neurogenesis in MG recapitulates this developmental cascade at the chromatin level. We used pseudotime to sort cells in the predicted lineage from progenitors to RGCs in the E14 retina and compared this with a similar analysis of MG-derived RGCs, and then analyzed the changes in motif enrichment in accessible chromatin as a function of pseudotime in a “cascade map” (Fig. 6, I and J) (28). This analysis revealed key differences between the developmental process of RGCs and the process of generating RGCs from MG. Normal development of RGCs shows a downregulation of progenitor TF motifs (Rax and Sox2) in accessible regions, followed by a transient increase in regions with bHLH motifs (Atoh7), and finally the induction of regions with mature RGC TF motifs (Pou4f1 / 2 / 3, Ebf1, and Onecut). This has recently been described in both mice and humans (28, 29). In contrast, in MGs reprogrammed to generate neurons using IPA, sequential changes in motif representation in accessible chromatin showed clear divergence from normal development. Although progenitor gene motifs were reduced as cells acquired an RGC-like identity, progenitor TFs were apparently never completely downregulated, as their motifs persisted in accessible regions of MG-derived RGCs ( Figure 6J In addition, although MG-derived RGCs showed an increase in accessible bHLH motifs and a significant increase in the representation of Pou4f1 / 2 / 3 motifs in accessible chromatin, TFs that may be located downstream of Pou4f1 / 3, such as Onecut and Ebf, were not activated in turn ( Figure 6J ). This epigenomic analysis suggests that RGC-like generation in IPA-treated MGs is imperfect, in part because accessibility to glial and progenitor regulatory regions is maintained. Consistent with this notion, the progenitor / MG marker Sox2 was still detectable by immunofluorescence 3 weeks after IPA reprogramming in GFP+ cells with neuronal morphology ( Figure 6K ).

[0200] Atoh1 enhances the ability of IPA to induce RGC-like cells from MG

[0201] scRNA and scATAC-seq analyses revealed that, although IPA-induced neurons resemble RGCs, these cells lack several features of mature RGCs. One hypothesis as to why this might be the case is the persistence of glial and progenitor genes and chromatin accessibility (i.e., Sox, Rax, Vsx2, etc.) in MG-derived neurons. Recently, we reported that Atoh-class TFs can potently stimulate neurogenesis when combined with Ascl1 (13); the Ascl1:Atoh1 combination enabled nearly 80% of transgenic expressing MGs to acquire a neural identity. Therefore, we tested whether combining Atoh1 with IPA reprogramming could improve RGC regeneration from MG in vivo.

[0202] We crossed mice containing tetracycline-inducible Atoh1 with the IPA strain and performed the regeneration paradigm described in Figure 1; we then performed scRNA-seq and IHC analysis of the MG progeny as described above (Figure 7, A and B). Consistent with our previous findings using Atoh1, IPA and expression of Atoh1 in MG resulted in the majority of MG progeny acquiring a neuronal identity (13). IHC revealed that the majority of GFP+ MG-derived neurons expressed the ganglion / amarine marker HuC / D and lacked expression of the bipolar marker Otx2 ( Figure 7C Adding Atoh1 to the IPA factor combination also bypassed the requirement for retinal injury-induced MG neurogenesis ( Figures 14A-14D We next performed scRNA-seq on regenerating cells treated with the IPA:Atoh1 condition and integrated them with cells from the IPA-only group to determine whether the addition of Atoh1 could improve RGC generation after retinal injury ( Fig.7D ). This analysis showed that MG-derived neurons from IPA:Atoh1 mice were most similar to MG-derived RGCs from IPA conditions, with smaller proportions of bipolar cells, cones, and amacrine cells also present ( Fig.7D as well as Fig.14E and 14F ).

[0203] To assess whether Atoh1 overexpression reduces the progenitor identity of MG-derived neurons after IPA expression, we formed a subset of RGC-like cells from each dataset for comparison. DGE analysis showed that under IPA:Atoh conditions, RGC-like cells showed a greater decrease in progenitor and glial gene expression than similar cells under IPA only conditions ( Fig. 7E This was further confirmed by GO analysis, where IPA:Atoh1 reduced genes associated with neural progenitor cells compared to the IPA-only condition ( Figure 7F ), which is consistent with the hypothesis that Atoh1 promotes the maturation of RGC-like cells.

[0204] We have previously demonstrated that Ascl1:Atoh1 can generate immature RGC-like cells from MG (13). We hypothesized that the addition of the IPA factor to Atoh1 might induce markers of more mature RGCs. We compared IPA:Atoh1 to the previously generated Ascl1:Atoh1 dataset and found similar cell type composition ( Figure 7G and Figures 14E to 14H However, we found that adding IPA to Atoh1 increased RGC-like cells by 15% and resulted in a corresponding decrease in bipolar and progenitor-like cells ( Figure 14G and 14H DGE analysis showed that the addition of IPA to Ascl1:Atoh1 resulted in the enrichment of RGC genes such as Map2 and Thy1 ( Figure 7H ). In addition, we found that the top GO terms enriched in IPA:Atoh1 were related to neuronal projection development and axonogenesis ( Fig.7I ), many of which are specifically involved in axon guidance (e.g., Epha8 and Sema3e) and synaptogenesis (e.g., Syt1 and Snap25).

[0205] Table 2: Genes associated with peaks enriched in E14 RGCs relative to MG-derived RGC-like cells.

[0206]

[0207]

[0208] discuss

[0209] Adult non-mammalian vertebrates can regenerate neurons in many regions of the central nervous system (CNS). For example, after tail amputation in young frogs and some adult urodendrons, radial glial cells of the spinal cord acquire gene expression patterns similar to those of neuronal progenitors and continue to proliferate and regenerate a seemingly normal spinal cord (30). Similarly, in the zebrafish retina and brain, glial cells respond to injury by activating a progenitor-like gene expression program of TF (31). These glial-derived progenitors undergo multiple rounds of mitosis, and their progeny differentiate into a range of neuronal types that can restore brain and retinal function (32).

[0210] Re-expression of developmentally active TFs in adult mammalian glial cells triggers a regenerative process in these cells that resembles regeneration in fish and amphibians in many ways. For example, after retinal injury, transgenic overexpression of the proneural TF Ascl1 combined with histone deacetylase inhibition stimulates MGs to acquire a progenitor-like state with the ability to generate bipolar neurons (4). MG-derived neurons differentiate to the point where they can form synapses with surrounding neural circuits and respond to light. In addition to lineage tracing of neurons to verify their glial origin, we used EdU labeling to show their adult origin and analyzed cells using scRNA-seq and scATAC-seq to visualize intermediate states between glial progenitors and regenerating neurons (5, 12, 13). Together, this demonstrates that TFs can reprogram glial cells in the adult CNS to generate neurons.

[0211] Although Ascl1 induces MG to adopt many characteristics of retinal progenitors, including proliferative neurogenesis and transcriptional and epigenetic landscapes similar to developmental progenitors, not all developmentally appropriate Ascl1 targets are induced in MG-derived progenitors, and the neuronal output of Ascl1MG is primarily restricted to bipolar neurons (5). Therefore, we reasoned that additional TFs may be required to properly direct MG-derived progenitors to specific neuronal types. This is particularly important for endogenous regenerative strategies, as most blinding diseases are the result of defects in specific neuronal subtypes. For example, glaucoma is primarily caused by RGC death.

[0212] RGCs are generated during development by a TF cascade characterized by the initial expression of Atoh7 and the downstream expression of other TFs, such as Pou4f1 / 2 and Islet1 (33). Atoh7 is required for correct RGC fate by inducing these downstream stabilizing transcription factors (34-36). Two of these downstream TFs, Pou4f2 and Islet1, are required for correct RGC fate specification (18, 37, 38), and ectopic expression of Pou4f2 and Islet1 in Atoh7 null retinas is sufficient to rescue RGC fate (19). By leveraging the extensive knowledge of normal transcriptional regulation of RGC fate, we were able to test whether members of this TF cascade could reinitiate the genesis of these cells from Ascl1-induced MG-derived progenitors.

[0213] Here we report that the RGC fate-inducing factors Islet1 and Pou4f2, as well as Ascl1, induce MG neurogenesis toward an RGC-like fate. Using IHC, we found that MG-derived neurons from IPA-expressing MG expressed protein markers and morphological features of RGCs. At a physiological level, these IPA-induced MG-derived neurons displayed neuronal resting membrane potentials and a subset generated voltage-gated action potentials, consistent with an RGC-like fate. Finally, our molecular analyses using scRNA-seq and scATAC-seq showed that IPA-induced MG-derived neurons were most similar to immature RGC cells at the transcriptomic and epigenetic levels.

[0214] Although expression of the IPA transgene in MGs collectively reprograms a subset of cells to an RGC-like fate, this combination does not activate all TFs required for mature RGCs. We found in both scRNA-seq and scATAC-seq datasets that some mature RGC genes could not be induced; instead, MG-derived neurons retained expression of some progenitor / glial genes, resulting in a chromatin landscape intermediate between progenitors and RGCs. In addition to IPA, the progenitor state in MGs could be suppressed by expression of Atoh1, consistent with a role for atonal TFs in promoting neuronal differentiation, and this allowed MG-derived RGCs to further differentiate. In summary, our results indicate that ectopic expression of developmental TFs that are essential for cell-type-specific trajectories can reprogram MGs to the same trajectory. This suggests a holistic approach in which complementary and sequential roles of TFs in the development of specific neuronal types can be used to regenerate more mature neurons. It remains to be seen whether expression of TFs in their normal developmental sequence will provide a more effective reprogramming strategy.

[0215] Interestingly, MG-derived RGC-like neurons were most commonly found in the inner nuclear layer (INL) rather than the ganglion cell layer (GCL). Although only a small fraction of RGCs are typically found in the INL of mice, most RGCs migrate to the GCL. The inner plexiform layer may provide a barrier to the migration of RGC-like cells, or they may lack cues to migrate appropriately. Although scRNA-seq data showed that MG-derived RGC-like cells expressed many RGC genes, they may lack some key migratory programs. Despite this, some of these cells still connect to existing neural circuits and respond to light, and may establish appropriate connections without an appropriate soma location.

[0216] Although we also did not observe robust axon outgrowth toward the optic nerve, we found that many genes important for axon outgrowth and guidance are expressed in RGC-like cells. It is possible that some key guidance factors are not expressed in these cells or that the adult retinal environment no longer expresses guidance factors required to guide axons to the optic nerve head. RGC transplantation experiments found that in some cases RGC axons projected ectopically, suggesting that the microenvironment of the adult retina may not fully reflect that of the developing retina; future studies are needed to better understand the interplay between cell-autonomous and non-autonomous factors in regeneration.

[0217] Over the past decade, many reports have shown that other developmental TFs, such as Sox2, Pax6, NeuroD1, Neurog2, and Ascl1, can stimulate glial neurogenesis in vitro and in the brain (39). However, the interpretation of many of these reports has been obscured by the finding that lineage tracing of new neurons using existing adeno-associated virus (AAV) paradigms is unreliable and often results in the misidentification of endogenous neurons as glial-derived (40, 41). Nevertheless, several early studies using alternative approaches to trace glial-derived neurons have demonstrated that reprogramming of astrocytes into neurons is possible and that proneural TFs, including Ascl1, are neurogenic stimulants (42–45). Therefore, the strategy of combining TFs from specific neuronal lineages, as we have used in the retina, may be applicable throughout the CNS.

[0218] Overall, this study provides a proof of concept that TFs used to specify cell fate during development can be exploited to initiate the acquisition of similar fates by reprogrammed glial cells in the adult CNS. The redesign of cell fate acquisition during endogenous regenerative strategies will be key to directing neural replacement tailored for specific neurodegenerative diseases.

[0219] References

[0220] 1. L. Todd, TAReh, Cold Spring Harb. Perspect. Biol. 14, a040816 (2022).

[0221] 2. A. Hamon, et al. Dev. Dyn. 245, 727-738 (2016).

[0222] 3. A. Bringmann, et al. Prog. Retin. Eye Res. 28, 423-451 (2009).

[0223] 4.N.L.Jorstad,etal.Nature 548,103-107(2017).

[0224] 5.N.L.Jorstad,et al.Cell Rep.30,2195-2208.e5(2020).

[0225] 6.T.Hoang,et al.Science 370,eabb8598(2020).

[0226] 7.E.M.Rueda,et al.Cell Rep.27,1637-1649.e6(2019).

[0227] 8.K.Yao,et al.Cell Rep.17,165-178(2016).

[0228] 9.K.Yao,et al.Nature 560,484-488(2018).

[0229] 10.J.Pollak,et al.Development 140,2619-2631(2013).

[0230] 11.Y.Ueki,et al.Proc、Natl.Acad.Sci.U.S.A.112,13717-13722(2015).

[0231] 12.L.Todd,et al.Cell Rep.33,108507(2020).

[0232] 13.L.Todd,et al.Cell Rep.37,109857(2021).

[0233] 14.C.Powell,et al.Sci.Rep.6,24851(2016).

[0234] 15.F.D.D'Orazi,et al.J.Comp.Neurol.528,2816-2830(2020).

[0235] 16.G.Beykin,et al.Prog.Retin.Eye Res.80,100875(2021).

[0236] 17.Y.Elshatory,et al.J.Comp.Neurol.503,182-197(2007).

[0237] 18.L.Gan,et al.Proc.Natl.Acad.Sci.USA93,3920-3925(1996).

[0238] 19.F.Wu,et al.Proc.Natl.Acad.Sci.USA112.E1559-E1568(2015).

[0239] 20.A.Usui,et al.Development 140,740-750(2013).

[0240] 21.BSClark,et al.Neuron 102,1111-1126.e5(2019).

[0241] 22.T.Stuart,et al.Cell 177,1888-1902.e21(2019).

[0242] 23.KCChang,et al.J.Neurosci.37,4967-4981(2017).

[0243] 24.Y.Jiang,et al.J.Biol.Chem.288,18429-18438(2013).

[0244] 25.TAReh,et al.J.Neurobiol.24,949-958(1993).

[0245] 26.Y.-R.Peng,et al.Neuron 95,869-883e866(2017).

[0246] 27.EABassett,et al.Trends Neurosci.35,565-573(2012).

[0247] 28.C.Finkbeiner,et al.Cell Rep.38,110294(2022).

[0248] 29.P.Lyu,et al.Cell Rep.37,109994(2021).

[0249] 30.ADKakebeen,et al.eLife 9,e52648(2020).

[0250] 31.BWLindsey et al.Prog.Neurobiol.170,99-114(2018).

[0251] 32.D.Goldman,Nat.Rev.Neurosci.15,431-442(2014).

[0252] 33.F.Wu,et al.Nat.Commun.12,1465(2021).

[0253] 34.N.LBrown,et al.Development 128,2497-2508(2001).

[0254] 35.JNKay,et al.Neuron 30,725-736(2001).

[0255] 36.J.Brodle-Kommit,et al.Sci.Adv.7,eabe4983(2021).

[0256] 37.X.Mu,et al.Proc.Natl.Acad.Sci.USA105,6942-6947(2008).

[0257] 38.L.Pan,et al.Development 135,1981-1990(2008).

[0258] 39.R.Bocchi,et al.Neuron 110,366-393(2022).

[0259] 40.S.Blackshaw,et al.J.Clin.Invest.131,e146134(2021).

[0260] 41.L.-L.Wang,et al.Cell 184,5465-5481.e16(2021).

[0261] 42.C.Lentini,et al.Cell Stem Cell 28.2104-2121.e10(2021).

[0262] 43.O.Torper,et al.Proc.Natl.Acad.Sci.U.SA.110,7038-7043(2013).

[0263] 44. C. Heinrich, et al. Stem Cell Rep. 3, 1000-1014 (2014).

[0264] 45. N. Mattugini, et al. Neuron 103, 1086-1095.e5 (2019).

[0265] 46. ​​A. Butler, et al. Nat. Biotechnol. 36, 411-420 (2018).

[0266] 47. ATSatpathy, et al. Nat. Biotechnol. 37, 925-936 (2019).

[0267] 48. T. Stuart, et al. Nat. Methods 18, 1333-1341 (2021).

[0268] 49. Y. Zhang, et al. Genome Biol. 9, R137 (2018).

[0269] 50. S. Neph, et al. Bioinformatics 28, 1919-1920 (2012).

[0270] 51. J. Cao, et al. Nature 566, 496-502 (2019).

[0271] 52. CYMcLean, et al. Nat. Biotechnol. 28, 495-501 (2010).

[0272] 53. NMTran, et al. Neuron 104, 1039-1055.e12 (2019).

[0273] Example 2: Use of transcription factors specific to developing retinal cells to promote the generation of RGCs.

[0274] To determine which transcription factors help promote the generation of retinal ganglion cells from Müller glia, we used lentivirus to induce the expression of several transcription factors that are expressed in developing retinal cells but not in Müller glia. Müller glia were grown in cell culture and infected with the virus. The cells were then cultured for 5-7 days and then processed for single-cell RNAseq to determine their fate. We found that, of all the factors tested, Onecut1 ( Fig.15The arrows in the figure can induce Müller glia to generate new cells with the expression of RGC characteristic genes, including Nefm, Nefl, Ebf1 and Gap43 ( Fig.16 ).

[0275] Example 3: AAV-delivered reprogramming transcription factors can induce neurogenesis.

[0276] Figures 17A-17C The protocol used to demonstrate the tracing of HuC / D+ neuronal lineage from MG by tdTomato is described. Immunofluorescence labeling shows successful delivery of reprogramming transcription factors, resulting in reprogramming of MG cells to HuC / D+ neurons. Fig.18 As shown, both Atoh1 and Atoh7 delivered by AAV can induce HuC / D+ neurons. The efficiency of Atoh1 and Atoh7 is roughly the same. Under these conditions, the efficiency of AAV delivery of reprogramming factors is much lower than transgene expression.

[0277] We tested five additional RGC transcription factors: Irx2, Irx5, Neurod2, Ebf1, and Tcf3. Among them, Irx2 and Neurod2 promoted axonal growth in IPA-reprogrammed MGs ( Fig.19 ). This suggests that some RGC transcription factors can increase some specific RGC genes.

[0278] Example 4: Human Muller glial cells can be generated in vitro from fetal retina or pluripotent stem cells

[0279] Fig. 20A Schematic illustration of the protocol for generating retinal organoids (ROs) from embryonic stem cells (ESCs). Fig. 20B Images of MG development over time in retinal organoids labeled with RLBP1 and SOX2 are shown. Fig. 20C The upper panel shows a schematic scheme for generating retinal spheres (RS), and the lower panel shows images of retinal spheres made from several fetal retinas and cultured for different periods of time as labeled. Fig.20D Characterization of MG in RS with RLBP1, VSX2, SOX9, SOX2, and GFAP is shown.

[0280] Human MG can be reprogrammed with ASCL1 to generate neurons in dissociated culture (Figure 22). These neurons express some pan-neuronal markers, such as DCX and TUJ1, but are not yet mature. Human MG can be derived from retinal organoids or fetal human retinas; both glial sources respond similarly to ASCL1.

[0281] Example 5: Promoter driving expression in Muller glial cells

[0282] Fig.23Characterization of the ShH10 capsid and RLBP1 promoter in non-human primate (NHP) Muller glial cells is described in . We found that the human RLBP1 promoter was able to drive expression in Muller glial cells (mouse, human or NHP) very well, but its size of 2.8kB was too cumbersome for 2-3 reprogramming TFs. So we started to study a smaller promoter: HES1. The HES1 promoter provides a good alternative to the RLBP1 promoter. The HES1 promoter is only 337bp and is expressed in adult Muller glial cells and retinal progenitor cells. In addition, the expression of HES1 is increased after ASCL1 infection. As shown in Figure 25, the HES1 promoter drives very good expression specifically in Muller glial cells in human retinal organoids. Seven days after infection with the Hes1-GFP construct, GFP is expressed in HES1 and Sox9 positive cells. The construct is specific at 7 days. Fig.25C showed that GFP was not expressed in OTX2-positive cells (HES1-GF), however we did obtain GFP / OTX2-positive cells after ASCL1 expression (reprogrammed cells; Fig.25D ). The HES1 promoter also directs GFP expression in MG in dissociated cultures of adult NHPs, such as Fig.26 shown.

[0283] This small and specific promoter offers the potential to drive TF reprogramming in Muller glia with a single AAV virus. Fig.24 The viruses shown, as well as more complex "self-regulatory loop" viruses, allow us to track reprogrammed cells over time. The HES1 promoter is shown to be specific. The related HES5 provides an alternative.

[0284] Throughout this application, various publications are cited. The disclosures of these publications are incorporated herein by reference in their entireties in order to more fully describe the state of the art to which this invention pertains.

[0285] From the foregoing it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor, wherein the RGC transcription factor is selected from the group consisting of Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, Tcf3 and a combination thereof.

2. The nucleic acid molecule of claim 1, wherein the RGC transcription factor is selected from the group consisting of Onecut1, Pou4f2 and Islet1, and combinations thereof.

3. The nucleic acid molecule of claim 1, wherein the RGC transcription factor is Onecut1.

4. The nucleic acid molecule of claim 1, wherein the RGC transcription factors comprise Pou4f2 and / or Islet1.

5. The nucleic acid molecule of claim 1, wherein the RGC transcription factor is selected from the group consisting of Irx2, Irx5, Neurod2, Ebf1, Tcf3, and combinations thereof.

6. The nucleic acid molecule of claim 1, wherein the RGC transcription factors comprise Irx2 and / or Neurod2.

7. The nucleic acid molecule of any one of claims 1 to 6, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding a proneural basic helix-loop-helix (bHLH) transcription factor selected from Ascl1, Atoh1 and Atoh7.

8. The nucleic acid molecule of any one of claims 1 to 7, wherein the nucleic acid sequence further comprises a promoter sequence operably linked to the nucleic acid sequence encoding the RGC transcription factor.

9. The nucleic acid molecule of claim 8, wherein the promoter sequence is a retinal ganglion cell (RGC) specific promoter.

10. The nucleic acid molecule of claim 8, wherein the promoter sequence is HES1, RLBP1 or GLAST.

11. A composition comprising the nucleic acid molecule according to any one of claims 1 to 10.

12. The composition of claim 11, further comprising a histone deacetylase (HDAC) inhibitor.

13. The composition of claim 12, wherein the HDAC inhibitor is trichostatin A (TSA), (ex) / romidepsin, (ex) / belinostat, (ex) / panobinostat and / or (ex) / vorinostat.

14. A method of stimulating regeneration of retinal ganglion cells in a subject, the method comprising: The composition of claim 11 is administered to the retina of the subject, thereby stimulating retinal ganglion cell regeneration.

15. The method of claim 14, wherein the composition comprises a first nucleic acid molecule encoding a proneural bHLH transcription factor, and a second nucleic acid molecule encoding a RGC transcription factor selected from the group consisting of Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, Tcf3, and combinations thereof.

16. The method of claim 14, wherein the administration comprises a first administration of a composition comprising a first nucleic acid molecule encoding a proneural bHLH transcription factor, and a second administration at a subsequent time point of a composition comprising a second nucleic acid molecule encoding an RGC transcription factor selected from the group consisting of: Onecut1, Pou4f2, Islet1, Islet1, Irx2, Irx5, Neurod2, Ebf1, Tcf3, and combinations thereof.

17. The method of claim 14, wherein the subject is treated for retinal disease, retinal damage or degeneration.

18. The method of claim 14, wherein the subject is an adult.

19. The method of claim 14, wherein a vector comprises the nucleic acid molecule.

20. The method of claim 19, wherein the vector is a non-viral vector or a viral vector.

21. The method of claim 20, wherein the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

22. The method of claim 14, wherein the administration to the retina is intravitreal or subretinal injection.

23. The method of claim 15, wherein the proneural bHLH transcription factor and the RGC transcription factor are expressed as a fusion protein.

24. The method of claim 15, wherein the first nucleic acid molecule and / or the second nucleic acid molecule is mRNA.

25. The method of any one of claims 15 to 24, wherein the proneural bHLH transcription factor comprises Ascl1, Atoh1, or Atoh7.

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